RAB23 facilitates clathrin-coated nascent vesicle formation at the plasma membrane and modulates cell signaling

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Rakibul Hasan, Maarit Takatalo, Pekka Nieminen, Ritva Rice, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4539384/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Apr, 2025 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted 4 You are reading this latest preprint version Abstract RAB23 is known to regulate several growth factors signaling during organogenesis. RABs and other small GTPases function as molecular switches during cellular membrane trafficking. However, what has not been established is how RAB23 functions during cellular membrane trafficking and how this influences cell signaling. To address this, we characterized RAB23’s localization in the endocytic pathway and determined the route of endocytosis. We find that RAB23 interacts with β-adaptin (AP2β1) subunit of the clathrin adaptor protein 2 (AP-2) complex, suggesting RAB23’s involvement in clathrin-dependent endocytosis at the plasma membrane. Our results show that RAB23 might function at multiple steps during clathrin-coated nascent vesicle formation. We find that RAB23 interacts with clathrin assembly protein PICALM, vesicle curvature protein endophilin A2, and a protein linked with vesicle scission, cortactin. To understand the functionality of RAB23, we performed time-lapse live cell imaging of transferrin uptake, which showed that clathrin-dependent endocytosis is affected in RAB23 deficient osteoprogenitors with inefficient cargo internalization. Our results show that deficiency of RAB23 reduced the interaction between β-adaptin and clathrin. We demonstrate that vesicle formation upon BMP stimulation and subsequent signal transduction is aberrant in RAB23-deficient cells. We further show evidence by providing microarray data-driven hypergeometric test of differentially expressed genes in WT and RAB23-deficient samples which suggests RAB23’s participation in vesicle formation, endocytosis and cell signaling. Collectively, our data indicate a role for RAB23 in vesicle formation, membrane trafficking, and cell signaling. Adaptor protein Coat protein Endocytosis RAB23 Vesicle Signaling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Ras-associated binding 23 (RAB23) protein belongs to the Rab-GTPase family of proteins and is expressed during embryonic development and in the adult brain [ 1 – 3 ]. RAB and other small GTPase family (Arf and Rho) proteins function as key molecular switches by hydrolyzing active GTP-bound to inactive GDP-bound forms during the formation, transport, docking and fusion of vesicles in the endocytic, recycling and in the secretory pathways [ 4 , 5 ]. RAB family proteins also known as signal transducers control intercellular communication by restricting ligand secretion and by controlling cargo internalization, for example, ligand-bound receptors [ 6 – 9 ]. Activated RABs utilize cytoskeleton proteins, motor proteins and effector proteins during vesicle transportation and docking to the destined cellular compartment [ 10 , 11 ]. RABs function by recruiting adaptor proteins, phosphatases, kinases, other RABs, actin filaments and microtubules [ 12 ]. RABs are found in almost every organelle and determine transport specificity and organelle membrane identity [ 13 , 14 ]. For instance, RAB5 is localized at the plasma membrane, clathrin-coated vesicles (CCV) and early endosomes and regulates early vesicle fusion [ 15 ]. RAB24 is localized at endoplasmic reticulum and is involved in autophagosome formation [ 16 ]. RAB23 has been localized to the plasma membrane and in the endocytic pathway [ 17 ]. Mutations in RAB23 cause Carpenter syndrome (CS), which is characterized by developmental defects in the heart, neural tube, and skeleton (MIM# 201000, Acrocephalopolysyndactyly type II) [ 18 – 21 ]. We have previously shown how RAB23 regulates skeletogenesis by suppressing aberrant ossification in the calvarial sutures [ 22 ]. This developmental phenotype was affected through the negative regulation of fibroblast growth factor (FGF) and Hedgehog (Hh) signaling via the signal transducers pERK1/2 and GLI1. We have also shown that RAB23 regulates musculoskeletal development through TGFβR and BMP signaling [ 23 ]. In addition, RAB23 regulates neural tube and cardiac development through Hh and Nodal signaling, respectively [ 2 , 24 ]. Also, aberrant RAB23 signaling results in the formation, invasion and metastasis of many different tumors [ 25 , 26 ]. Even though RAB23 is known to regulate several growth factors signaling what is not known is how RAB23 regulates vesicle trafficking and how this might influence growth factor signaling. Endocytic vesicles allow cells to uptake extracellular substances: ligands, receptors, soluble molecules, proteins and lipids by membrane internalization [ 27 – 29 ]. These vesicles can be recycled back to the plasma membrane, or become mature into a late endosome and eventually undergo lysosomal degradation along with its cargo [ 30 ]. Vesicles that form at the plasma membrane go through clathrin (coat protein)-dependent, caveolae-dependent, or independent routes of membrane internalization [ 27 , 30 ]. Clathrin-mediated endocytosis is a well-characterized pathway, which utilizes adaptor protein 2 (AP-2) complex at the plasma membrane [ 31 , 32 ]. AP-2 is a heterotetrameric complex, consisting of α-adaptin (1 and 2), β2, µ2 and σ2 subunits [ 33 , 34 ]. Clathrin is also involved in endosome formation at the trans- Golgi network, which utilizes adaptor protein 1 (AP-1) complex [ 35 , 36 ]. Clathrin-dependent vesicle formation is a multi-step process that starts with AP-2 mediated recognition of cargo at the membrane site, where AP-2 recognizes and interacts with the cargo and G proteins and forms the inner adaptor layer, followed by clathrin recruitment takes place to form the cage-like outer layer that wraps the adaptor layer. Membrane curvature takes place simultaneously at this stage. Finally, the nascent vesicle undergoes neck scission and detaches from the membrane [ 28 , 30 , 37 ]. In this study, we aimed to understand how RAB23 is involved in cellular membrane trafficking thereby its involvement in cell signaling. We demonstrate that RAB23 may be involved in multiple steps during clathrin-dependent nascent vesicle formation; cargo recognition with AP-2, clathrin assembly, membrane bending and scission. We show that RAB23 deficiency causes a reduction in the interaction between AP-2 (β-adaptin) and clathrin. Our results show that deficiency of RAB23 affects vesicle formation and membrane internalization within the endocytic pathway and impairs BMP2 signaling. Furthermore, by analyzing microarray expression data from WT and RAB23 deficient samples, we provide evidence that RAB23 is involved in vesicle formation, membrane trafficking and TGF-beta receptor signaling pathway. Collectively, our data indicate a role for RAB23 in nascent vesicle formation, cargo internalization and the regulation of cell signaling. Materials and Methods Reagent/Resource table Reagent/Resource Reference or Source Identifier Antibodies Endosomal Marker Antibody Sampler Kit: Rabbit anti-EEA1 Rabbit anti-RAB5 Rabbit anti-RAB7 Rabbit anti-RAB11 Rabbit anti-Clathrin Rabbit anti-Caveolin 1 Cell signaling technology Cat#12666 Rabbit anti-Clathrin Cell signaling technology Cat#4796 Rabbit anti-LC3A/B Cell signaling technology Cat#12741 Mouse anti-Clathrin (X22) Abcam Cat#2731 Mouse anti-β actin Abcam Cat#ab8226 Rabbit anti-β actin Abcam Cat#ab8227 Rabbit anti-RAB23 Proteintech Cat#11101-1-AP Rabbit anti-AP2B1 (β-adaptin) Proteintech Cat#15690-1-AP Mouse anti-AP2α1 (α-adaptin 1) (C-5) Santa Cruz Biotechnology Cat#sc-398024 Mouse anti-AP2α2 (α-adaptin 2) (F-12) Santa Cruz Biotechnology Cat#sc-55497 Rabbit anti-Transferrin Proteintech Cat#17435-1-AP Rabbit anti-VAMP8 Proteintech Cat#15546-1-AP Mouse anti-HA Sigma-Aldrich Cat#H3663 Rabbit anti-pSMAD1/5/8 Millipore Cat#AB3848 Mouse anti-αTubulin Sigma-Aldrich Cat#T6199 Mouse anti-SH3GL1, Endophilin II (A-11) Santa Cruz Biotechnology Cat#sc-365704 Mouse anti-CALM (A-2), PICALM Santa Cruz Biotechnology Cat#sc-271224 Mouse anti-Cortactin (H-5) Santa Cruz Biotechnology Cat#sc-55579 Rabbit anti-GFP Invitrogen Cat#A-11122 Transferrin from Human Serum, Alexa 594 Conjugate Thermo Fisher Scientific Cat# T13343 Goat anti-rabbit IgG (H + L), Alexa 488 Thermo Fisher Scientific Cat#A-11008 Goat anti-mouse IgG (H + L), Alexa 546 Thermo Fisher Scientific Cat#A-110003 Goat anti-mouse IgG (H + L), Alexa 488 Thermo Fisher Scientific Cat#A-11001 Goat anti-rabbit IgG (H + L), Alexa 647 Thermo Fisher Scientific Cat#A-21245 Goat anti-rabbit 680LT Invitrogen Cat#925-68021 Goat anti-rabbit 800CW Invitrogen Cat#925-32211 Goat anti-mouse IRDye 800CW Invitrogen Cat#925-32210 Chemicals, Peptides, and Recombinant Proteins BMP2 R&D Cat#355-BM-010 4% PFA in PBS Thermo Fisher Scientific Cat#15424389 Hoechst 33342 Thermo Fisher Scientific Cat# H3570 Pierce IP lysis buffer Thermo Fisher Scientific Cat#87787 Prolong gold antifade Thermo Fisher Scientific Ref: P36934 PageRuler Plus prestained protein ladder Thermo Fisher Scientific Cat#26620 Odyssey blocking buffer LI-COR 927-40100 Pierce protease inhibitor Thermo Fisher Scientific Cat#A32955 Pierce phosphatase inhibitor Thermo Fisher Scientific Cat#A32957 FuGene ® 6 Transfection Reagent Promega Cat# E2691 Protein G Mag sepharose GE Healthcare Cat#28944008 Critical Commercial Assays iST GFP-Trap test kit Chromotek gtak-iST-8 Pierce BCA protein assay kit Thermo Fisher Scientific Cat#23225 Cell Brite Biotium Cat#30024 Cell Brite Biotium Cat#30021 Experimental Models: Cell Lines Human MG-63 Sigma Cat# 86051601 Mouse Calvaria derived (CD) cells [ 22 ] https://elifesciences.org/articles/55829 Experimental Models: Organisms/Strains Rab23opb2 mice C57Bl/6 [ 22 ] https://elifesciences.org/articles/55829 Recombinant DNA Plasmid: pEGFP-C1 BD Biosciences Clontech Cat#6084-1 Plasmid: RAB23-pEGFP-C1 This paper N/A Plasmid: HA-RAB23-pcDNA 3.1 (-) This paper N/A Software and Algorithms Fiji ImageJ National Institute of Health https://fiji.sc/ JACoP National Institute of Health https://fiji.sc/ Chipster 3.16.0 CSC https://chipster.csc.fi/ Odyssey infrared imaging system LI-COR Biosciences Model 9120 Other 4–20% Mini-PROTEAN TGX Gels Biorad Cat#456–1094 DMEM (Low glucose) Life Technologies Cat#11885084 DMEM (High glucose) Life Technologies Cat#41965039 Opti-MEM Life Technologies Cat#30985047 Trypsin EDTA Life Technologies Cat#25200056 FBS Life Technologies Cat#10270106 Gibco Sodium Pyruvate (100 mM) Thermo Fisher Scientific Cat#11360039 BSA Sigma-Aldrich Cat#A3059 Penicillin/Streptomycin Lonza Catalog#DE17-602E Ascorbic acid β-glyceraldehyde Triton X-100 Sigma-Aldrich Cat#T8787 Goat serum Life Technologies Cat# PCN5000 Cell line and maintenance Human osteosarcoma MG-63 cell line (Sigma; 86051601) was used for GFP-Trap assay (Chromotek; gtak-iST-8), protein immunoprecipitation and protein co-localization studies. Cells were cultured in DMEM containing low glucose (Life Technologies; 11885084) and supplemented with 10% FBS (Life Technologies; 10270106), glutamine, penicillin, streptomycin (Lonza; DE17-602E) and maintained at 37°C with 5% CO 2 . Mouse primary cells Mouse calvaria derived (CD) primary cells were obtained from Wt and Rab23 −/− embryos at E15.5. Generation of Rab23 −/− mouse, and CD primary cell isolation procedure and maintenance have been described previously [ 22 , 38 ]. Primary cells have been maintained in DMEM containing high-glucose (Life Technologies; 41965039) and supplemented with 10% FBS, glutamine, penicillin, streptomycin and maintained at 37°C with 5% CO 2 . The second passage of cells was used for experiments. Starved cells were stimulated with osteogenic medium containing β-glycerophosphate (50 µg/ml), ascorbic acid (25 µg/ml) and BMP2 (25 ng/ml) in addition to the growth medium containing FBS. Expression vectors Human full-length RAB23 coding region was conjugated with HA in HA-pcDNA3.1 expression vector. HA-RAB23 pcDNA3.1 (this paper) was used for HA-RAB23 protein expression, protein co-immunoprecipitation and cell immunostaining studies. HA-empty vector was used as control for cell immunostaining. Human full-length RAB23 coding region was conjugated with EGFP in EGFP-pcDNA3.1 expression vector. RAB23-pEGFP-C1 (this paper) was used for EGFP-RAB23 protein expression, protein co-immunoprecipitation using GFP-Trap assay and cell immunostaining studies. EGFP expression vector pEGFP-C1 (Clontech; 6084-1) were used for protein co-immunoprecipitation using GFP-Trap assay and cell immunostaining. Plasmid transfection FuGene 6 Transfection Reagent (Promega; E2691) was used for transient plasmid transfections according to the manufacturer’s protocol. Transfection reagents were pipetted in Opti-MEM medium (Life Technology; 30985047), MG-63 Cells were allowed to grow 48 hours after transfection for transient expression of RAB23 protein with HA tag and with GFP tag. Control GFP-empty vector and HA-empty vector was used to express GFP and HA proteins. iST GFP-Trap assay GFP-Trap based co-immunoprecipitation was performed using GFP-Trap A agarose beads (iST GFP-Trap Test Kit, Kit for AP-MS sample preparation of GFP-fusion proteins, Chromotek; gtak-iST-8,). MG-63 cells were transfected with control GFP and GFP-RAB23 plasmid constructs. After transfection, cells were allowed to grow for 48 hours followed by 1 hour starvation and stimulation with osteogenic medium (DMEM with 10% FBS, 50 µg/ml β-glyceraldehyde, 50 µg/ml ascorbic acid, 25 ng/ml BMP2) for 10 minutes, and the subsequent procedure was carried out according to the manufacturer’s protocol. IP lysis buffer, IP wash I and II buffers were prepared according to the manufacturer’s recommendation. The eluted samples were further processed for SDS-PAGE for western blotting analysis using anti-GFP, anti-β actin, anti-Cortactin, anti-PICALM and anti-Endophilin A2 antibody. Co-immunoprecipitation , sample precipitation and analysis For co-immunoprecipitation experiments using anti-HA antibody, MG-63 cells were transfected using HA-RAB23 pcDNA3.1 expression vector. After 48 hours of transfection cells were starved for 1 hour followed by stimulated with osteogenic medium (DMEM with 10% FBS, 50 µg/ml β-glyceraldehyde, 50 µg/ml ascorbic acid, 25 ng/ml BMP2) for 10 minutes. For co-immunoprecipitation using β-adaptin antibody, E15.5 Wt and Rab23 −/− mouse calvaria derived primary cells were used. The second passage of cells was used for co-immunoprecipitation. Cells were serum starved for 1 hour followed by BMP2 was added at a concentration of 75 ng/ml and kept at 37°C for 5 minutes. In both cases, cells were lysed for 20 min at 4°C by Pierce IP lysis buffer (Thermo Fisher Scientific; 87787) added with protease inhibitor (Thermo Fisher Scientific; A32955) and phosphatase inhibitor cocktails (Thermo Fisher Scientific; A32957). Protein lysates were clarified using centrifugation at 14,000 rpm for 15 minutes at 4°C. Input protein samples were taken and stored. Supernatant fractions from control samples (un-transfected) and transfected samples were kept with mouse primary IgG and mouse anti-HA antibody, respectively for co-immunoprecipitation at 4°C for overnight. Wt and Rab23 −/− mouse primary calvaria derived samples were kept with rabbit anti-β-adaptin antibody for co-immunoprecipitation. Next day protein samples were transferred into fresh tubes and protein G Mag sepharose 4 Fast Flow (GE healthcare; 28944008) was added on the protein sample for 6 hours at 4°C. After several washing steps of the beads, co-immunoprecipitated proteins (anti-HA antibody bound) were eluted twice by 2.5% acetic acid. Eluted protein volumes were precipitated using chloroform/methanol precipitation procedure. Precipitated samples were dried using SpeedVac concentrator (Thermo Fisher Scientific). Dry precipitated protein samples were re-solubilized in reduced SDS sample buffer. Anti-β-adaptin bound protein samples were eluted in 2x samples buffer boiled at 95° C for 6 minutes and analyzed by 4–20% SDS–PAGE (Biorad; 456–1094) and western blotting with rabbit primary anti-β-adaptin, anti-α adaptin 1, anti-α adaptin 2, anti-clathrin,anti-RAB23 and anti-β actin antibodies. Fluorescent intensity was detected using the infrared Odyssey System (Li-Cor Biosciences; 9120). BMP2 stimulation and analysis of pSMAD and VAMP8 levels WT and Rab23 −/− calvaria derived primary cells were isolated at embryonic day E15.5 and cultured on DMEM growth medium containing 10% FBS for several days. After reaching to 80–90% confluency cells were trypsinized and equal number of WT and Rab23 −/− cells were plated on growth medium for BMP2 stimulation study. After 24 hours, cells were serum starved for 1 hour for BMP2 (R&D; 355-BM-010) stimulation. After a mild wash, BMP2 was added at a concentration of 75 ng/ml and kept at 37°C for 5 and 10 minutes, followed by cells were fixed and immunostained with anti-clathrin and anti-β adaptin antibody for vesicle formation, and immunostained with pSMAD1/5/8 to analyze the BMP2 signals in WT and Rab23 −/− samples. Confocal microscopy was performed for imaging. pSMAD1/5/8 data is presented as pSMAD1/5/8 positive nuclei divided by all nuclei. Western blotting using anti-pSMAD1/5/8 and anti-VAMP8 antibody was used to detect the level of pSMAD1/5/8 and VAMP8 and normalized against α-tubulin and β-actin, respectively. Cell lysis , protein extraction , quantification and western blotting Cells were lysed with RIPA buffer containing 0.1% SDS together with protease inhibitor and phosphatase inhibitor on ice. After brief sonication (10 seconds, twice) cell lysates were centrifuged at 14,000 rpm for 10 minutes at 4˚C to collect the protein supernatant. By using BCA protein assay kit protein concentrations were measured for western blotting analysis. Equal amount of proteins from wild type and Rab23 −/− cells were subjected to prepare to separate on SDS-PAGE under reduced gel electrophoresis (4–20% Mini-PROTEAN TGX Gels, Bio-Rad; 456–1094). After transferring to nitrocellulose membrane, membranes were blocked by odyssey blocking buffer at room temperature for 3 hours. Membranes were then incubated with primary antibodies and kept overnight at 4°C. Fluorophore-conjugated goat anti-rabbit or anti-mouse secondary antibodies were used against primary antibody at room temperature for 1 hour. β-actin (for VAMP8) and α-Tubulin was used to normalize protein expressions. Membranes were scanned using an Odyssey infrared imaging system (Odyssey; LI-COR Biosciences, model 9120). Band intensity was determined using ImageJ software (NIH). Transferrin uptake assay WT and Rab23 −/− calvaria derived primary cells were isolated at embryonic day E15.5 and cultured for several days. After reaching to 80–90% confluency cells were trypsinized and equal number of cells were plated for transferrin uptake study. After 48 hours of culture cells were serum starved for 1 hours, after a mild wash Alexa Fluor™ 594 Conjugate transferrin (Thermo Fisher Scientific; T13343) was added at 25 µg/ml with 0.1% FBS and kept at 37°C for 5, 10, 30, 45, 60 or 120 minutes. Cells were washed several times and lysed by ice cold RIPA buffer containing 0.1% SDS. Cell’s lysates were briefly sonicated and centrifuged to recover protein fractions. Protein concentration was measured using BCA protein assay kit and equal amount of WT and Rab23 −/− proteins were prepared for western blot analysis. For transferrin uptake in WT and Rab23 −/− CD cells and subsequent cell immunostaining analysis, transferrin was pulsed for 5, 10 and 30 minutes in the above-mentioned conditions followed by washing and fixed in 4% paraformaldehyde for 20 minutes and continued for cell immunostaining after staining with Cell Brite plasma membrane dye (Biotium; 30024). Transferrin uptake was quantified from the images as intensity by ImageJ. For quantification of transferrin uptake at the live cell periphery, time-lapse images were segmented at the beginning and at the end of the time lapse. Followed by intensity of the transferrin was measured in the segmented images using ImageJ tool without interfering/adjusting the transferrin intensity. Data presented as the intensity of the segmented image of the first time-lapse divided by the intensity of the image of the first time-lapse as 1 at the beginning of the time-lapse. After 5 minutes, the intensity of the image of last time-lapse divided by the intensity of the image of first time-lapse. Flow cytometry Flow cytometry was performed to understand the transferrin uptake by Wt and RAB23 deficient cells. WT and Rab23 −/− calvaria derived primary cells were isolated at embryonic day E15.5 and cultured until the confluency reached to 80–90%. Thereafter, cells were trypsinized and equal number of cells were plated for transferrin uptake study. After 48 hours of culture cells were serum starved for 1 hours, after a mild wash Alexa Fluor™ 594 Conjugate transferrin (Thermo Fisher Scientific; T13343) was added at 25 µg/ml with 0.1% FBS and kept at 37°C for 5, 10 and 30 minutes. After brief washes with PBS, cells were trypsinized and centrifused to remove the trypsin and then resuspended in PBS for flow cytometric detection and quantification of Alexa fluor 594. The normalization was performed accordingly by Wt and RAB23 deficient cells that kept without transferrin. The flow cytometry analysis was performed at the HiLife Flow Cytometry Unit, University of Helsinki with Novocyte Quanteon flow cytometer. AlexaFluor 594 was detected with 561nm laser excitation and 615/20 BP filter. Cells population was gated on FCS/SSC by excluding the debris only. NovoExpress was used as analysis software. Immunostaining , co-localization and confocal microscopy Immunostaining was performed for vesicle characterization on MG-63 cells, BMP2 related, transferrin uptake related and co-localization dynamics related studies on WT and Rab23 −/− CD cells. MG-63 cells were grown on coverslips for 24 hours and transfected with either HA-RAB23 pcDNA3.1, HA-empty vector, RAB23-pEGFP-C1 or GFP-empty expression vector for another 48 hours. For BMP2,transferrin uptake and co-localization dynamics related studies, E15.5 WT and Rab23 −/− CD cells were grown on cover slip and carried out the BMP2,transferrin uptake and co-localization dynamics studies at different time points at 37°C. For co-localization dynamics studies cells were starved for 1 hour followed by stimulated with osteogenic medium (DMEM with 10% FBS, 50 µg/ml β-glyceraldehyde, 50 µg/ml ascorbic acid, 25 ng/ml BMP2) for 5 and 10 minutes. In these experiments, cells were fixed in 4% PFA (Thermo Fischer Scientific; 15424389) for 20 minutes and permeabilized by 0.5% Triton-X-100 (Sigma-Aldrich; T8787) diluted in PBS for 5–10 minutes. Cells were blocked with 5% goat serum (Life Technologies; PCN5000) diluted in blocking buffer (5% BSA and 0.1% Tween-20 in PBS) for 1 hour at room temperature followed by primary antibodies (including anti-GFP antibody) incubation at 4°C overnight in blocking buffer (LI-COR; 927-40100). Next day cells were incubated with Alexa fluor-conjugated secondary antibodies diluted in blocking buffer for 1h at RT. Cells were counterstained with Hoechst 33342 (Thermo Fisher Scientific; H3570) and mounted with ProLong Gold Antifade (Thermo Fisher Scientific; P36934). Confocal microscopy was performed at the Biomedicum imaging unit, University of Helsinki. Confocal microscopy images were obtained with an inverted confocal microscope system (Zeiss LSM 880) using a Plan apochromatic 63x/1.4 NA oil objective. Excitation was achieved using diode 405nm and argon multiline 350/488/532/561/633 nm lasers in confocal microscopy analysis. All images were taken at RT and analyzed with Fiji ImageJ 1.50b (64-bit) software. ImageJ plugin tool JACoP was used to study the Pearsons co-localization co-efficient. While determining Pearsons’s correlation coefficient we have provided total signal of each co-localization pair. The Pearsons method of correlation co-efficient used the total signal and determined the co-localization co-efficient. Live cell time-lapse imaging Time-lapse microscopy was performed at the Biomedicum imaging unit, University of Helsinki. Time-lapse imaging was performed to study transferrin internalization dynamics in presence of membrane dye in Wt and Rab23 −/− CD primary cells. Initially, membrane was stained with Cell Brite membrane dye (Biotium; 30021) followed by 5-minute transferrin (red) pulse, washing the cells and time-lapse imaging for 5 minutes (frame rate 1-s interval). Zeiss LSM 880 Confocal microscopy based time-lapse images were obtained with an inverted confocal microscope system using a Plan apochromatic 63x/1.4 NA oil objective with sequential dual laser excitation at 594 nm and 488 nm. Bioinformatics analysis Image J software was used for image processing. Image J plugging tool JACoP was used for Pearson’s co-localization studies. Windows movie maker, Image J and VEED.IO were used to process the videos presented in this article. Hypergeometric test for consensuspathDB, Gene Ontology analysis for KEGG, molecular and biological functions of microarray obtained data have been performed by Chipster 3.16.0 [ 39 ]. Statistical analysis Paired student t -test has been applied to perform the statistics of all the data obtained from western blotting, co-localization. Data are represented as mean ± SD and P -value less than 0.05 considered as statistically significant. Results Localization of RAB23 in the endocytic pathway RAB23 is proposed to be involved in the endocytic pathway, however, little is known about the precise location during endocytosis. To understand this, we performed protein co-localization studies of RAB23 with endocytic pathway-specific vesicle markers EEA1, RAB5, RAB7, RAB11 and LC3A/B. RAB23 was over-expressed in human osteosarcoma MG-63 cells by using N-terminally HA-tagged full-length RAB23, HA-RAB23 pcDNA3.1 expression vector. HA-empty vector was used as a control for co-localization of HA protein with these markers. After 48 hours of transfection, cells were starved for 1 hour followed by stimulation with osteogenic medium for 10 minutes. Cells were then fixed and immunostained with anti-HA and anti-EEA1, anti-HA and anti-RAB5, anti-HA and anti-RAB7, anti-HA and anti-RAB11, and anti-HA and anti-LC3A/B antibodies followed by confocal microscopy. We found that HA-RAB23 co-localizes with the early endosomal markers EEA1 and RAB5 at different cellular locations including in the cell periphery (Fig. 1 a). HA-RAB23 co-localizes with late endosomal marker RAB7 and autophagy marker LC3 A/B mostly in the cytoplasmic region (Fig. 1 a). HA-RAB23 showed very low or no co-localization with recycling endosomal marker RAB11 (Fig. 1 a). We found that control HA protein did not show any co-localization with any of these markers (Fig. S1 a). Quantification by Pearson’s correlation coefficient also showed low interaction (Fig. S1 c). Next, we quantified the co-localizations of HA-RAB23 with the vesicle markers. We found that HA-RAB23 highly co-localized with the early endosomal marker EEA1 and moderately with RAB5 (Fig. 1 a, b). HA-RAB23 showed very strong co-localization with the late endosomal marker RAB7 and the autophagy marker LC3A/B (Fig. 1 a, b). However, HA-RAB23 showed no or very low level of co-localization with the recycling endosome marker RAB11 (Fig. 1 b). RAB23’s co-localization with EEA1 and RAB5 indicates that RAB23 may participate in early vesicle formation. RAB23 co-localization with the late endosomal marker RAB7 and the autophagy marker LC3A/B collectively suggests the involvement of RAB23 in the late endocytic pathway. Association of RAB23 to clathrin-mediated endocytosis We aimed to pinpoint the endocytic route where RAB23 might be functional, specifically in receptor-mediated or receptor-independent pathways. We performed co-localization analysis of HA-RAB23 with clathrin, a marker for the receptor-mediated endocytic route and caveolin 1, a marker for receptor-independent route of endocytosis. RAB23 expression in these cells was over-expressed using the HA-RAB23 pcDNA3.1 expression vector and the HA-empty vector was used for control co-localization studies. Cells were immunostained with anti-HA and anti-clathrin, anti-HA and anti-caveolin 1 antibodies, followed by confocal microscopy was performed. Co-localization images show that HA-RAB23 strongly co-localizes with clathrin at different cellular locations including in the cell periphery and with caveolin 1 showed low co-localizations (Fig. 1 c). Quantification of the co-localizations shows a high correlation coefficient of HA-RAB23 with clathrin and low correlation coefficient of HA-RAB23 with caveolin 1 (Fig. S2 ). However, the control HA protein did not show any co-localizations with any of these proteins (Fig. S1 b) and showed a low Pearsons correlation coefficient (Fig. S1 c). RAB23 interacts with β-adaptin subunit (AP2β1) of the clathrin adaptor protein 2 (AP-2) complex Since RAB23 co-localized with the early endosomal markers EEA1 and RAB5 and with clathrin, we tested whether RAB23 shows any protein-protein interaction with clathrin. We performed protein co-immunoprecipitation using anti-HA antibody on MG-63 osteoblastic cells that were transfected with HA-RAB23 pcDNA3.1 expression vector. After 48 hours of transfection, RAB23 expression was analyzed by western blotting using anti-RAB23 antibody that recognized endogenous RAB23 and overexpressed HA-tagged RAB23 (Fig. S3 ). Protein co-immunoprecipitation using control IgG and anti-HA antibody followed by western blotting using anti-clathrin and anti-RAB23 antibody did not recognize clathrin but recognized HA-RAB23 indicating that RAB23 shows no interaction with clathrin coat (Fig. S4 ). Clathrin is involved in the very early steps of nascent vesicle formation in receptor-mediated endocytosis where the adaptor protein 2 (AP-2) complex first becomes recruited at the plasma membrane followed by clathrin coat assembly which takes place around AP-2 to form double-layered vesicle. AP-2 is a heterotetrameric complex, consisting of α-adaptin (1 and 2) β2, α2 and σ2 subunits [ 33 , 34 ]. And studies in mammals show that the appendage of the β2 subunit (β-adaptin) specifically interacts with clathrin [ 40 – 42 ]. To determine whether RAB23 co-localizes and/or interacts with α-adaptin (1 and 2) and β2 subunits of AP-2, we performed co-localization analysis of GFP-RAB23 with α-adaptin 1 (AP2α1), α-adaptin 2 (AP2α2) and HA-RAB23 with β-adaptin (AP2β1) subunits of AP-2 in MG-63 cells transfected with either GFP-RAB23 or HA-RAB23 pcDNA3.1 expression vectors. For control co-localization GFP-empty vector and HA-empty vector were used. Cells were stained with anti-HA, anti-GFP, anti-α-adaptin 1, anti-α-adaptin 2 and anti-β-adaptin antibody. Confocal microscopy images show that β-adaptin co-localizes with HA-RAB23 at the cell periphery (Fig. 1 c) and showed no co-localizations with control HA protein (Fig. S1 b). Quantification of the co-localizations of HA-RAB23 with β-adaptin indicates strong co-localization of HA-RAB23 with the β-adaptin (Fig. S2 ). Further protein co-immunoprecipitation analysis using control IgG and anti-HA antibody on MG-63 cells (transfected with HA-RAB23 pcDNA3.1 expression vector) followed by western blotting against β-adaptin detected a protein band in the sample lane at the level (105 kDa) similar to the transfected and un-transfected input β-adaptin protein level (Fig. 1 d). Western blotting against-RAB23 detected HA-RAB23 protein band (⁓30 kDa) in the sample lane and input lanes but not in the control IgG lane. Western blotting against β-actin detected the β-actin protein band (⁓42 kDa) in the input lanes. (Fig. 1 d). GFP-RAB23 co-localizes with α-adaptin 1 at the cell periphery (Fig. 1 e) and showed little or no co-localization with α-adaptin 2 (Fig. 1 e). None of the α-adaptin 1 and α-adaptin 2 proteins showed co-localization with control GFP (Fig. S5 ). Subsequent protein co-immunoprecipitation followed by western blotting against α-adaptin 1 and α-adaptin 2 failed to detect α-adaptin 1 (Fig. 1 f) and α-adaptin 2 (Fig. 1 g) respectively in the sample lane. Internal control β actin was detected in the input lanes when immunoblotted against β-actin (Fig. 1 g, f). Collectively, these findings indicate that HA-RAB23 interacts with the β-adaptin subunit of the adaptor protein 2 (AP-2) complex (Fig. 1 h). RAB23, β-adaptin and clathrin show triple co-localization If RAB23 interacts with β-adaptin then RAB23, β-adaptin and clathrin might show collective co-localizations during AP-2/clathrin mediated vesicle formation. To investigate this hypothesis, we performed triple co-localization analysis for RAB23, β-adaptin and clathrin. RAB23 was over-expressed in MG-63 cells using GFP-RAB23 expression plasmid where GFP was N-terminally tagged with RAB23. Control GFP was also over-expressed in these cells using GFP plasmid. Cells were stained with anti-β adaptin and anti-clathrin antibodies together with GFP-RAB23 or with control GFP. Analysis of triple co-localization at the cell periphery and subsequent quantification suggests that GFP-RAB23, β-adaptin and clathrin show triple co-localization at the cell periphery (Fig. 2 a, Fig. S6 ). However, co-localization analysis using anti-β adaptin and anti-clathrin together with control GFP did very low triple co-localization at the cell periphery (Fig. 2 b, Fig. S6 ). These findings indicate that RAB23 may participate in early membrane internalization in the AP-2/clathrin route. Exploring possible roles of RAB23 during clathrin-coated nascent vesicle formation AP-2/clathrin vesicle formation is a multi-step process, which initiates after recognition of the cargo by AP-2, followed by clathrin recruitment, curvature formation, scission and then detachment of the early vesicle from the membrane [ 28 ]. We aimed to understand which of these stages RAB23 might be involved. To do so, we performed co-localization analysis of GFP-RAB23 with PICALM/AP180 (Phosphatidylinositol-binding clathrin assembly protein); a protein required for clathrin binding and assembly [ 43 , 44 ]. GFP-RAB23 with BAR domain-containing protein endophilin, which is required for membrane bending and curvature [ 45 ]. GFP-RAB23 with cortactin, which is recruited to the clathrin during vesicle scission [ 46 ]. RAB23 was over-expressed using the GFP-RAB23 expression plasmid in MG-63 cells and GFP-empty vector was transfected for control co-localization After 48 hours of transfection, cells were fixed and stained against endophilin A2, PICALM and cortactin. Confocal microscopy-based analysis showed that endophilin A2, PICALM and cortactin co-localized with GFP-RAB23 at the periphery of the cell and in vesicle-like structures (Fig. 3 a). We quantified the co-localizations of GFP-RAB23 with endophilin A2, GFP-RAB23 with PICALM and GFP-RAB23 with cortactin (Fig. S7 ). However, the control GFP protein showed no co-localizations with any of these proteins (Fig. S8 a) Quantification by Pearson’s correlation coefficient showed low interaction (Fig. S8 b).We found that GFP-RAB23 strongly co-localized with endophilin A2 and cortactin and moderately with PICALM (Fig. 3 a, Fig. S7 ). To investigate whether RAB23 interacts with these proteins, we performed protein co-immunoprecipitation using GFP-Trap assay on MG-63 cells. These cells were transfected with GFP-RAB23 and control GFP expression plasmids to overexpress GFP-RAB23 and control GFP, respectively. Co-immunoprecipitation using GFP-Trap assay followed by western blotting against GFP on the eluted proteins obtained from control GFP-Trap assay and from GFP-RAB23-Trap assay that detected GFP and GFP-RAB23 (Fig. 3 b, lower panel). Western blotting using anti-PICALM, anti-endophilin A2 and anti-cortactin antibodies detected their corresponding bands 70 kDa, 45 kDa and 88 kDa, respectively, in the GFP-RAB23-Trapped sample to the same level of their input protein bands (Fig. 3 b, upper panel). However, these proteins did not show bands in the control GFP-Trapped sample (Fig. 3 b, upper panel). Western blotting against β-actin detected the β-actin protein band (⁓42 kDa) in the input lanes. These bands detection confirmed that PICALM, endophilin A2 and cortactin interact with RAB23. RAB23 regulates clathrin-mediated cargo internalization To test the functionality of RAB23 in clathrin-mediated endocytosis we investigated clathrin-coated vesicle formation and cargo internalization in mouse-derived WT and Rab23 −/− primary cells using the well-established transferrin model of ligand-receptor endocytosis. Transferrin is a glycoprotein that co-localizes with cytosolic RAB23 [ 17 ] and requires the clathrin assembly protein PICALM for its internalization [ 47 ]. We showed that PICALM interacts and co-localizes with RAB23 (Fig. 3 ). Here, we performed time-lapse microscopy in mouse WT and Rab23 −/− calvaria-derived (CD) primary cells which differentiate into osteoblasts [ 22 ]. After 24 hours of culture, cells were starved for 1 hour followed by transferrin uptaking experiment was performed. Initially, the membrane was stained with CellBrite membrane dye followed by a 5-minute transferrin (red) pulse (alexa-594 conjugated, 25 µg/ml), washing the cells and time-lapse imaging for 5 minutes (frame rate 1-s interval) by using Zeiss LSM880 microscope. Results show that WT cells efficiently internalized transferrin from the cell periphery (Fig. 4 a, Video 1). In RAB23 deficient cells transferrin patches persisted longer at the cell periphery (Fig. 4 a, Video 2) also, many transferrin patches that were internalized often expelled to the cell periphery (Fig. 4 a, Video 2). We initially quantified the internalized transferrin at different time points by western blotting and by Flow cytometry. Serum-starved WT and Rab23 −/− calvaria derived (CD) primary cells were treated with transferrin (alexa-594 conjugated, 25 µg/ml) and incubated for 5, 10, 30, 45, 60 and 120 minutes at 37°C. Western blotting using anti-transferrin antibody showed reduced transferrin uptake in Rab23 −/− cells at all-time points and that transferrin uptake never reached the wild-type levels (Fig. 4 b, c). By using flow cytometry, we analyzed transferrin uptake by these cells at 5, 10 and 30 minutes. Similar to western blotting, flow cytometry results show that Rab23 −/− cells uptake reduced transferrin compared to Wt cells at each time point (Fig. S9 ). We then analyzed transferrin accumulation at the cell periphery in WT and Rab23 −/− cells by using time-lapse images (Fig. 4 d, e). Here, we found that in WT cells transferrin was efficiently internalized from the cell periphery and reduced over time (Fig. 4 d, Video 3). Whereas Rab23 −/− cells showed an accumulation of transferrin at the cell periphery (Fig. 4 d, Video 4). Quantification of transferrin accumulation at the cell periphery over this time period of time-lapse showed that RAB23 deficient cells retain more transferrin at the cell periphery compared to WT cells (Fig. 4 f). These observations are compatible with our findings that RAB23 may be involved in multiple steps during nascent vesicle formation at the cell membrane (Fig. 1 – 3 ), and that deficiency of RAB23 showed an effect on the transferrin vesicle formation and internalization (Fig. 4 ). Transferrin patches co-localize with AP-2 ( β-adaptin) for longer in the absence of RAB23 To understand the dynamics of the decreased AP-2/clathrin regulated transferrin internalization in Rab23 −/− cells, we performed transferrin co-localization with the cargo recognition marker, AP-2 (β-adaptin). Serum-starved WT and Rab23 −/− calvaria derived (CD) primary cells were treated with transferrin (alexa-594 conjugated, 25 µg/ml) and incubated for 5 and 10 minutes at 37°C. Cells were then fixed, immunostained with anti β-adaptin and confocal microscopy was performed to capture the images (Fig. 4 g). Co-localization between β-adaptin and transferrin represents that WT cells had a higher co-localization coefficient at 5 minutes compared to the Rab23 −/− cells (Fig. 4 g, h). At 10 minutes of incubation, transferrin patches showed more co-localization with β-adaptin in Rab23 −/− cells in comparison to WT cells (Fig. 4 g, h). This indicated that in the absence of RAB23, transferrin patches spend a longer time bound to AP-2. Moreover, we found that transferrin uptake in the presence of the membrane dye (cellbrite blue) showed transferrin retention at the cell periphery and less internalization in Rab23 −/− cells at 5, 10 and 30 minutes compared to WT cells (Fig. S10 ). Analysis of RAB23 requirements in the assembly of clathrin-coat to the AP-2 (β-adaptin) Clathrin-coated vesicle formation at the plasma membrane initiates AP-2 mediated cargo selection, followed by the recruitment of PICALM, which plays a critical role in clathrin assembly [ 48 – 50 ]. Our results show that RAB23 co-localizes with clathrin and interacts with β-adaptin and PICALM (Fig. 1 – 3 ). We show that deficiency of RAB23 affects internalization of transferrin (Fig. 4 ). Next, we asked if RAB23 is involved in multiple steps of early vesicle formation, deficiency of RAB23 might show an effect on co-localization of the major proteins involved in vesicle formation. To address this question, we aimed to analyze the dynamics of co-localization of the RAB23 interacted proteins at the cell surface in response to osteogenic medium for 5 and 10 minutes on starved WT and Rab23 −/− CD primary cells (Fig. 5 ). Here, we assessed the co-localization of the proteins; PICALM and clathrin, clathrin and AP-2 which we showed involved in clathrin-mediated early steps of nascent vesicle formation together with RAB23 (Fig. 3 ). Immunostaining and subsequent quantification of co-localization between PICALM and clathrin, AP-2 and clathrin showed reduced co-localization co-efficient in Rab23 −/− cells compared to WT cells at 5 minutes (Fig. 5 a). The early stage (5 minutes) feedback affected the dynamics of vesicle formation at 10 minutes. At this time, we found reduced co-localization of clathrin to AP-2 in Rab23 −/− cells (Fig. 5 b). Our results thus suggest that RAB23 might be involved in several assembly steps during nascent vesicle formation (Fig. 5 c). Rab23 −/− cells show aberrant vesicle formation upon BMP2 stimulation and show reduced interaction between AP-2 (β-adaptin) and clathrin Differential uptake of transferrin in Rab23 −/− cells indicates a possible functional abnormality of AP-2/clathrin mediated ligand-receptor internalization which could result in abnormalities in signaling. Studies have shown that TGFβR/BMPR internalizes through the clathrin route and resides in the EEA1-positive compartment for pSMAD activation [ 51 – 53 ]. We have previously shown that RAB23 regulates TGFβR/BMPR signaling in musculoskeletal development and patterning [ 23 ]. Here, we wanted to understand whether WT and Rab23 −/− cells show differential formation of vesicles upon BMP2 stimulation. We immunostained and subsequently analyzed the co-localization of β-adaptin and clathrin in WT and Rab23 −/− primary cells. Cells were starved followed by stimulated with BMP2 for 5 and 10 minutes. Here, we found that upon BMP2 stimulation for 5 minutes, WT cells efficiently formed vesicles in the cell periphery and also showed vesicle-like structures (Fig. 6 a). However, Rab23 −/− cells showed aberrant formation of vesicles in the cell periphery and showed aberrant vesicle-like structures (Fig. 6 a). This phenomenon of vesicle formation decreased at 10 minutes upon BMP2 stimulation in both the cell types (Fig. 6 a). Quantification of vesicle or vesicle-like structure showed that Rab23 −/− cells showed significantly reduced number of vesicles or vesicle-like structures in these time points when compared to WT cells (Fig. 6 b). To understand, if deficiency of RAB23 affected the interaction between AP-2 (β-adaptin) and clathrin, we performed protein co-immunoprecipitation using control anti-IgG and anti-β-adaptin antibodies on the samples obtained from mouse Wt and Rab23 −/− calvaria derived primary cells. Cells were serum starved for 1 hour followed by BMP2 was added at a concentration of 75 ng/ml in the culture medium and kept at 37°C for 5 minutes. Western blotting on co-immunoprecipitated samples using anti-β-adaptin and anti-clathrin antibodies detected the β-adaptin (105 kDa) band and clathrin (190 kDa) band at the same molecular weight as that of the input β-adaptin and input clathrin protein in Wt and Rab23 −/− samples (Fig. 6 c, d). Quantification of interaction between the β-adaptin subunit of AP-2 and clathrin showed that the interaction clathrin/β-adaptin was drastically reduced in Rab23 −/− samples, indicating that RAB23 is required for efficient interaction between β-adaptin and clathrin. RAB23 deficiency causes reduced expression of PICALM endocytic target R-SNARE protein VAMP8 We show that PICALM, which is an endocytic clathrin adaptor protein, interacts with RAB23 (Fig. 3 ). As PICALM is known for its endocytic function of R-SNARE proteins VAMP2, 3 and 8 [ 49 ], we therefore aimed to understand if RAB23 works at least partly by modulating PICALM recruitments. We might predict less R-SNARE protein level in RAB23 deficient cells. In this regard, Wt and Rab23 −/− calvaria-derived primary cells were serum starved for 1 hour followed by BMP2 was added at a concentration of 75 ng/ml in the culture medium and kept at 37°C for 5 minutes. Western blotting was performed to detect VAMP8 (15 kDa) and β-actin (42 kDa) in these samples (Fig. S11 a). After quantification, we found that Rab23 −/− samples, showed decreased level of VAMP8 compared to Wt samples (Fig. S11 b). RAB23 deficiency caused altered pSMAD 1/5/8 activation upon BMP2 stimulation Our results showed differential formation of vesicles upon BMP2 stimulation in Wt and Rab23 −/− cells and showed reduced interaction between AP-2 and clathrin in Rab23 −/− cells. We next analyzed pSMAD1/5/8 level in starved WT and Rab23 −/− cells by immunostaining and immunoblotting upon BMP2 stimulation for 5 and 10 minutes. Immunostaining using anti-pSMAD1/5/8 antibody and subsequent counting of pSMAD1/5/8 positive cells compared to all cells showed a reduction of pSMAD1/5/8 signal in Rab23 −/− cells compared to WT cells at 5 and 10 minutes (Fig. 7 a, b). Immunoblotting using anti-pSMAD1/5/8 and anti α-Tubulin antibody and subsequent quantification of pSMAD1/5/8 against α-Tubulin showed a reduction of pSMAD1/5/8 level in Rab23 −/− cells compared to WT cells at 5 and 10 minutes (Fig. 7 c, d). These findings indicate that RAB23 may regulate BMP2 signaling. Evidence that RAB23 regulates vesicle biogenesis and signaling receptor activity We have previously shown that BMPs regulate osteogenesis and suture morphogenesis [ 54 ]. Also, RAB23 negatively regulates FGF and Hedgehog signaling in mouse calvarial bone and suture development where we performed a microarray-based gene expression analysis on WT and Rab23 −/− mouse (E15.5) calvarial bones and sutural tissues, which revealed 223 genes were significantly differential expressed [ 22 ]. In this current study, we analyzed the differentially expressed genes by Chipster [ 39 ], a bioinformatic tool to perform hypergeometric test for ConsensusPathDB to understand the functions of the genes that were differentially expressed. We found that RAB23 regulated several differentially expressed genes, which are involved in vesicle-mediated transport and membrane transport in the cell (Fig. 8 a, Table 1 ). This analysis also showed that several genes are involved in the TGFβ receptor signaling pathway (Fig. 8 a, Table 1 ). Upon performing a hypergeometric test for KEGG ontology for the over-representing genes, we found that several genes are involved in endocytosis and regulation of actin cytoskeleton (Fig. 8 b). Further GO (Gene Ontology) analysis for molecular function and biological processes of the underrepresenting genes showed that RAB23 regulates genes that have molecular transducer activity and signaling receptor activity including G-protein coupled receptor activity (Fig. 8 c) and G-protein coupled receptor signaling pathway as biological processes (Fig. 8 d). Discussion RAB-GTPases act as master regulators in the endocytic and secretory pathways [ 55 ]. RAB23 is known to localize to the plasma membrane and the endocytic pathway [ 17 ]. However, multiple endocytic routes exist and the function of RAB23 in the context of membrane trafficking is largely unknown. Our data suggest that RAB23 functions in the clathrin-dependent route where RAB23 may participate in AP-2/clathrin-coated nascent vesicle formation at the plasma membrane (Fig. 1 ). Clathrin-coated nascent vesicle formation is an upstream event of early endosome which starts with AP-2 mediated cargo recognition, followed by clathrin coat assembly and subsequently vesicle scission [ 28 , 30 ]. These nascent vesicles then start docking and fuse with EEA1-positive early endosomes [ 56 ]. Our study reveals that during clathrin-coated nascent vesicle biogenesis at the plasma membrane, RAB23 may function at multiple steps and thus, deficiency of RAB23 affects vesicle formation, internalization, transport and cell signaling. By hypergeometric analysis of microarray data obtained from differentially expressed genes in WT and RAB23 deficient mouse primary cells, we provide further evidence that RAB23 is involved in vesicle formation, endocytosis and cell signaling. Vesicle transport keeps cargo identity intact by forming membrane-bound structures, and at the same time, it is essential to ferry the cargo from one cellular compartment to the target destination [ 57 , 58 ]. Vesicle coats consist of an inner adaptor protein layer that recognizes and interacts with the cargo and G proteins, and a cage-like outer layer that wraps the adaptor layer [ 59 , 60 ]. Upon binding with cargo and G proteins, adaptor proteins form a “prebudding complex”, the inner layer of vesicle. Subsequently, coat proteins are recruited to the prebudding complex to form a cage-like structure, known as the second layer of the vesicle. Finally, a GTPase-mediated hydrolysis, for instance driven by a RAB protein, detachs the nascent vesicle from the cell membrane [ 28 ]. Several other proteins including kinases are also involved in this process [ 12 , 61 ]. Here, we show that in the clathrin-coated endocytic vesicle formation RAB23 interacts with β-adaptin subunit of the adaptor protein 2 (AP-2) complex but not with α-adaptin 1 and 2 (Fig. 1 ). There might be several reasons for this discrepancy. Firstly, the heterotetrametric AP-2 adaptor, which is a big complex of subunits; α-adaptin (⁓110 kDa), β-adaptin (⁓110 kDa), µ2-subunit (⁓50 kDa) and σ2 (⁓17 kDa) might be too big protein (⁓300 kDa) for a small vesicle protein antibody like RAB23 (⁓30 kDa) to pull down. α and µ2 are among the two subunits of AP-2 that are membrane bound and they might have interactions with many other proteins [ 62 ]. Even though RAB23 might have an interaction with these subunits through β-adaptin the only strong interaction subunit (β-adaptin) might come across with RAB23. Secondly, how the subunits of heterotetrameric AP-2 adaptor complex are assembled and dissemble might be another issue and the conformational changes which are highly dynamic. RAB23 might interact with β-adaptin in a conformational state where the complex might loosely become interconnected within themselves and may show only interaction with β-adaptin before their compact assembly or when they are loosely interconnected during conformational changes. In addition to β-adaptin, our result further demonstrates that RAB23 interacts with the clathrin assembly protein PICALM, BAR domain-containing protein endophilin A2 and vesicle scission protein cortactin (Fig. 1 – 3 ). This collectively suggests that the small GTPase protein RAB23 might be involved in multiple steps during clathrin-coated nascent vesicle formation. Clathrin-mediated endocytosis is involved in several important cellular processes, including cargo sorting to the endosome at the plasma membrane and trans- Golgi network-mediated secretion of proteins [ 63 , 64 ]. Perturbation of clathrin in multicellular organisms causes lethality [ 65 , 66 ]. Also, genetically removing the clathrin-dependent core adaptor protein AP-2 results in embryonic lethality in worms, flies and mice [ 29 , 67 , 68 ]. And in the absence of AP-2, the endocytic patch at the plasma membrane takes a significantly longer time to produce vesicles, many patches are unable to form vesicles, retake cargo at the cell membrane and some patches are stacked at the membrane [ 69 ]. Similar to AP-2 disruption, we show that RAB23-deficient cells exhibit reduced transferrin internalization, retention of transferrin at the cell surface and spend longer time at the cell periphery, and some patches retake to the cell membrane as shown by time-lapse live cell imaging (Fig. 4 , Videos 1–4). We demonstrate that transferrin patches are retained in the first step of nascent endosome marked by β-adaptin subunit of AP-2 and take a longer time to become endocytosed (Fig. 4 ). Furthermore, we show that the dynamics of co-localization between β-adaptin of AP-2 and clathrin are aberrant in RAB23 deficient cells (Fig. 5 ). These findings suggest that RAB23, AP-2 and clathrin functions together during endocytic patch formation and efficient patch internalization. Clathrin-mediated transferrin uptake also has been shown affected by several of kinases (92) when they are knocked down, which is an indication that clathrin-mediated endocytosis utilizes numerous proteins for efficient cargo internalization [ 61 ]. We demonstrate that RAB23 deficiency affected the number of vesicle formations with aberrant morphology upon BMP2 stimulation and altered BMP2 signaling. A previous study showed that endocytic clathrin adaptor PICALM directs endocytosis of R-SNARE (VAMP2, 3 and 8) [ 49 ]. Our study showed that PICALM interacts with RAB23 (Fig. 3 ) and that deficiency of RAB23 reduced the expression of VAMP8 (Fig. S11 ). We also show that deficiency of RAB23 causes reduced protein interaction between clathrin and AP-2 (Fig. 6 ). This abnormality of ligand-receptor endocytosis may not be restricted to transferrin, R-SNARE or BMP2 signaling through BMP receptors that we have shown in this study, we speculate that a common mechanism for several growth factors signaling pathways including Hedgehog signaling through ciliary vesicle formation, FGF and TGFβR signaling. RAB23 deficiency could alter many cellular signals that pass through the AP-2/clathrin route. The clathrin route allows selective internalization of various metabolites carrying receptors [ 70 , 71 ]. In RAB23-deficient mice, which mimic Carpenter syndrome in humans, the lack of functional RAB23 results in overt FGF10, Hh and Nodal signaling with consequent misexpression of downstream signal transducers (pERK1/2, Gli1, Lefty1/2 and Pitx2). This leads to overt osteogenesis at the growing bone ends in the developing skull, defective dorsal cell type specification during neural tube closure and abnormal left-right patterning of the heart [ 2 , 22 , 24 , 72 ]. We have shown that RAB23 regulates musculoskeletal development through TGFβR and BMPR signaling [ 23 ]. These phenotypes go hand in hand with our findings that RAB23 modulates adaptor protein-mediated assembly of clathrin during the early steps of endocytosis, which has been shown to regulate growth factor-receptor signaling [ 29 , 73 , 74 ]. In summary, here we show a role for RAB23 in clathrin-mediated nascent vesicle formation and endocytosis. Our results show that in the endocytic pathway, RAB23 co-localizes with early endosomal markers EEA1 and RAB5. RAB23 also co-localizes with late endosomal marker RAB7 and autophagy marker LC3 A/B, reminiscing the previous finding that RAB23 is involved in autophagosome formation during group A streptococcus infection [ 75 ] Our results show, RAB23 interacts with adaptor proteins AP-2 and participates in the clathrin-coated vesicle formation. Clathrin-coated vesicle formation is a multi-step process that includes prebudding, clathrin assembly, curvature formation, and detachment of the nascent vesicle. RAB23 interaction with AP-2, PICALM, endophilin A2 and cortactin as well as co-localization with clathrin is required for proper vesicle formation and subsequent cargo internalization as well as cell signaling (Fig. 9 ). Our data suggest mechanistic insights into the cellular membrane trafficking functions of RAB23 in mammalian cells and shows that RAB23 may play a role at multiple steps during clathrin-coated nascent vesicle formation and endocytosis. Abbreviations AP-1 Adaptor protein 1 AP-2 Adaptor protein 2 AP2β1 β-adaptin subunit of Adaptor protein 2 complex AP2α1 α-adaptin 1 subunit of Adaptor protein 2 complex AP2α2 α-adaptin 2 subunit of Adaptor protein 2 complex BMP Bone morphogenetic protein CCV Clathrin-coated vesicle CDC Calvaria derived cells Co-IP Co-immunoprecipitation CS Carpenter syndrome EEA1 Early endosomal antigen 1 FGF Fibroblast growth factor GEO Gene expression omnibus GFP Green fluorescence protein GO Gene ontology HH Hedgehog PICALM Phosphatidylinositol-binding clathrin assembly protein RAB23 Ras-associated binding 23 TGFβ Transforming growth factor β VAMP8 Vesicle associated membrane protein 8 WT Wild type Declarations Acknowledgments We would like to thank Vesa Olkkonen, Johan Peränen and Maria Sanz Navarro for their critical and constructive discussion on this research project. We also thank Airi Sinkko and Anne Kivimäki and Johanna Pispa for their excellent technical help. Author contributions M. R. H., M. T., P. N, T. M., D. P. R. designed the experiments. M. R. H., M. T., T. M., D. P. R. generated and processed the mice. M. R. H., M. T., R. R., P. N., T. M., D. P. R. wrote and approved the manuscript. M. R. H., M. T., T. M., P. N. performed the experiments. M. R. H. performed time-lapse imaging. D.P.R conceived the study, supervised the experimental design and interpretation. Funding This work was supported by the Academy of Finland (257472), Biocentrum Helsinki,Helsinki University Hospital (TYH2019250, TYH2021333), FINDOS-Helsinki and Sigrid Jusélius Foundation (4702957). Data availability No data were generated from this study. For analysis of differentially expressed genes in microarray, we have utilized the MIAME-compliant microarray data that has already been deposited in the GEO database. GEO accession GSE140884. The dataset link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE140884. We have deposited this data in our previous study [22] Materials availability Plasmids generated in this study are available from the lead contact. Ethics declarations Competing interests The authors declare that they have no competing interests exist. Ethics statement This animal study was reviewed and approved by the Helsinki University Hospital, and the Southern Finland Council Animal Welfare and Ethics Committee. Consent to participate Not applicable Consent for publishing Not applicable References Olkkonen, V.M., et al., Isolation of a Mouse Cdna-Encoding Rab23, a Small Novel Gtpase Expressed Predominantly in the Brain. Gene, 1994. 138 (1-2): p. 207-211. Eggenschwiler, J.T., E. Espinoza, and K.V. Anderson, Rab23 is an essential negative regulator of the mouse Sonic hedgehog signalling pathway. Nature, 2001. 412 (6843): p. 194-8. Guo, A., et al., Open brain gene product Rab23: expression pattern in the adult mouse brain and functional characterization. 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Cellular Microbiology, 2012. 14 (8): p. 1149-1165. Table Table 1. Pathways and gene list Pathways Genes Vesicle-mediated transport Ank3, Ap2b1, Arfip2, Chmp4b, Copz2, Galnt1, Gosr2, Kdelr2, Kif1a, Kif23, Rab3a, Sec31a, Sparc, Syt1, Txndc5 Genes functions in the cell surface Ap2b1, Atp1b1, Cav1, Grb7, Pik3r1, Rab3a, Syt1, Tgfb1 Tgf-beta receptor signaling Ap2b1. Ccnd1, Cav1, Mef2c, Pik3r1, Sparc, Tgfb1, Vdr Supplementary Files FigureS1.jpg FigureS2.jpg FigureS3.jpg FigureS4.jpg FigureS5.jpg FigureS6.jpg FigureS7.jpg FigureS8.jpg FigureS9.jpg FigureS10.jpg FigureS11.jpg SupportinginformationHasanetal.pdf Video1.Wtcell.mp4 Video2.Rab23deficientcell.mp4 Video3.Wtcellperiphery.mp4 Video4.RAB23deficientcellperiphery.mp4 Cite Share Download PDF Status: Published Journal Publication published 22 Apr, 2025 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted Reviewers agreed at journal 24 Apr, 2024 Reviewers invited by journal 24 Apr, 2024 Editor assigned by journal 24 Apr, 2024 First submitted to journal 24 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4539384","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":311296273,"identity":"7acb9609-4c75-42c2-820b-ff8f3db0bd0b","order_by":0,"name":"Md. Rakibul Hasan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIie3QMWsCMRTA8XcU0uXOrC9c0a8QOVBL/TB3iy4OuhSH0maKix/gBOln6OR85YEuUlehHSrCuXRztIPp9ehSckOnDvkv4Q0/8hIAl+tfxoAAOiCLYZxBDTzlKQmcVRMsyToz8zcRuoJc/BBPfxFzKgChLKA9WSY00gjty9XuOHp8qzNOepcOAWsWcrXuZTQz5Ho6iMLZIo8YJpPmkwS0vQWhrygwRGYDCIMFJRo9Ld4l3FsJP5Rkc9ifgjk9aP5cEPstaBYryDZuhYGimEGiReVimMeUvqAvtx+tG39JTY2GpBLthPei4/C2W5eb/v7Vv6MG56tcTD+72FAWU+b//heXy+Vy/b0zZmZUPwigPncAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-5477-6934","institution":"University of Helsinki Faculty of Medicine: Helsingin Yliopisto Laaketieteellinen tiedekunta","correspondingAuthor":true,"prefix":"","firstName":"Md.","middleName":"Rakibul","lastName":"Hasan","suffix":""},{"id":311296274,"identity":"62dff864-3d4d-40d9-87da-90857183c65f","order_by":1,"name":"Maarit Takatalo","email":"","orcid":"","institution":"University of Helsinki Faculty of Medicine: Helsingin Yliopisto Laaketieteellinen tiedekunta","correspondingAuthor":false,"prefix":"","firstName":"Maarit","middleName":"","lastName":"Takatalo","suffix":""},{"id":311296275,"identity":"2fb38685-6248-4ecf-abf8-ad692d6aaebf","order_by":2,"name":"Pekka Nieminen","email":"","orcid":"","institution":"University of Helsinki Faculty of Medicine: Helsingin Yliopisto Laaketieteellinen tiedekunta","correspondingAuthor":false,"prefix":"","firstName":"Pekka","middleName":"","lastName":"Nieminen","suffix":""},{"id":311296276,"identity":"951de7d3-59c5-4453-ab32-31603112fcda","order_by":3,"name":"Ritva Rice","email":"","orcid":"","institution":"University of Helsinki Faculty of Medicine: Helsingin Yliopisto Laaketieteellinen tiedekunta","correspondingAuthor":false,"prefix":"","firstName":"Ritva","middleName":"","lastName":"Rice","suffix":""},{"id":311296277,"identity":"62f0719e-765c-492c-90b6-a906c85fd3fc","order_by":4,"name":"Tuija Mustonen","email":"","orcid":"","institution":"University of Helsinki Faculty of Medicine: Helsingin Yliopisto Laaketieteellinen tiedekunta","correspondingAuthor":false,"prefix":"","firstName":"Tuija","middleName":"","lastName":"Mustonen","suffix":""},{"id":311296278,"identity":"cd82d00a-8ecc-4843-84b3-dce4c113af3f","order_by":5,"name":"David P Rice","email":"","orcid":"https://orcid.org/0000-0001-9301-3078","institution":"University of Helsinki Faculty of Medicine: Helsingin Yliopisto Laaketieteellinen tiedekunta","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"P","lastName":"Rice","suffix":""}],"badges":[],"createdAt":"2024-06-06 10:31:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4539384/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4539384/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00018-025-05694-w","type":"published","date":"2025-04-22T15:58:07+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59124096,"identity":"2b8059f2-7a7e-46c9-bbd9-2ecb43bc12aa","added_by":"auto","created_at":"2024-06-26 15:20:15","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11847436,"visible":true,"origin":"","legend":"\u003cp\u003eHA-RAB23 co-localizes with early and late endosomal markers and interacts with the β-adaptin subunit of the clathrin adaptor protein 2 (AP-2) complex\u003c/p\u003e\n\u003cp\u003e(a, b) Co-localization analysis (a) of HA-RAB23 with endocytic pathway-specific vesicle markers EEA1, RAB5, RAB7, RAB11 and with autophagy marker LC3 A/B in MG-63 cells. Images show HA-RAB23 (red) co-localizes with the early endosomal markers EEA1 and RAB5 (green) in different locations in the cell including in the cell periphery (inset). HA-RAB23 shows robust co-localizations with late endosomal marker RAB7 (green) and autophagy marker LC3 A/B (green). HA-RAB23 shows little or no co-localization with recycling endosomal marker RAB11. Scale bar: 10 µm. Quantification (b) of HA-RAB23 co-localizations with EEA1, RAB5, RAB7, LC3 A/B and RAB11 using Pearson’s correlation coefficient r = 0-0.19 (very low co-localization), r = 0.2-0.39 (low co-localization), r = 0.4-0.59 (moderate correlation), r = 0.6 – 0.79 (high correlation) and r = 0.8 – 1.0 (very high correlation). n = 3 (total number of cells 25-30).\u003c/p\u003e\n\u003cp\u003e(c) Co-localization analysis of HA-RAB23 with endocytic route-specific markers clathrin, caveolin 1 and β-adaptin subunit of the clathrin adaptor protein 2 (AP-2) complex in MG-63 cells. Images show robust co-localization of HA-RAB23 (red) with clathrin (green) and β-adaptin (green), HA-RAB23 shows low co-localization with caveolin 1 (green). Scale bar: 10 µm. n = 3 (total number of cells 20-25).\u003c/p\u003e\n\u003cp\u003e(d) Protein co-immunoprecipitation using IgG and anti-HA antibody on un-transfected and transfected MG-63 cells, respectively. Co-immunoprecipitation followed by western blotting using anti-β adaptin antibody detected the β-adaptin band in the anti-HA co-immunoprecipitated sample at the same molecular weight (105 kDa) to that of input β-adaptin protein observed in transfected and un-transfected cells. Western blotting using anti-RAB23 antibody detected HA-RAB23 protein in the anti-HA co-immunoprecipitated sample at 30 kDa. Western blotting using anti-β actin antibody detected β-actin protein in the un-transfected and transfected inputs at 42 kDa (n=4 independent blots). PM; plasma membrane.\u003c/p\u003e\n\u003cp\u003e(e) Co-localization of GFP-RAB23 with AP-2 subunit α-adaptin 1 and α-adaptin 2 in MG-63 cells transfected with RAB23-pEGFP-C1 expression vector. Images show that GFP-RAB23 co-localizes with α-adaptin 1 in the cell periphery. GFP-RAB23 shows little or no co-localizations with α-adaptin 2. Nuclear staining (blue). n = 3 (total number of cells ⁓20). Scale bar, 10 µm.\u003c/p\u003e\n\u003cp\u003e(f) Protein co-immunoprecipitation using IgG and anti-HA antibody on un-transfected and transfected (HA-RAB23 pcDNA3.1 expression plasmid) MG-63 cells, followed by western blotting using anti-α-adaptin 1 antibody failed to detect α-adaptin 1 protein band (105 kDa) in the co-immunoprecipitated sample. Western blotting using anti-RAB23 antibody detected HA-RAB23 protein in the anti-HA co-immunoprecipitated sample at 30 kDa. Western blotting using anti-β actin antibody detected β-actin protein in the un-transfected and transfected inputs at 42 kDa. (n=2 independent blots).\u003c/p\u003e\n\u003cp\u003e(g) Protein co-immunoprecipitation using IgG and anti-HA antibody on un-transfected and transfected (HA-RAB23 pcDNA3.1 expression plasmid) MG-63 cells, followed by western blotting using anti-α-adaptin 2 antibody failed to detect α-adaptin 2 protein band (105 kDa) in co-immunoprecipitated sample. Western blotting using anti-RAB23 antibody detected HA-RAB23 protein in the anti-HA co-immunoprecipitated sample at 30 kDa. Western blotting using anti-β actin antibody detected β-actin protein in the un-transfected and transfected inputs at 42 kDa. (n=2 independent blots).\u003c/p\u003e\n\u003cp\u003e(h) Model suggests RAB23, clathrin and AP-2 might function at the cell membrane.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539384/v1/ac2eafb9e485c0ac68f0b65a.jpg"},{"id":59125376,"identity":"3bab000f-54c1-418e-8a8c-f389e4f46614","added_by":"auto","created_at":"2024-06-26 15:28:15","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":10232297,"visible":true,"origin":"","legend":"\u003cp\u003eRAB23, clathrin and AP-2 co-localize during nascent vesicle formation\u003c/p\u003e\n\u003cp\u003e(a) Triple co-localization of GFP-RAB23 with clathrin and β-adaptin subunit of the clathrin adaptor protein 2 (AP-2) in MG-63 cells transfected with RAB23-pEGFP-C1 expression vector. Images show RAB23 (green) co-localizes together with clathrin (red) and the β-adaptin (magenta) and forms a complex (arrow, inset) in the cell periphery. n = 3 (total number of cells 12-15). Scale bar, 10 µm.\u003c/p\u003e\n\u003cp\u003e(b) Control co-localization of GFP with clathrin and β-adaptin subunit of the clathrin adaptor protein 2 (AP-2) in MG-63 cells transfected with EGFP-pcDNA3.1 expression vector. GFP (green) showed no co-localization with clathrin (red) and the β-adaptin (magenta). n = 3 (total number of cells 12-15). Scale bar, 10 µm.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539384/v1/a2e334c266619fd38992b3b0.jpg"},{"id":59124088,"identity":"cfb490ef-eed2-4069-b250-b1df84d7f60f","added_by":"auto","created_at":"2024-06-26 15:20:15","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5452996,"visible":true,"origin":"","legend":"\u003cp\u003eRAB23 co-localizes and interacts with endophilin A2, PICALM and cortactin\u003c/p\u003e\n\u003cp\u003e(a) Co-localization analysis of GFP-RAB23 with clathrin-dependent nascent vesicle markers endophilin A2, PICALM and cortactin in MG-63 cells transfected with RAB23-pEGFP-C1 expression vector. Images show that GFP-RAB23 co-localizes with endophilin A2, PICALM and cortactin in the cell periphery. Co-localization of GFP-RAB23 with endophilin A2 and GFP-RAB23 with cortactin shows RAB23 localizes in the vesicle-like structure (inset). Nuclear staining (blue). n = 3 (total number of cells 25-30). Scale bar, 10 µm.\u003c/p\u003e\n\u003cp\u003e(b) Protein co-immunoprecipitation using GFP-Trapped assay on control GFP and GFP-RAB23 expression plasmid transfected in MG-63 cells, followed by western blotting using anti-endophilin A2, anti-PICALM and anti-cortactin shows corresponding protein band at 45 kDa, 70 kDa and 88 kDa, respectively in GFP-RAB23-Trapped lanes (upper panel) but not in control GFP lane. These detected proteins in the GFP-RAB23-Trapped lane showed similar molecular weight levels to their corresponding input protein level. Co-immunoprecipitation of control GFP-Trap and GFP-RAB23-Trap assays were validated by western blotting using anti-GFP antibody showing GFP and GFP-RAB23 protein bands at 27 kDa and 55 kDa, respectively (lower panel). Western blotting using anti-β actin antibody detected β-actin protein in the GFP-Trap and GFP-RAB23-Trap inputs at 42 kDa (n=2 independent blots).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539384/v1/c6857ad9d56635e1f589ccbb.jpg"},{"id":59125387,"identity":"93a92cba-06b5-4db7-8a17-3623bd97a601","added_by":"auto","created_at":"2024-06-26 15:28:16","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":12557482,"visible":true,"origin":"","legend":"\u003cp\u003eRAB23 deficiency changes the pattern of transferrin distribution and reduces transferrin uptake\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;(a) Time-lapse imaging of transferrin internalization dynamics in the presence of membrane dye (Cellbrite green) in WT (Linked with Video 1) and Rab23-/- (Linked with Video 2) cells. Cells were starved for 1 hour in growth medium containing 0.1% FBS. Initially, membrane was stained with membrane dye (green) followed by 5 minutes transferrin (red) pulse. Cells were then washed, and time-lapse imaging was performed for 5 minutes (frame rate 1-s interval) with sequential dual laser excitation at 594 nm and 488 nm. The first time frame indicates the starting time (T=5 min) of the time-lapse, the mid-time frame indicates when the time reaches 7.5 minutes and the last time frame indicates the end time (T=10 min) of the time-lapse. WT cells show robust internalization of transferrin (arrow, Video 1) while Rab23-/- cells retain transferrin at the cell membrane (Video 2), Or, after being initial internalization of transferrin, many transferrin patches repulse from cytoplasm to the periphery of the cell (arrow, last time frame). The dash line indicates the boundary of the cell. 10-12 cells in each group (n = 3 independent experiments). Scale bar, 10 µm.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;(b, c) Western blotting (b) and subsequent quantifications (c) show uptake of transferrin by cultured WT and Rab23-/- mouse calvaria-derived primary cells at 5, 10, 30, 45, 60 and 120 minutes. Cells were starved and allowed to uptake transferrin with 0.1% FBS. α-Tubulin was used for normalizing the transferrin level. n = 3 independent experiments. Data represented as mean ± SD, paired Student’s t-test was used. Statistical significance was defined as a P˂0.05 (*), ˂0.02 (**) and ˂0.005 (***).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;(d-f) Time-lapse imaging of transferrin (white dots in black background) accumulation pattern in the periphery of WT (Linked with Video 3) and Rab23-/- (Linked with Video 4) cells. Cells were starved for 1 hour in growth medium containing 0.1% FBS followed by 5 minutes transferrin pulse. Cells were then washed, and time-lapse imaging was performed for 5 minutes (frame rate 1-s interval) with laser excitation at 594 nm. The first time frame indicates the starting time (T=5 min) of the time-lapse and the last time frame indicates the end time (10 min) of the time-lapse. Segmented cell periphery shows transferrin (white dots, T=5 and 10 mins). In WT cells, transferrin internalized (white and green arrows) efficiently from the cell periphery and the intensity of transferrin reduced after 5 minutes (T=10, dotted rectangles) while Rab23-/- cells transferrin internalized (white and green arrows) inefficiently from the cell periphery and transferrin retain at the cell periphery at this time (T=10, dotted rectangles) (d). Scale bar: 20 µm. A model image represents the cell periphery (cellbrite green) in the segmented cell (e). Quantification of transferrin uptake in the cell periphery that showed in WT and Rab23-/- cells by time-lapse imaging. Cells were initially segmented to define the cell periphery and quantified the intensity of transferrin in the first frame (T=5) and last frame (T=10 minutes) of time-lapse. Rab23-/- cells retain more transferrin at the cell periphery compared to WT cells (f). 10-12 cells in each group (n = 3 independent experiments). Data represented as mean ± SD, paired Student’s t-test was used. Statistical significance was defined as a P˂0.05 (*), ˂0.02 (**).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;(g, h) Co-localization (g) analysis of transferrin (red) and AP-2 (β-adaptin, green) at 5 and 10 minutes in WT and Rab23-/- primary cells. Cells were starved and allowed to uptake transferrin with 0.1% FBS. WT cells show more transferrin and AP-2 positive patches at the cell periphery at 5 mins (upper inset, arrow) and many patches already internalized at this time (lower inset), while Rab23-/- cells show less co-localized transferrin with AP-2 at the cell periphery (upper inset). At 10 minutes Rab23-/- cells show more co-localization of transferrin with AP-2 (yellow, arrow) compared to WT cells. n = 3 independent experiments (total number of cells 25-30). Scale bar, 10 µm. Quantification (h) of transferrin co-localizations with AP-2 at 5- and 10 minutes using Pearson’s correlation coefficient r = 0-0.19 (very low co-localization), r = 0.2-0.39 (low co-localization), r = 0.4-0.59 (moderate correlation).\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539384/v1/a1e90fa296f1e3edcb1f3ce1.jpg"},{"id":59124100,"identity":"46cfc7fc-15f0-43e5-b3f2-a84e7338a79d","added_by":"auto","created_at":"2024-06-26 15:20:15","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":9927444,"visible":true,"origin":"","legend":"\u003cp\u003eRAB23 facilitates the assembly of clathrin-coat to the adaptor protein AP-2 (β-adaptin)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;(a, b) Dynamics of co-localization between PICALM and clathrin, AP-2 and clathrin for 5 and 10 minutes in WT and Rab23-/- cells. Cells were initially starved followed by stimulated with the osteogenic medium. At 5 minutes (A) PICALM and clathrin, AP-2 and clathrin showed less co-localization coefficient in Rab23-/- cells compared to WT cells (a). n = 3 independent experiments (total number of cells ≈ 30). At 10 minutes (b), AP-2 and clathrin showed significantly less co-localization coefficient in Rab23-/- cells when compared to WT cells. n = 3 independent experiments (total number of cells ≈ 30). Scale bar, 10 µm. Data represented as mean ± SD, paired Student’s t-test was used. Statistical significance was defined as a P˂0.05 (*).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;(c) A model delineating clathrin-mediated vesicle biogenesis at the plasma membrane upon binding with ligand (red) to receptor (i, green). Vesicle biogenesis starts with the recruitment of adaptor protein-2 (AP-2, sky blue). AP-2 interacts with ligand-receptor or cargoes and forms a prebudding structure (ii). Clathrin assembly protein PICALM (black) recruits clathrin (yellow) and forms a cage-like vesicle coat layer around the AP-2 mediated prebudding layer (iii). RAB23 regulates multiple steps: assembly of PICALM and AP-2 (blue), assembly of PICALM and clathrin (blue) and assembly of AP-2 and clathrin (blue) during nascent vesicle formation.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539384/v1/6ffdd93ec40987f40a320a7f.jpg"},{"id":59125388,"identity":"18f7315b-6ce0-4978-819f-966c0654712b","added_by":"auto","created_at":"2024-06-26 15:28:16","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":10044474,"visible":true,"origin":"","legend":"\u003cp\u003eRAB23 deficiency causes aberrant vesicle formation and alters BMP2 signaling\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;(a, b) Co-localization (a) analysis of clathrin (red) and AP-2 (β-adaptin, green) upon unstimulated and BMP2 stimulated WT and Rab23-/- primary cells. Cells were starved and stimulated with BMP2 containing medium supplemented with 0.1% FBS for 5 and 10 minutes. BMP2 unstimulated cells received only fresh growth medium containing 0.1% FBS. Both WT and Rab23-/- unstimulated primary cells showed co-localization of clathrin and AP-2, however, upon BMP2 stimulation for 5 minutes WT cells showed robust formation of the vesicle (i, ii) and vesicle like structure in the cell periphery, Rab23-/- cells lacked the robustness and showed aberrant vesicle like structure (i´, ii´). At 10 minutes of BMP2 stimulation, WT cells showed a reduced number of vesicles and vesicle-like structures compared to 5 minutes of BMP2 stimulation in WT cells, whereas Rab23-/- cells showed a drastic reduction of such structures at this time point. Scale bar, 20 µm. Quantification (b) of vesicle and vesicle-like structure in WT and Rab23-/- primary cells without and with BMP2 stimulation for 5 and 10 minutes. (n=3) (total number of cells ≈ 45). White arrowhead indicates vesicle-like structure. Data represented as mean ± SD, paired Student’s t-test was used. Statistical significance was defined as a P˂0.05 (*), ˂0.02 (**) and ˂0.005 (***).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;(c, d) Protein co-immunoprecipitation (c) using IgG and AP-2 (β-adaptin) antibody on samples obtained from mouse Wt and Rab23-/- calvaria derived primary cells. Before co-immunoprecipitation cells were serum starved for 1 hour followed by BMP2 was added at a concentration of 75 ng/ml in the culture medium and kept at 37°C for 5 minutes. Western blotting on co-immunoprecipitated samples using anti-β-adaptin and anti-clathrin antibodies detected the β-adaptin (105 kDa) band and clathrin (190 kDa) band at the same molecular weight as that of the input β-adaptin and input clathrin protein in Wt and Rab23-/- samples but not in the control IgG immunoprecipitated sample. Western blotting using anti-β actin antibody detected β-actin protein in the Wt and Rab23-/- inputs at 42 kDa. Quantification (d) of interaction (clathrin/β-adaptin subunit of AP-2) (n=3 independent blots). Data represented as mean ± SD, paired Student’s t-test was used. Statistical significance was defined as a P˂0.05 (*).\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539384/v1/c6c5a56e9fb43e0fbeb42aa2.jpg"},{"id":59125385,"identity":"3a0ad9a4-09b9-4efb-8bfd-bfd5ac604c41","added_by":"auto","created_at":"2024-06-26 15:28:15","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5026313,"visible":true,"origin":"","legend":"\u003cp\u003eRAB23 deficiency alters BMP2 signaling\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;(a, b) Immunostaining (a) analysis of pSMAD1/5/8 in WT and Rab23-/- primary cells on unstimulated and BMP2 stimulation. Cells were starved and stimulated with BMP2 containing medium supplemented with 0.1% FBS for 5 and 10 minutes. BMP2 unstimulated cells received only fresh growth medium containing 0.1% FBS. WT cells showed significantly higher pSMAD1/5/8 levels at both time points compared to Rab23-/- cells. Scale bar, 20 µm. Quantification (b) of pSMAD1/5/8 positive WT and Rab23-/- primary cells upon BMP2 stimulation for 5 and 10 minutes. n=3) (total number of cells ≈ several hundred). Data represented as mean ± SD, paired Student’s t-test was used. Statistical significance was defined as a P˂0.05 (*), ˂0.02 (**) and ˂0.005 (***).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;(c, d) Western blotting (c) and subsequent quantifications (d) of pSMAD1/5/8 upon BMP2 stimulation in WT and Rab23-/- mouse calvaria-derived primary cells for 5 and 10 minutes. Cells were starved and stimulated with BMP2. α-Tubulin was used for normalizing the pSMAD1/5/8 level. n = 3. Data represented as mean ± SD, paired Student’s t-test was used. Statistical significance was defined as a P˂0.05 (*) and ˂0.02 (**).\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539384/v1/47f40d1f2bc66efc5e218d8a.jpg"},{"id":59124109,"identity":"7d03875b-d5f7-4c48-8325-a66d3d3daea4","added_by":"auto","created_at":"2024-06-26 15:20:16","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":5392169,"visible":true,"origin":"","legend":"\u003cp\u003eMicroarray-based hypergeometric analysis of differentially expressed genes in WT and Rab23-/- samples suggest RAB23 involvement in vesicle biogenesis and endocytosis\u003c/p\u003e\n\u003cp\u003e(a) Microarray analysis of differentially expressed genes (223 genes, P˂0.05) in WT and RAB23 deficient (Rab23-/-) calvaria-derived samples followed by hypergeometric test for consensuspathDB shows a number of genes involved in vesicle-mediated transport, membrane transport and several genes show the involvement in TGF-beta receptor signaling pathway.\u003c/p\u003e\n\u003cp\u003e(b) Hypergeometric test for over representing genes (P˂0.05) search for KEGG Ontology shows a number of differentially expressed genes are involved in the regulation of actin cytoskeleton and endocytosis.\u003c/p\u003e\n\u003cp\u003e(c) Hypergeometric test for Gene Ontology (GO: molecular function) of the under-representing genes (P˂0.05) shows a number of genes involved in molecular transducer, signaling receptor and G-protein coupled receptor activity.\u003c/p\u003e\n\u003cp\u003e(d) Hypergeometric test for Gene Ontology (GO: biological function) of the under-representing genes (P˂0.05) shows a number of genes involved in the G-protein coupled receptor signaling pathway.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539384/v1/15df1fcec58a4b587f80d9be.jpg"},{"id":59124108,"identity":"082eae8e-7207-43bd-bd5d-f48c142c813d","added_by":"auto","created_at":"2024-06-26 15:20:16","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":2535570,"visible":true,"origin":"","legend":"\u003cp\u003eRAB23 regulation of clathrin-coated nascent vesicle formation\u003c/p\u003e\n\u003cp\u003eThe model represents how RAB23 is involved in multiple steps of AP-2/clathrin-coated nascent vesicle formation at the plasma membrane (PM). The cargo is initially recognized by AP-2 at the plasma membrane (PM), followed by clathrin assembly and curvature formation takes place by PICALM and BAR domain-containing protein endophilin A2. Cortactin is recruited before the dynamin-mediated detachment of the nascent vesicle. Deficiency of RAB23 affects this process of vesicle formation, vesicle internalization and subsequently leads to aberrant cell signaling.\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539384/v1/8e0c483da13a673a948753aa.jpg"},{"id":81569776,"identity":"297a3053-a595-4be8-ac97-fbb5d33e6dbb","added_by":"auto","created_at":"2025-04-28 16:11:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17922953,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4539384/v1/6c2fb8f2-1505-4652-a9d3-4e307eb3b374.pdf"},{"id":59124095,"identity":"5f3a0a7c-0fad-46d2-9acd-387a3aa383b6","added_by":"auto","created_at":"2024-06-26 15:20:15","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":8598981,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539384/v1/610118096ccc7dc79fcdd3bb.jpg"},{"id":59124090,"identity":"c5ee49fa-060a-4a4e-8b24-c1fc66afee47","added_by":"auto","created_at":"2024-06-26 15:20:15","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2073275,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539384/v1/8a7d9b618be2efa73869662c.jpg"},{"id":59124093,"identity":"000f6854-7ca6-4763-ac79-e287b5516447","added_by":"auto","created_at":"2024-06-26 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15:20:15","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":3664715,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539384/v1/66dabba5107902681cfd57b5.jpg"},{"id":59124097,"identity":"a49edecd-af7b-4e49-8ec7-d46618298870","added_by":"auto","created_at":"2024-06-26 15:20:15","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":2040616,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539384/v1/dec0614a3c737f4f1bead499.jpg"},{"id":59124104,"identity":"191070b6-6f3a-4bcd-aa86-2987768ddb26","added_by":"auto","created_at":"2024-06-26 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15:28:17","extension":"jpg","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":2279476,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539384/v1/72fd4e211ec3bb63c4452ada.jpg"},{"id":59125389,"identity":"f7124ee2-00c6-4230-96be-acf29c192bed","added_by":"auto","created_at":"2024-06-26 15:28:17","extension":"pdf","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":961526,"visible":true,"origin":"","legend":"","description":"","filename":"SupportinginformationHasanetal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4539384/v1/532bd872255c15fbb368441a.pdf"},{"id":59124110,"identity":"d831bde3-c042-4c8c-99e3-325da77574fe","added_by":"auto","created_at":"2024-06-26 15:20:17","extension":"mp4","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":4496976,"visible":true,"origin":"","legend":"","description":"","filename":"Video1.Wtcell.mp4","url":"https://assets-eu.researchsquare.com/files/rs-4539384/v1/39ddba228374ad38559ec37d.mp4"},{"id":59125378,"identity":"324a82e6-2d43-4a87-b592-cfc16bf60820","added_by":"auto","created_at":"2024-06-26 15:28:15","extension":"mp4","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":4451880,"visible":true,"origin":"","legend":"","description":"","filename":"Video2.Rab23deficientcell.mp4","url":"https://assets-eu.researchsquare.com/files/rs-4539384/v1/3ffb924991a8414679854ce8.mp4"},{"id":59124101,"identity":"cb48aa77-3d64-46b4-b63c-888082a5d9f1","added_by":"auto","created_at":"2024-06-26 15:20:16","extension":"mp4","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":927819,"visible":true,"origin":"","legend":"","description":"","filename":"Video3.Wtcellperiphery.mp4","url":"https://assets-eu.researchsquare.com/files/rs-4539384/v1/ce485346e3b6cd7e0e3c4cf5.mp4"},{"id":59124103,"identity":"d7a0e1ce-d0db-47f2-b1fa-e680492d6c3e","added_by":"auto","created_at":"2024-06-26 15:20:16","extension":"mp4","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":1236022,"visible":true,"origin":"","legend":"","description":"","filename":"Video4.RAB23deficientcellperiphery.mp4","url":"https://assets-eu.researchsquare.com/files/rs-4539384/v1/a2d7c4aa43d0491cca523dcd.mp4"}],"financialInterests":"","formattedTitle":"RAB23 facilitates clathrin-coated nascent vesicle formation at the plasma membrane and modulates cell signaling","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRas-associated binding 23 (RAB23) protein belongs to the Rab-GTPase family of proteins and is expressed during embryonic development and in the adult brain [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. RAB and other small GTPase family (Arf and Rho) proteins function as key molecular switches by hydrolyzing active GTP-bound to inactive GDP-bound forms during the formation, transport, docking and fusion of vesicles in the endocytic, recycling and in the secretory pathways [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. RAB family proteins also known as signal transducers control intercellular communication by restricting ligand secretion and by controlling cargo internalization, for example, ligand-bound receptors [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Activated RABs utilize cytoskeleton proteins, motor proteins and effector proteins during vesicle transportation and docking to the destined cellular compartment [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. RABs function by recruiting adaptor proteins, phosphatases, kinases, other RABs, actin filaments and microtubules [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. RABs are found in almost every organelle and determine transport specificity and organelle membrane identity [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. For instance, RAB5 is localized at the plasma membrane, clathrin-coated vesicles (CCV) and early endosomes and regulates early vesicle fusion [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. RAB24 is localized at endoplasmic reticulum and is involved in autophagosome formation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. RAB23 has been localized to the plasma membrane and in the endocytic pathway [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMutations in \u003cem\u003eRAB23\u003c/em\u003e cause Carpenter syndrome (CS), which is characterized by developmental defects in the heart, neural tube, and skeleton (MIM# 201000, Acrocephalopolysyndactyly type II) [\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. We have previously shown how RAB23 regulates skeletogenesis by suppressing aberrant ossification in the calvarial sutures [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. This developmental phenotype was affected through the negative regulation of fibroblast growth factor (FGF) and Hedgehog (Hh) signaling via the signal transducers pERK1/2 and GLI1. We have also shown that RAB23 regulates musculoskeletal development through TGFβR and BMP signaling [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In addition, RAB23 regulates neural tube and cardiac development through Hh and Nodal signaling, respectively [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Also, aberrant RAB23 signaling results in the formation, invasion and metastasis of many different tumors [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Even though RAB23 is known to regulate several growth factors signaling what is not known is how RAB23 regulates vesicle trafficking and how this might influence growth factor signaling.\u003c/p\u003e \u003cp\u003eEndocytic vesicles allow cells to uptake extracellular substances: ligands, receptors, soluble molecules, proteins and lipids by membrane internalization [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. These vesicles can be recycled back to the plasma membrane, or become mature into a late endosome and eventually undergo lysosomal degradation along with its cargo [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Vesicles that form at the plasma membrane go through clathrin (coat protein)-dependent, caveolae-dependent, or independent routes of membrane internalization [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Clathrin-mediated endocytosis is a well-characterized pathway, which utilizes adaptor protein 2 (AP-2) complex at the plasma membrane [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. AP-2 is a heterotetrameric complex, consisting of α-adaptin (1 and 2), β2, \u0026micro;2 and σ2 subunits [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Clathrin is also involved in endosome formation at the \u003cem\u003etrans-\u003c/em\u003eGolgi network, which utilizes adaptor protein 1 (AP-1) complex [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Clathrin-dependent vesicle formation is a multi-step process that starts with AP-2 mediated recognition of cargo at the membrane site, where AP-2 recognizes and interacts with the cargo and G proteins and forms the inner adaptor layer, followed by clathrin recruitment takes place to form the cage-like outer layer that wraps the adaptor layer. Membrane curvature takes place simultaneously at this stage. Finally, the nascent vesicle undergoes neck scission and detaches from the membrane [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we aimed to understand how RAB23 is involved in cellular membrane trafficking thereby its involvement in cell signaling. We demonstrate that RAB23 may be involved in multiple steps during clathrin-dependent nascent vesicle formation; cargo recognition with AP-2, clathrin assembly, membrane bending and scission. We show that RAB23 deficiency causes a reduction in the interaction between AP-2 (β-adaptin) and clathrin. Our results show that deficiency of RAB23 affects vesicle formation and membrane internalization within the endocytic pathway and impairs BMP2 signaling. Furthermore, by analyzing microarray expression data from WT and RAB23 deficient samples, we provide evidence that RAB23 is involved in vesicle formation, membrane trafficking and TGF-beta receptor signaling pathway. Collectively, our data indicate a role for RAB23 in nascent vesicle formation, cargo internalization and the regulation of cell signaling.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReagent/Resource table\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReagent/Resource\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReference or Source\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIdentifier\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eAntibodies\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEndosomal Marker Antibody Sampler Kit:\u003c/p\u003e \u003cp\u003eRabbit anti-EEA1\u003c/p\u003e \u003cp\u003eRabbit anti-RAB5\u003c/p\u003e \u003cp\u003eRabbit anti-RAB7\u003c/p\u003e \u003cp\u003eRabbit anti-RAB11\u003c/p\u003e \u003cp\u003eRabbit anti-Clathrin\u003c/p\u003e \u003cp\u003eRabbit anti-Caveolin 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell signaling technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#12666\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRabbit anti-Clathrin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell signaling technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#4796\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRabbit anti-LC3A/B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell signaling technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#12741\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMouse anti-Clathrin (X22)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#2731\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMouse anti-β actin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#ab8226\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRabbit anti-β actin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#ab8227\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRabbit anti-RAB23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteintech\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#11101-1-AP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRabbit anti-AP2B1 (β-adaptin)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteintech\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#15690-1-AP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMouse anti-AP2α1 (α-adaptin 1) (C-5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSanta Cruz Biotechnology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#sc-398024\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMouse anti-AP2α2 (α-adaptin 2) (F-12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSanta Cruz Biotechnology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#sc-55497\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRabbit anti-Transferrin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteintech\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#17435-1-AP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRabbit anti-VAMP8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteintech\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#15546-1-AP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMouse anti-HA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSigma-Aldrich\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#H3663\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRabbit anti-pSMAD1/5/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMillipore\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#AB3848\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMouse anti-αTubulin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSigma-Aldrich\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#T6199\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMouse anti-SH3GL1, Endophilin II (A-11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSanta Cruz Biotechnology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#sc-365704\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMouse anti-CALM (A-2), PICALM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSanta Cruz Biotechnology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#sc-271224\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMouse anti-Cortactin (H-5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSanta Cruz Biotechnology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#sc-55579\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRabbit anti-GFP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInvitrogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#A-11122\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTransferrin from Human Serum, Alexa 594 Conjugate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermo Fisher Scientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat# T13343\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGoat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L), Alexa 488\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermo Fisher Scientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#A-11008\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGoat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L), Alexa 546\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermo Fisher Scientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#A-110003\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGoat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L), Alexa 488\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermo Fisher Scientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#A-11001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGoat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L), Alexa 647\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermo Fisher Scientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#A-21245\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGoat anti-rabbit 680LT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInvitrogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#925-68021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGoat anti-rabbit 800CW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInvitrogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#925-32211\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGoat anti-mouse IRDye 800CW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInvitrogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#925-32210\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eChemicals, Peptides, and Recombinant Proteins\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMP2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u0026amp;D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#355-BM-010\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4% PFA in PBS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermo Fisher Scientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#15424389\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHoechst 33342\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermo Fisher Scientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat# H3570\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePierce IP lysis buffer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermo Fisher Scientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#87787\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProlong gold antifade\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermo Fisher Scientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRef: P36934\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePageRuler Plus prestained protein ladder\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermo Fisher Scientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#26620\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOdyssey blocking buffer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLI-COR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e927-40100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePierce protease inhibitor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermo Fisher Scientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#A32955\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePierce phosphatase inhibitor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermo Fisher Scientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#A32957\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFuGene \u0026reg; 6 Transfection Reagent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePromega\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat# E2691\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtein G Mag sepharose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGE Healthcare\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#28944008\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCritical Commercial Assays\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eiST GFP-Trap test kit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChromotek\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003egtak-iST-8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePierce BCA protein assay kit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermo Fisher Scientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#23225\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCell Brite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiotium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#30024\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCell Brite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiotium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#30021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eExperimental Models: Cell Lines\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuman MG-63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSigma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat# 86051601\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMouse Calvaria derived (CD) cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://elifesciences.org/articles/55829\u003c/span\u003e\u003cspan address=\"https://elifesciences.org/articles/55829\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eExperimental Models: Organisms/Strains\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eRab23opb2\u003c/em\u003e mice C57Bl/6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://elifesciences.org/articles/55829\u003c/span\u003e\u003cspan address=\"https://elifesciences.org/articles/55829\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRecombinant DNA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlasmid: pEGFP-C1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBD Biosciences Clontech\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#6084-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlasmid: RAB23-pEGFP-C1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThis paper\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlasmid: HA-RAB23-pcDNA 3.1 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThis paper\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSoftware and Algorithms\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFiji ImageJ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNational Institute of Health\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://fiji.sc/\u003c/span\u003e\u003cspan address=\"https://fiji.sc/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJACoP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNational Institute of Health\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://fiji.sc/\u003c/span\u003e\u003cspan address=\"https://fiji.sc/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChipster 3.16.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://chipster.csc.fi/\u003c/span\u003e\u003cspan address=\"https://chipster.csc.fi/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOdyssey infrared imaging system\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLI-COR Biosciences\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eModel 9120\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eOther\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u0026ndash;20% Mini-PROTEAN TGX Gels\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiorad\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#456\u0026ndash;1094\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMEM (Low glucose)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLife Technologies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#11885084\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMEM (High glucose)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLife Technologies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#41965039\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOpti-MEM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLife Technologies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#30985047\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTrypsin EDTA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLife Technologies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#25200056\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFBS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLife Technologies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#10270106\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGibco Sodium Pyruvate (100 mM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermo Fisher Scientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#11360039\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBSA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSigma-Aldrich\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#A3059\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePenicillin/Streptomycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLonza\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCatalog#DE17-602E\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAscorbic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-glyceraldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTriton X-100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSigma-Aldrich\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat#T8787\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGoat serum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLife Technologies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCat# PCN5000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell line and maintenance\u003c/h2\u003e \u003cp\u003eHuman osteosarcoma MG-63 cell line (Sigma; 86051601) was used for GFP-Trap assay (Chromotek; gtak-iST-8), protein immunoprecipitation and protein co-localization studies. Cells were cultured in DMEM containing low glucose (Life Technologies; 11885084) and supplemented with 10% FBS (Life Technologies; 10270106), glutamine, penicillin, streptomycin (Lonza; DE17-602E) and maintained at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMouse primary cells\u003c/h2\u003e \u003cp\u003eMouse calvaria derived (CD) primary cells were obtained from Wt and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e embryos at E15.5. Generation of \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mouse, and CD primary cell isolation procedure and maintenance have been described previously [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Primary cells have been maintained in DMEM containing high-glucose (Life Technologies; 41965039) and supplemented with 10% FBS, glutamine, penicillin, streptomycin and maintained at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. The second passage of cells was used for experiments. Starved cells were stimulated with osteogenic medium containing β-glycerophosphate (50 \u0026micro;g/ml), ascorbic acid (25 \u0026micro;g/ml) and BMP2 (25 ng/ml) in addition to the growth medium containing FBS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eExpression vectors\u003c/h2\u003e \u003cp\u003eHuman full-length RAB23 coding region was conjugated with HA in HA-pcDNA3.1 expression vector. HA-RAB23 pcDNA3.1 (this paper) was used for HA-RAB23 protein expression, protein co-immunoprecipitation and cell immunostaining studies. HA-empty vector was used as control for cell immunostaining. Human full-length RAB23 coding region was conjugated with EGFP in EGFP-pcDNA3.1 expression vector. RAB23-pEGFP-C1 (this paper) was used for EGFP-RAB23 protein expression, protein co-immunoprecipitation using GFP-Trap assay and cell immunostaining studies. EGFP expression vector pEGFP-C1 (Clontech; 6084-1) were used for protein co-immunoprecipitation using GFP-Trap assay and cell immunostaining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid transfection\u003c/h2\u003e \u003cp\u003eFuGene 6 Transfection Reagent (Promega; E2691) was used for transient plasmid transfections according to the manufacturer\u0026rsquo;s protocol. Transfection reagents were pipetted in Opti-MEM medium (Life Technology; 30985047), MG-63 Cells were allowed to grow 48 hours after transfection for transient expression of RAB23 protein with HA tag and with GFP tag. Control GFP-empty vector and HA-empty vector was used to express GFP and HA proteins.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eiST GFP-Trap assay\u003c/h2\u003e \u003cp\u003eGFP-Trap based co-immunoprecipitation was performed using GFP-Trap A agarose beads (iST GFP-Trap Test Kit, Kit for AP-MS sample preparation of GFP-fusion proteins, Chromotek; gtak-iST-8,). MG-63 cells were transfected with control GFP and GFP-RAB23 plasmid constructs. After transfection, cells were allowed to grow for 48 hours followed by 1 hour starvation and stimulation with osteogenic medium (DMEM with 10% FBS, 50 \u0026micro;g/ml β-glyceraldehyde, 50 \u0026micro;g/ml ascorbic acid, 25 ng/ml BMP2) for 10 minutes, and the subsequent procedure was carried out according to the manufacturer\u0026rsquo;s protocol. IP lysis buffer, IP wash I and II buffers were prepared according to the manufacturer\u0026rsquo;s recommendation. The eluted samples were further processed for SDS-PAGE for western blotting analysis using anti-GFP, anti-β actin, anti-Cortactin, anti-PICALM and anti-Endophilin A2 antibody.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCo-immunoprecipitation\u003c/b\u003e, \u003cb\u003esample precipitation and analysis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor co-immunoprecipitation experiments using anti-HA antibody, MG-63 cells were transfected using HA-RAB23 pcDNA3.1 expression vector. After 48 hours of transfection cells were starved for 1 hour followed by stimulated with osteogenic medium (DMEM with 10% FBS, 50 \u0026micro;g/ml β-glyceraldehyde, 50 \u0026micro;g/ml ascorbic acid, 25 ng/ml BMP2) for 10 minutes. For co-immunoprecipitation using β-adaptin antibody, E15.5 Wt and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mouse calvaria derived primary cells were used. The second passage of cells was used for co-immunoprecipitation. Cells were serum starved for 1 hour followed by BMP2 was added at a concentration of 75 ng/ml and kept at 37\u0026deg;C for 5 minutes. In both cases, cells were lysed for 20 min at 4\u0026deg;C by Pierce IP lysis buffer (Thermo Fisher Scientific; 87787) added with protease inhibitor (Thermo Fisher Scientific; A32955) and phosphatase inhibitor cocktails (Thermo Fisher Scientific; A32957). Protein lysates were clarified using centrifugation at 14,000 rpm for 15 minutes at 4\u0026deg;C. Input protein samples were taken and stored. Supernatant fractions from control samples (un-transfected) and transfected samples were kept with mouse primary IgG and mouse anti-HA antibody, respectively for co-immunoprecipitation at 4\u0026deg;C for overnight. Wt and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mouse primary calvaria derived samples were kept with rabbit anti-β-adaptin antibody for co-immunoprecipitation. Next day protein samples were transferred into fresh tubes and protein G Mag sepharose 4 Fast Flow (GE healthcare; 28944008) was added on the protein sample for 6 hours at 4\u0026deg;C. After several washing steps of the beads, co-immunoprecipitated proteins (anti-HA antibody bound) were eluted twice by 2.5% acetic acid. Eluted protein volumes were precipitated using chloroform/methanol precipitation procedure. Precipitated samples were dried using SpeedVac concentrator (Thermo Fisher Scientific). Dry precipitated protein samples were re-solubilized in reduced SDS sample buffer. Anti-β-adaptin bound protein samples were eluted in 2x samples buffer boiled at 95\u0026deg; C for 6 minutes and analyzed by 4\u0026ndash;20% SDS\u0026ndash;PAGE (Biorad; 456\u0026ndash;1094) and western blotting with rabbit primary anti-β-adaptin, anti-α adaptin 1, anti-α adaptin 2, anti-clathrin,anti-RAB23 and anti-β actin antibodies. Fluorescent intensity was detected using the infrared Odyssey System (Li-Cor Biosciences; 9120).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eBMP2 stimulation and analysis of pSMAD and VAMP8 levels\u003c/h2\u003e \u003cp\u003eWT and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e calvaria derived primary cells were isolated at embryonic day E15.5 and cultured on DMEM growth medium containing 10% FBS for several days. After reaching to 80\u0026ndash;90% confluency cells were trypsinized and equal number of WT and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells were plated on growth medium for BMP2 stimulation study. After 24 hours, cells were serum starved for 1 hour for BMP2 (R\u0026amp;D; 355-BM-010) stimulation. After a mild wash, BMP2 was added at a concentration of 75 ng/ml and kept at 37\u0026deg;C for 5 and 10 minutes, followed by cells were fixed and immunostained with anti-clathrin and anti-β adaptin antibody for vesicle formation, and immunostained with pSMAD1/5/8 to analyze the BMP2 signals in WT and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e samples. Confocal microscopy was performed for imaging. pSMAD1/5/8 data is presented as pSMAD1/5/8 positive nuclei divided by all nuclei. Western blotting using anti-pSMAD1/5/8 and anti-VAMP8 antibody was used to detect the level of pSMAD1/5/8 and VAMP8 and normalized against α-tubulin and β-actin, respectively.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell lysis\u003c/b\u003e, \u003cb\u003eprotein extraction\u003c/b\u003e, \u003cb\u003equantification and western blotting\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCells were lysed with RIPA buffer containing 0.1% SDS together with protease inhibitor and phosphatase inhibitor on ice. After brief sonication (10 seconds, twice) cell lysates were centrifuged at 14,000 rpm for 10 minutes at 4˚C to collect the protein supernatant. By using BCA protein assay kit protein concentrations were measured for western blotting analysis. Equal amount of proteins from wild type and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells were subjected to prepare to separate on SDS-PAGE under reduced gel electrophoresis (4\u0026ndash;20% Mini-PROTEAN TGX Gels, Bio-Rad; 456\u0026ndash;1094). After transferring to nitrocellulose membrane, membranes were blocked by odyssey blocking buffer at room temperature for 3 hours. Membranes were then incubated with primary antibodies and kept overnight at 4\u0026deg;C. Fluorophore-conjugated goat anti-rabbit or anti-mouse secondary antibodies were used against primary antibody at room temperature for 1 hour. β-actin (for VAMP8) and α-Tubulin was used to normalize protein expressions. Membranes were scanned using an Odyssey infrared imaging system (Odyssey; LI-COR Biosciences, model 9120). Band intensity was determined using ImageJ software (NIH).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eTransferrin uptake assay\u003c/h2\u003e \u003cp\u003eWT and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e calvaria derived primary cells were isolated at embryonic day E15.5 and cultured for several days. After reaching to 80\u0026ndash;90% confluency cells were trypsinized and equal number of cells were plated for transferrin uptake study. After 48 hours of culture cells were serum starved for 1 hours, after a mild wash Alexa Fluor\u0026trade; 594 Conjugate transferrin (Thermo Fisher Scientific; T13343) was added at 25 \u0026micro;g/ml with 0.1% FBS and kept at 37\u0026deg;C for 5, 10, 30, 45, 60 or 120 minutes. Cells were washed several times and lysed by ice cold RIPA buffer containing 0.1% SDS. Cell\u0026rsquo;s lysates were briefly sonicated and centrifuged to recover protein fractions. Protein concentration was measured using BCA protein assay kit and equal amount of WT and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e proteins were prepared for western blot analysis. For transferrin uptake in WT and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e CD cells and subsequent cell immunostaining analysis, transferrin was pulsed for 5, 10 and 30 minutes in the above-mentioned conditions followed by washing and fixed in 4% paraformaldehyde for 20 minutes and continued for cell immunostaining after staining with Cell Brite plasma membrane dye (Biotium; 30024). Transferrin uptake was quantified from the images as intensity by ImageJ. For quantification of transferrin uptake at the live cell periphery, time-lapse images were segmented at the beginning and at the end of the time lapse. Followed by intensity of the transferrin was measured in the segmented images using ImageJ tool without interfering/adjusting the transferrin intensity. Data presented as the intensity of the segmented image of the first time-lapse divided by the intensity of the image of the first time-lapse as 1 at the beginning of the time-lapse. After 5 minutes, the intensity of the image of last time-lapse divided by the intensity of the image of first time-lapse.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eFlow cytometry was performed to understand the transferrin uptake by Wt and RAB23 deficient cells. WT and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e calvaria derived primary cells were isolated at embryonic day E15.5 and cultured until the confluency reached to 80\u0026ndash;90%. Thereafter, cells were trypsinized and equal number of cells were plated for transferrin uptake study. After 48 hours of culture cells were serum starved for 1 hours, after a mild wash Alexa Fluor\u0026trade; 594 Conjugate transferrin (Thermo Fisher Scientific; T13343) was added at 25 \u0026micro;g/ml with 0.1% FBS and kept at 37\u0026deg;C for 5, 10 and 30 minutes. After brief washes with PBS, cells were trypsinized and centrifused to remove the trypsin and then resuspended in PBS for flow cytometric detection and quantification of Alexa fluor 594. The normalization was performed accordingly by Wt and RAB23 deficient cells that kept without transferrin. The flow cytometry analysis was performed at the HiLife Flow Cytometry Unit, University of Helsinki with Novocyte Quanteon flow cytometer. AlexaFluor 594 was detected with 561nm laser excitation and 615/20 BP filter. Cells population was gated on FCS/SSC by excluding the debris only. NovoExpress was used as analysis software.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunostaining\u003c/b\u003e, \u003cb\u003eco-localization and confocal microscopy\u003c/b\u003e\u003c/p\u003e \u003cp\u003eImmunostaining was performed for vesicle characterization on MG-63 cells, BMP2 related, transferrin uptake related and co-localization dynamics related studies on WT and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e CD cells. MG-63 cells were grown on coverslips for 24 hours and transfected with either HA-RAB23 pcDNA3.1, HA-empty vector, RAB23-pEGFP-C1 or GFP-empty expression vector for another 48 hours. For BMP2,transferrin uptake and co-localization dynamics related studies, E15.5 WT and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e CD cells were grown on cover slip and carried out the BMP2,transferrin uptake and co-localization dynamics studies at different time points at 37\u0026deg;C. For co-localization dynamics studies cells were starved for 1 hour followed by stimulated with osteogenic medium (DMEM with 10% FBS, 50 \u0026micro;g/ml β-glyceraldehyde, 50 \u0026micro;g/ml ascorbic acid, 25 ng/ml BMP2) for 5 and 10 minutes. In these experiments, cells were fixed in 4% PFA (Thermo Fischer Scientific; 15424389) for 20 minutes and permeabilized by 0.5% Triton-X-100 (Sigma-Aldrich; T8787) diluted in PBS for 5\u0026ndash;10 minutes. Cells were blocked with 5% goat serum (Life Technologies; PCN5000) diluted in blocking buffer (5% BSA and 0.1% Tween-20 in PBS) for 1 hour at room temperature followed by primary antibodies (including anti-GFP antibody) incubation at 4\u0026deg;C overnight in blocking buffer (LI-COR; 927-40100). Next day cells were incubated with Alexa fluor-conjugated secondary antibodies diluted in blocking buffer for 1h at RT. Cells were counterstained with Hoechst 33342 (Thermo Fisher Scientific; H3570) and mounted with ProLong Gold Antifade (Thermo Fisher Scientific; P36934). Confocal microscopy was performed at the Biomedicum imaging unit, University of Helsinki. Confocal microscopy images were obtained with an inverted confocal microscope system (Zeiss LSM 880) using a Plan apochromatic 63x/1.4 NA oil objective. Excitation was achieved using diode 405nm and argon multiline 350/488/532/561/633 nm lasers in confocal microscopy analysis. All images were taken at RT and analyzed with Fiji ImageJ 1.50b (64-bit) software. ImageJ plugin tool JACoP was used to study the Pearsons co-localization co-efficient. While determining Pearsons\u0026rsquo;s correlation coefficient we have provided total signal of each co-localization pair. The Pearsons method of correlation co-efficient used the total signal and determined the co-localization co-efficient.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLive cell time-lapse imaging\u003c/h2\u003e \u003cp\u003eTime-lapse microscopy was performed at the Biomedicum imaging unit, University of Helsinki. Time-lapse imaging was performed to study transferrin internalization dynamics in presence of membrane dye in Wt and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e CD primary cells. Initially, membrane was stained with Cell Brite membrane dye (Biotium; 30021) followed by 5-minute transferrin (red) pulse, washing the cells and time-lapse imaging for 5 minutes (frame rate 1-s interval). Zeiss LSM 880 Confocal microscopy based time-lapse images were obtained with an inverted confocal microscope system using a Plan apochromatic 63x/1.4 NA oil objective with sequential dual laser excitation at 594 nm and 488 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatics analysis\u003c/h2\u003e \u003cp\u003eImage J software was used for image processing. Image J plugging tool JACoP was used for Pearson\u0026rsquo;s co-localization studies. Windows movie maker, Image J and VEED.IO were used to process the videos presented in this article. Hypergeometric test for consensuspathDB, Gene Ontology analysis for KEGG, molecular and biological functions of microarray obtained data have been performed by Chipster 3.16.0 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003ePaired student \u003cem\u003et\u003c/em\u003e-test has been applied to perform the statistics of all the data obtained from western blotting, co-localization. Data are represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and \u003cem\u003eP\u003c/em\u003e-value less than 0.05 considered as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eLocalization of RAB23 in the endocytic pathway\u003c/h2\u003e\n \u003cp\u003eRAB23 is proposed to be involved in the endocytic pathway, however, little is known about the precise location during endocytosis. To understand this, we performed protein co-localization studies of RAB23 with endocytic pathway-specific vesicle markers EEA1, RAB5, RAB7, RAB11 and LC3A/B. RAB23 was over-expressed in human osteosarcoma MG-63 cells by using N-terminally HA-tagged full-length RAB23, HA-RAB23 pcDNA3.1 expression vector. HA-empty vector was used as a control for co-localization of HA protein with these markers. After 48 hours of transfection, cells were starved for 1 hour followed by stimulation with osteogenic medium for 10 minutes. Cells were then fixed and immunostained with anti-HA and anti-EEA1, anti-HA and anti-RAB5, anti-HA and anti-RAB7, anti-HA and anti-RAB11, and anti-HA and anti-LC3A/B antibodies followed by confocal microscopy. We found that HA-RAB23 co-localizes with the early endosomal markers EEA1 and RAB5 at different cellular locations including in the cell periphery (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). HA-RAB23 co-localizes with late endosomal marker RAB7 and autophagy marker LC3 A/B mostly in the cytoplasmic region (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). HA-RAB23 showed very low or no co-localization with recycling endosomal marker RAB11 (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). We found that control HA protein did not show any co-localization with any of these markers (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003ea). Quantification by Pearson\u0026rsquo;s correlation coefficient also showed low interaction (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003ec). Next, we quantified the co-localizations of HA-RAB23 with the vesicle markers. We found that HA-RAB23 highly co-localized with the early endosomal marker EEA1 and moderately with RAB5 (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). HA-RAB23 showed very strong co-localization with the late endosomal marker RAB7 and the autophagy marker LC3A/B (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). However, HA-RAB23 showed no or very low level of co-localization with the recycling endosome marker RAB11 (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). RAB23\u0026rsquo;s co-localization with EEA1 and RAB5 indicates that RAB23 may participate in early vesicle formation. RAB23 co-localization with the late endosomal marker RAB7 and the autophagy marker LC3A/B collectively suggests the involvement of RAB23 in the late endocytic pathway.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eAssociation of RAB23 to clathrin-mediated endocytosis\u003c/h2\u003e\n \u003cp\u003eWe aimed to pinpoint the endocytic route where RAB23 might be functional, specifically in receptor-mediated or receptor-independent pathways. We performed co-localization analysis of HA-RAB23 with clathrin, a marker for the receptor-mediated endocytic route and caveolin 1, a marker for receptor-independent route of endocytosis. RAB23 expression in these cells was over-expressed using the HA-RAB23 pcDNA3.1 expression vector and the HA-empty vector was used for control co-localization studies. Cells were immunostained with anti-HA and anti-clathrin, anti-HA and anti-caveolin 1 antibodies, followed by confocal microscopy was performed. Co-localization images show that HA-RAB23 strongly co-localizes with clathrin at different cellular locations including in the cell periphery and with caveolin 1 showed low co-localizations (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec). Quantification of the co-localizations shows a high correlation coefficient of HA-RAB23 with clathrin and low correlation coefficient of HA-RAB23 with caveolin 1 (Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e). However, the control HA protein did not show any co-localizations with any of these proteins (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eb) and showed a low Pearsons correlation coefficient (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003ec).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eRAB23 interacts with \u0026beta;-adaptin subunit (AP2\u0026beta;1) of the clathrin adaptor protein 2 (AP-2) complex\u003c/h2\u003e\n \u003cp\u003eSince RAB23 co-localized with the early endosomal markers EEA1 and RAB5 and with clathrin, we tested whether RAB23 shows any protein-protein interaction with clathrin. We performed protein co-immunoprecipitation using anti-HA antibody on MG-63 osteoblastic cells that were transfected with HA-RAB23 pcDNA3.1 expression vector. After 48 hours of transfection, RAB23 expression was analyzed by western blotting using anti-RAB23 antibody that recognized endogenous RAB23 and overexpressed HA-tagged RAB23 (Fig. \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e). Protein co-immunoprecipitation using control IgG and anti-HA antibody followed by western blotting using anti-clathrin and anti-RAB23 antibody did not recognize clathrin but recognized HA-RAB23 indicating that RAB23 shows no interaction with clathrin coat (Fig. \u003cspan class=\"InternalRef\"\u003eS4\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eClathrin is involved in the very early steps of nascent vesicle formation in receptor-mediated endocytosis where the adaptor protein 2 (AP-2) complex first becomes recruited at the plasma membrane followed by clathrin coat assembly which takes place around AP-2 to form double-layered vesicle. AP-2 is a heterotetrameric complex, consisting of \u0026alpha;-adaptin (1 and 2) \u0026beta;2, \u0026alpha;2 and \u0026sigma;2 subunits [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. And studies in mammals show that the appendage of the \u0026beta;2 subunit (\u0026beta;-adaptin) specifically interacts with clathrin [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e]. To determine whether RAB23 co-localizes and/or interacts with \u0026alpha;-adaptin (1 and 2) and \u0026beta;2 subunits of AP-2, we performed co-localization analysis of GFP-RAB23 with \u0026alpha;-adaptin 1 (AP2\u0026alpha;1), \u0026alpha;-adaptin 2 (AP2\u0026alpha;2) and HA-RAB23 with \u0026beta;-adaptin (AP2\u0026beta;1) subunits of AP-2 in MG-63 cells transfected with either GFP-RAB23 or HA-RAB23 pcDNA3.1 expression vectors. For control co-localization GFP-empty vector and HA-empty vector were used. Cells were stained with anti-HA, anti-GFP, anti-\u0026alpha;-adaptin 1, anti-\u0026alpha;-adaptin 2 and anti-\u0026beta;-adaptin antibody. Confocal microscopy images show that \u0026beta;-adaptin co-localizes with HA-RAB23 at the cell periphery (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec) and showed no co-localizations with control HA protein (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eb). Quantification of the co-localizations of HA-RAB23 with \u0026beta;-adaptin indicates strong co-localization of HA-RAB23 with the \u0026beta;-adaptin (Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e). Further protein co-immunoprecipitation analysis using control IgG and anti-HA antibody on MG-63 cells (transfected with HA-RAB23 pcDNA3.1 expression vector) followed by western blotting against \u0026beta;-adaptin detected a protein band in the sample lane at the level (105 kDa) similar to the transfected and un-transfected input \u0026beta;-adaptin protein level (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed). Western blotting against-RAB23 detected HA-RAB23 protein band (⁓30 kDa) in the sample lane and input lanes but not in the control IgG lane. Western blotting against \u0026beta;-actin detected the \u0026beta;-actin protein band (⁓42 kDa) in the input lanes. (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e\n \u003cp\u003eGFP-RAB23 co-localizes with \u0026alpha;-adaptin 1 at the cell periphery (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ee) and showed little or no co-localization with \u0026alpha;-adaptin 2 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ee). None of the \u0026alpha;-adaptin 1 and \u0026alpha;-adaptin 2 proteins showed co-localization with control GFP (Fig. \u003cspan class=\"InternalRef\"\u003eS5\u003c/span\u003e). Subsequent protein co-immunoprecipitation followed by western blotting against \u0026alpha;-adaptin 1 and \u0026alpha;-adaptin 2 failed to detect \u0026alpha;-adaptin 1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ef) and \u0026alpha;-adaptin 2 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eg) respectively in the sample lane. Internal control \u0026beta; actin was detected in the input lanes when immunoblotted against \u0026beta;-actin (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eg, f).\u003c/p\u003e\n \u003cp\u003eCollectively, these findings indicate that HA-RAB23 interacts with the \u0026beta;-adaptin subunit of the adaptor protein 2 (AP-2) complex (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eh).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eRAB23, \u0026beta;-adaptin and clathrin show triple co-localization\u003c/h2\u003e\n \u003cp\u003eIf RAB23 interacts with \u0026beta;-adaptin then RAB23, \u0026beta;-adaptin and clathrin might show collective co-localizations during AP-2/clathrin mediated vesicle formation. To investigate this hypothesis, we performed triple co-localization analysis for RAB23, \u0026beta;-adaptin and clathrin. RAB23 was over-expressed in MG-63 cells using GFP-RAB23 expression plasmid where GFP was N-terminally tagged with RAB23. Control GFP was also over-expressed in these cells using GFP plasmid. Cells were stained with anti-\u0026beta; adaptin and anti-clathrin antibodies together with GFP-RAB23 or with control GFP. Analysis of triple co-localization at the cell periphery and subsequent quantification suggests that GFP-RAB23, \u0026beta;-adaptin and clathrin show triple co-localization at the cell periphery (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea, Fig. \u003cspan class=\"InternalRef\"\u003eS6\u003c/span\u003e). However, co-localization analysis using anti-\u0026beta; adaptin and anti-clathrin together with control GFP did very low triple co-localization at the cell periphery (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb, Fig. \u003cspan class=\"InternalRef\"\u003eS6\u003c/span\u003e). These findings indicate that RAB23 may participate in early membrane internalization in the AP-2/clathrin route.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eExploring possible roles of RAB23 during clathrin-coated nascent vesicle formation\u003c/h2\u003e\n \u003cp\u003eAP-2/clathrin vesicle formation is a multi-step process, which initiates after recognition of the cargo by AP-2, followed by clathrin recruitment, curvature formation, scission and then detachment of the early vesicle from the membrane [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. We aimed to understand which of these stages RAB23 might be involved. To do so, we performed co-localization analysis of GFP-RAB23 with PICALM/AP180 (Phosphatidylinositol-binding clathrin assembly protein); a protein required for clathrin binding and assembly [\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]. GFP-RAB23 with BAR domain-containing protein endophilin, which is required for membrane bending and curvature [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e]. GFP-RAB23 with cortactin, which is recruited to the clathrin during vesicle scission [\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e]. RAB23 was over-expressed using the GFP-RAB23 expression plasmid in MG-63 cells and GFP-empty vector was transfected for control co-localization After 48 hours of transfection, cells were fixed and stained against endophilin A2, PICALM and cortactin. Confocal microscopy-based analysis showed that endophilin A2, PICALM and cortactin co-localized with GFP-RAB23 at the periphery of the cell and in vesicle-like structures (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). We quantified the co-localizations of GFP-RAB23 with endophilin A2, GFP-RAB23 with PICALM and GFP-RAB23 with cortactin (Fig. \u003cspan class=\"InternalRef\"\u003eS7\u003c/span\u003e). However, the control GFP protein showed no co-localizations with any of these proteins (Fig. \u003cspan class=\"InternalRef\"\u003eS8\u003c/span\u003ea) Quantification by Pearson\u0026rsquo;s correlation coefficient showed low interaction (Fig. \u003cspan class=\"InternalRef\"\u003eS8\u003c/span\u003eb).We found that GFP-RAB23 strongly co-localized with endophilin A2 and cortactin and moderately with PICALM (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea, Fig. \u003cspan class=\"InternalRef\"\u003eS7\u003c/span\u003e). To investigate whether RAB23 interacts with these proteins, we performed protein co-immunoprecipitation using GFP-Trap assay on MG-63 cells. These cells were transfected with GFP-RAB23 and control GFP expression plasmids to overexpress GFP-RAB23 and control GFP, respectively. Co-immunoprecipitation using GFP-Trap assay followed by western blotting against GFP on the eluted proteins obtained from control GFP-Trap assay and from GFP-RAB23-Trap assay that detected GFP and GFP-RAB23 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb, lower panel). Western blotting using anti-PICALM, anti-endophilin A2 and anti-cortactin antibodies detected their corresponding bands 70 kDa, 45 kDa and 88 kDa, respectively, in the GFP-RAB23-Trapped sample to the same level of their input protein bands (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb, upper panel). However, these proteins did not show bands in the control GFP-Trapped sample (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb, upper panel). Western blotting against \u0026beta;-actin detected the \u0026beta;-actin protein band (⁓42 kDa) in the input lanes. These bands detection confirmed that PICALM, endophilin A2 and cortactin interact with RAB23.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eRAB23 regulates clathrin-mediated cargo internalization\u003c/h2\u003e\n \u003cp\u003eTo test the functionality of RAB23 in clathrin-mediated endocytosis we investigated clathrin-coated vesicle formation and cargo internalization in mouse-derived WT and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e primary cells using the well-established transferrin model of ligand-receptor endocytosis. Transferrin is a glycoprotein that co-localizes with cytosolic RAB23 [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e] and requires the clathrin assembly protein PICALM for its internalization [\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e]. We showed that PICALM interacts and co-localizes with RAB23 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Here, we performed time-lapse microscopy in mouse WT and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e calvaria-derived (CD) primary cells which differentiate into osteoblasts [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. After 24 hours of culture, cells were starved for 1 hour followed by transferrin uptaking experiment was performed. Initially, the membrane was stained with CellBrite membrane dye followed by a 5-minute transferrin (red) pulse (alexa-594 conjugated, 25 \u0026micro;g/ml), washing the cells and time-lapse imaging for 5 minutes (frame rate 1-s interval) by using Zeiss LSM880 microscope. Results show that WT cells efficiently internalized transferrin from the cell periphery (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea, Video 1). In RAB23 deficient cells transferrin patches persisted longer at the cell periphery (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea, Video 2) also, many transferrin patches that were internalized often expelled to the cell periphery (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea, Video 2). We initially quantified the internalized transferrin at different time points by western blotting and by Flow cytometry. Serum-starved WT and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e calvaria derived (CD) primary cells were treated with transferrin (alexa-594 conjugated, 25 \u0026micro;g/ml) and incubated for 5, 10, 30, 45, 60 and 120 minutes at 37\u0026deg;C. Western blotting using anti-transferrin antibody showed reduced transferrin uptake in \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells at all-time points and that transferrin uptake never reached the wild-type levels (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb, c). By using flow cytometry, we analyzed transferrin uptake by these cells at 5, 10 and 30 minutes. Similar to western blotting, flow cytometry results show that \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells uptake reduced transferrin compared to Wt cells at each time point (Fig. \u003cspan class=\"InternalRef\"\u003eS9\u003c/span\u003e). We then analyzed transferrin accumulation at the cell periphery in WT and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells by using time-lapse images (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed, e). Here, we found that in WT cells transferrin was efficiently internalized from the cell periphery and reduced over time (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed, Video 3). Whereas \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells showed an accumulation of transferrin at the cell periphery (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed, Video 4). Quantification of transferrin accumulation at the cell periphery over this time period of time-lapse showed that RAB23 deficient cells retain more transferrin at the cell periphery compared to WT cells (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ef). These observations are compatible with our findings that RAB23 may be involved in multiple steps during nascent vesicle formation at the cell membrane (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e), and that deficiency of RAB23 showed an effect on the transferrin vesicle formation and internalization (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTransferrin patches co-localize with AP-2\u003c/strong\u003e (\u003cstrong\u003e\u0026beta;-adaptin) for longer in the absence of RAB23\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eTo understand the dynamics of the decreased AP-2/clathrin regulated transferrin internalization in \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells, we performed transferrin co-localization with the cargo recognition marker, AP-2 (\u0026beta;-adaptin). Serum-starved WT and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e calvaria derived (CD) primary cells were treated with transferrin (alexa-594 conjugated, 25 \u0026micro;g/ml) and incubated for 5 and 10 minutes at 37\u0026deg;C. Cells were then fixed, immunostained with anti \u0026beta;-adaptin and confocal microscopy was performed to capture the images (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eg). Co-localization between \u0026beta;-adaptin and transferrin represents that WT cells had a higher co-localization coefficient at 5 minutes compared to the \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eg, h). At 10 minutes of incubation, transferrin patches showed more co-localization with \u0026beta;-adaptin in \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells in comparison to WT cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eg, h). This indicated that in the absence of RAB23, transferrin patches spend a longer time bound to AP-2. Moreover, we found that transferrin uptake in the presence of the membrane dye (cellbrite blue) showed transferrin retention at the cell periphery and less internalization in \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells at 5, 10 and 30 minutes compared to WT cells (Fig. \u003cspan class=\"InternalRef\"\u003eS10\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003eAnalysis of RAB23 requirements in the assembly of clathrin-coat to the AP-2 (\u0026beta;-adaptin)\u003c/h2\u003e\n \u003cp\u003eClathrin-coated vesicle formation at the plasma membrane initiates AP-2 mediated cargo selection, followed by the recruitment of PICALM, which plays a critical role in clathrin assembly [\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e]. Our results show that RAB23 co-localizes with clathrin and interacts with \u0026beta;-adaptin and PICALM (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). We show that deficiency of RAB23 affects internalization of transferrin (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Next, we asked if RAB23 is involved in multiple steps of early vesicle formation, deficiency of RAB23 might show an effect on co-localization of the major proteins involved in vesicle formation. To address this question, we aimed to analyze the dynamics of co-localization of the RAB23 interacted proteins at the cell surface in response to osteogenic medium for 5 and 10 minutes on starved WT and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e CD primary cells (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). Here, we assessed the co-localization of the proteins; PICALM and clathrin, clathrin and AP-2 which we showed involved in clathrin-mediated early steps of nascent vesicle formation together with RAB23 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Immunostaining and subsequent quantification of co-localization between PICALM and clathrin, AP-2 and clathrin showed reduced co-localization co-efficient in \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells compared to WT cells at 5 minutes (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). The early stage (5 minutes) feedback affected the dynamics of vesicle formation at 10 minutes. At this time, we found reduced co-localization of clathrin to AP-2 in \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb). Our results thus suggest that RAB23 might be involved in several assembly steps during nascent vesicle formation (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eRab23\u003c/strong\u003e \u003csup\u003e\u0026nbsp;\u003cstrong\u003e\u0026minus;/\u0026minus;\u003c/strong\u003e\u0026nbsp;\u003c/sup\u003e \u003cstrong\u003ecells show aberrant vesicle formation upon BMP2 stimulation and show reduced interaction between AP-2 (\u0026beta;-adaptin) and clathrin\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eDifferential uptake of transferrin in \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells indicates a possible functional abnormality of AP-2/clathrin mediated ligand-receptor internalization which could result in abnormalities in signaling. Studies have shown that TGF\u0026beta;R/BMPR internalizes through the clathrin route and resides in the EEA1-positive compartment for pSMAD activation [\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e]. We have previously shown that RAB23 regulates TGF\u0026beta;R/BMPR signaling in musculoskeletal development and patterning [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. Here, we wanted to understand whether WT and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells show differential formation of vesicles upon BMP2 stimulation. We immunostained and subsequently analyzed the co-localization of \u0026beta;-adaptin and clathrin in WT and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e primary cells. Cells were starved followed by stimulated with BMP2 for 5 and 10 minutes. Here, we found that upon BMP2 stimulation for 5 minutes, WT cells efficiently formed vesicles in the cell periphery and also showed vesicle-like structures (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea). However, \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells showed aberrant formation of vesicles in the cell periphery and showed aberrant vesicle-like structures (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea). This phenomenon of vesicle formation decreased at 10 minutes upon BMP2 stimulation in both the cell types (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea). Quantification of vesicle or vesicle-like structure showed that \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells showed significantly reduced number of vesicles or vesicle-like structures in these time points when compared to WT cells (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e\n \u003cp\u003eTo understand, if deficiency of RAB23 affected the interaction between AP-2 (\u0026beta;-adaptin) and clathrin, we performed protein co-immunoprecipitation using control anti-IgG and anti-\u0026beta;-adaptin antibodies on the samples obtained from mouse Wt and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e calvaria derived primary cells. Cells were serum starved for 1 hour followed by BMP2 was added at a concentration of 75 ng/ml in the culture medium and kept at 37\u0026deg;C for 5 minutes. Western blotting on co-immunoprecipitated samples using anti-\u0026beta;-adaptin and anti-clathrin antibodies detected the \u0026beta;-adaptin (105 kDa) band and clathrin (190 kDa) band at the same molecular weight as that of the input \u0026beta;-adaptin and input clathrin protein in Wt and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e samples (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec, d). Quantification of interaction between the \u0026beta;-adaptin subunit of AP-2 and clathrin showed that the interaction clathrin/\u0026beta;-adaptin was drastically reduced in \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e samples, indicating that RAB23 is required for efficient interaction between \u0026beta;-adaptin and clathrin.\u003c/p\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003eRAB23 deficiency causes reduced expression of PICALM endocytic target R-SNARE protein VAMP8\u003c/h2\u003e\n \u003cp\u003eWe show that PICALM, which is an endocytic clathrin adaptor protein, interacts with RAB23 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). As PICALM is known for its endocytic function of R-SNARE proteins VAMP2, 3 and 8 [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e], we therefore aimed to understand if RAB23 works at least partly by modulating PICALM recruitments. We might predict less R-SNARE protein level in RAB23 deficient cells. In this regard, Wt and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e calvaria-derived primary cells were serum starved for 1 hour followed by BMP2 was added at a concentration of 75 ng/ml in the culture medium and kept at 37\u0026deg;C for 5 minutes. Western blotting was performed to detect VAMP8 (15 kDa) and \u0026beta;-actin (42 kDa) in these samples (Fig. \u003cspan class=\"InternalRef\"\u003eS11\u003c/span\u003ea). After quantification, we found that \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e samples, showed decreased level of VAMP8 compared to Wt samples (Fig. \u003cspan class=\"InternalRef\"\u003eS11\u003c/span\u003eb).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003eRAB23 deficiency caused altered pSMAD 1/5/8 activation upon BMP2 stimulation\u003c/h2\u003e\n \u003cp\u003eOur results showed differential formation of vesicles upon BMP2 stimulation in Wt and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells and showed reduced interaction between AP-2 and clathrin in \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells. We next analyzed pSMAD1/5/8 level in starved WT and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells by immunostaining and immunoblotting upon BMP2 stimulation for 5 and 10 minutes. Immunostaining using anti-pSMAD1/5/8 antibody and subsequent counting of pSMAD1/5/8 positive cells compared to all cells showed a reduction of pSMAD1/5/8 signal in \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells compared to WT cells at 5 and 10 minutes (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea, b). Immunoblotting using anti-pSMAD1/5/8 and anti \u0026alpha;-Tubulin antibody and subsequent quantification of pSMAD1/5/8 against \u0026alpha;-Tubulin showed a reduction of pSMAD1/5/8 level in \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells compared to WT cells at 5 and 10 minutes (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ec, d).\u003c/p\u003e\n \u003cp\u003eThese findings indicate that RAB23 may regulate BMP2 signaling.\u003c/p\u003e\n \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n \u003ch2\u003eEvidence that RAB23 regulates vesicle biogenesis and signaling receptor activity\u003c/h2\u003e\n \u003cp\u003eWe have previously shown that BMPs regulate osteogenesis and suture morphogenesis [\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e]. Also, RAB23 negatively regulates FGF and Hedgehog signaling in mouse calvarial bone and suture development where we performed a microarray-based gene expression analysis on WT and \u003cem\u003eRab23\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mouse (E15.5) calvarial bones and sutural tissues, which revealed 223 genes were significantly differential expressed [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. In this current study, we analyzed the differentially expressed genes by Chipster [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e], a bioinformatic tool to perform hypergeometric test for ConsensusPathDB to understand the functions of the genes that were differentially expressed. We found that RAB23 regulated several differentially expressed genes, which are involved in vesicle-mediated transport and membrane transport in the cell (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ea, Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). This analysis also showed that several genes are involved in the TGF\u0026beta; receptor signaling pathway (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ea, Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Upon performing a hypergeometric test for KEGG ontology for the over-representing genes, we found that several genes are involved in endocytosis and regulation of actin cytoskeleton (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eb). Further GO (Gene Ontology) analysis for molecular function and biological processes of the underrepresenting genes showed that RAB23 regulates genes that have molecular transducer activity and signaling receptor activity including G-protein coupled receptor activity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ec) and G-protein coupled receptor signaling pathway as biological processes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ed).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eRAB-GTPases act as master regulators in the endocytic and secretory pathways [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. RAB23 is known to localize to the plasma membrane and the endocytic pathway [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, multiple endocytic routes exist and the function of RAB23 in the context of membrane trafficking is largely unknown. Our data suggest that RAB23 functions in the clathrin-dependent route where RAB23 may participate in AP-2/clathrin-coated nascent vesicle formation at the plasma membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Clathrin-coated nascent vesicle formation is an upstream event of early endosome which starts with AP-2 mediated cargo recognition, followed by clathrin coat assembly and subsequently vesicle scission [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. These nascent vesicles then start docking and fuse with EEA1-positive early endosomes [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Our study reveals that during clathrin-coated nascent vesicle biogenesis at the plasma membrane, RAB23 may function at multiple steps and thus, deficiency of RAB23 affects vesicle formation, internalization, transport and cell signaling. By hypergeometric analysis of microarray data obtained from differentially expressed genes in WT and RAB23 deficient mouse primary cells, we provide further evidence that RAB23 is involved in vesicle formation, endocytosis and cell signaling.\u003c/p\u003e \u003cp\u003eVesicle transport keeps cargo identity intact by forming membrane-bound structures, and at the same time, it is essential to ferry the cargo from one cellular compartment to the target destination [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Vesicle coats consist of an inner adaptor protein layer that recognizes and interacts with the cargo and G proteins, and a cage-like outer layer that wraps the adaptor layer [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Upon binding with cargo and G proteins, adaptor proteins form a \u0026ldquo;prebudding complex\u0026rdquo;, the inner layer of vesicle. Subsequently, coat proteins are recruited to the prebudding complex to form a cage-like structure, known as the second layer of the vesicle. Finally, a GTPase-mediated hydrolysis, for instance driven by a RAB protein, detachs the nascent vesicle from the cell membrane [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Several other proteins including kinases are also involved in this process [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Here, we show that in the clathrin-coated endocytic vesicle formation RAB23 interacts with β-adaptin subunit of the adaptor protein 2 (AP-2) complex but not with α-adaptin 1 and 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). There might be several reasons for this discrepancy. Firstly, the heterotetrametric AP-2 adaptor, which is a big complex of subunits; α-adaptin (⁓110 kDa), β-adaptin (⁓110 kDa), \u0026micro;2-subunit (⁓50 kDa) and σ2 (⁓17 kDa) might be too big protein (⁓300 kDa) for a small vesicle protein antibody like RAB23 (⁓30 kDa) to pull down. α and \u0026micro;2 are among the two subunits of AP-2 that are membrane bound and they might have interactions with many other proteins [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Even though RAB23 might have an interaction with these subunits through β-adaptin the only strong interaction subunit (β-adaptin) might come across with RAB23. Secondly, how the subunits of heterotetrameric AP-2 adaptor complex are assembled and dissemble might be another issue and the conformational changes which are highly dynamic. RAB23 might interact with β-adaptin in a conformational state where the complex might loosely become interconnected within themselves and may show only interaction with β-adaptin before their compact assembly or when they are loosely interconnected during conformational changes. In addition to β-adaptin, our result further demonstrates that RAB23 interacts with the clathrin assembly protein PICALM, BAR domain-containing protein endophilin A2 and vesicle scission protein cortactin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This collectively suggests that the small GTPase protein RAB23 might be involved in multiple steps during clathrin-coated nascent vesicle formation.\u003c/p\u003e \u003cp\u003eClathrin-mediated endocytosis is involved in several important cellular processes, including cargo sorting to the endosome at the plasma membrane and \u003cem\u003etrans-\u003c/em\u003eGolgi network-mediated secretion of proteins [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Perturbation of clathrin in multicellular organisms causes lethality [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Also, genetically removing the clathrin-dependent core adaptor protein AP-2 results in embryonic lethality in worms, flies and mice [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. And in the absence of AP-2, the endocytic patch at the plasma membrane takes a significantly longer time to produce vesicles, many patches are unable to form vesicles, retake cargo at the cell membrane and some patches are stacked at the membrane [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Similar to AP-2 disruption, we show that RAB23-deficient cells exhibit reduced transferrin internalization, retention of transferrin at the cell surface and spend longer time at the cell periphery, and some patches retake to the cell membrane as shown by time-lapse live cell imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Videos 1\u0026ndash;4). We demonstrate that transferrin patches are retained in the first step of nascent endosome marked by β-adaptin subunit of AP-2 and take a longer time to become endocytosed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Furthermore, we show that the dynamics of co-localization between β-adaptin of AP-2 and clathrin are aberrant in RAB23 deficient cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These findings suggest that RAB23, AP-2 and clathrin functions together during endocytic patch formation and efficient patch internalization. Clathrin-mediated transferrin uptake also has been shown affected by several of kinases (92) when they are knocked down, which is an indication that clathrin-mediated endocytosis utilizes numerous proteins for efficient cargo internalization [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. We demonstrate that RAB23 deficiency affected the number of vesicle formations with aberrant morphology upon BMP2 stimulation and altered BMP2 signaling. A previous study showed that endocytic clathrin adaptor PICALM directs endocytosis of R-SNARE (VAMP2, 3 and 8) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Our study showed that PICALM interacts with RAB23 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and that deficiency of RAB23 reduced the expression of VAMP8 (Fig. \u003cspan refid=\"MOESM11\" class=\"InternalRef\"\u003eS11\u003c/span\u003e). We also show that deficiency of RAB23 causes reduced protein interaction between clathrin and AP-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This abnormality of ligand-receptor endocytosis may not be restricted to transferrin, R-SNARE or BMP2 signaling through BMP receptors that we have shown in this study, we speculate that a common mechanism for several growth factors signaling pathways including Hedgehog signaling through ciliary vesicle formation, FGF and TGFβR signaling. RAB23 deficiency could alter many cellular signals that pass through the AP-2/clathrin route.\u003c/p\u003e \u003cp\u003eThe clathrin route allows selective internalization of various metabolites carrying receptors [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. In RAB23-deficient mice, which mimic Carpenter syndrome in humans, the lack of functional RAB23 results in overt FGF10, Hh and Nodal signaling with consequent misexpression of downstream signal transducers (pERK1/2, Gli1, Lefty1/2 and Pitx2). This leads to overt osteogenesis at the growing bone ends in the developing skull, defective dorsal cell type specification during neural tube closure and abnormal left-right patterning of the heart [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. We have shown that RAB23 regulates musculoskeletal development through TGFβR and BMPR signaling [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. These phenotypes go hand in hand with our findings that RAB23 modulates adaptor protein-mediated assembly of clathrin during the early steps of endocytosis, which has been shown to regulate growth factor-receptor signaling [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn summary, here we show a role for RAB23 in clathrin-mediated nascent vesicle formation and endocytosis. Our results show that in the endocytic pathway, RAB23 co-localizes with early endosomal markers EEA1 and RAB5. RAB23 also co-localizes with late endosomal marker RAB7 and autophagy marker LC3 A/B, reminiscing the previous finding that RAB23 is involved in autophagosome formation during group A streptococcus infection [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e] Our results show, RAB23 interacts with adaptor proteins AP-2 and participates in the clathrin-coated vesicle formation. Clathrin-coated vesicle formation is a multi-step process that includes prebudding, clathrin assembly, curvature formation, and detachment of the nascent vesicle. RAB23 interaction with AP-2, PICALM, endophilin A2 and cortactin as well as co-localization with clathrin is required for proper vesicle formation and subsequent cargo internalization as well as cell signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Our data suggest mechanistic insights into the cellular membrane trafficking functions of RAB23 in mammalian cells and shows that RAB23 may play a role at multiple steps during clathrin-coated nascent vesicle formation and endocytosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAP-1 Adaptor protein 1\u0026nbsp;\u003cbr\u003eAP-2 Adaptor protein 2\u0026nbsp;\u003cbr\u003eAP2\u0026beta;1 \u0026beta;-adaptin subunit of Adaptor protein 2 complex\u0026nbsp;\u003cbr\u003eAP2\u0026alpha;1 \u0026alpha;-adaptin 1 subunit of Adaptor protein 2 complex\u0026nbsp;\u003cbr\u003eAP2\u0026alpha;2 \u0026alpha;-adaptin 2 subunit of Adaptor protein 2 complex\u0026nbsp;\u003cbr\u003eBMP Bone morphogenetic protein\u0026nbsp;\u003cbr\u003eCCV Clathrin-coated vesicle\u0026nbsp;\u003cbr\u003eCDC Calvaria derived cells\u0026nbsp;\u003cbr\u003eCo-IP Co-immunoprecipitation\u0026nbsp;\u003cbr\u003eCS Carpenter syndrome\u0026nbsp;\u003cbr\u003eEEA1 Early endosomal antigen 1\u0026nbsp;\u003cbr\u003eFGF Fibroblast growth factor\u0026nbsp;\u003cbr\u003eGEO Gene expression omnibus\u0026nbsp;\u003cbr\u003eGFP Green fluorescence protein\u0026nbsp;\u003cbr\u003eGO Gene ontology\u0026nbsp;\u003cbr\u003eHH Hedgehog\u0026nbsp;\u003cbr\u003ePICALM Phosphatidylinositol-binding clathrin assembly protein\u0026nbsp;\u003cbr\u003eRAB23 Ras-associated binding 23\u0026nbsp;\u003cbr\u003eTGF\u0026beta; Transforming growth factor \u0026beta;\u0026nbsp;\u003cbr\u003eVAMP8 Vesicle associated membrane protein 8\u0026nbsp;\u003cbr\u003eWT Wild type\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Vesa Olkkonen, Johan Per\u0026auml;nen and Maria Sanz Navarro for their critical and constructive discussion on this research project. We also thank Airi Sinkko and Anne Kivim\u0026auml;ki and Johanna Pispa for their excellent technical help.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM. R. H., M. T., P. N, T. M., D. P. R. designed the experiments. M. R. H., M. T., T. M., D. P. R. generated and processed the mice. M. R. H., M. T., R. R., P. N., T. M., D. P. R. wrote and approved the manuscript. M. R. H., M. T., T. M., P. N. performed the experiments. M. R. H. performed time-lapse imaging. D.P.R conceived the study, supervised the experimental design and interpretation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Academy of Finland (257472), Biocentrum Helsinki,Helsinki University Hospital (TYH2019250, TYH2021333), FINDOS-Helsinki and Sigrid Jus\u0026eacute;lius Foundation (4702957).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo data were generated from this study. For analysis of differentially expressed genes in microarray, we have utilized the MIAME-compliant microarray data that has already been deposited in the GEO database. GEO accession GSE140884. The dataset link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE140884. We have deposited this data in our previous study [22]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlasmids generated in this study are available from the lead contact.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests exist.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis animal study was reviewed and approved by the Helsinki University Hospital, and the Southern Finland Council Animal Welfare and Ethics Committee.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publishing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eOlkkonen, V.M., et al., \u003cem\u003eIsolation of a Mouse Cdna-Encoding Rab23, a Small Novel Gtpase Expressed Predominantly in the Brain.\u003c/em\u003e Gene, 1994. \u003cstrong\u003e138\u003c/strong\u003e(1-2): p. 207-211.\u003c/li\u003e\n \u003cli\u003eEggenschwiler, J.T., E. 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Goldstein, \u003cem\u003eRole of the coated endocytic vesicle in the uptake of receptor-bound low density lipoprotein in human fibroblasts.\u003c/em\u003e Cell, 1977. \u003cstrong\u003e10\u003c/strong\u003e(3): p. 351-364.\u003c/li\u003e\n \u003cli\u003ePearse, B.M.F., \u003cem\u003eCoated Vesicles from Human-Placenta Carry Ferritin, Transferrin, and Immunoglobulin-G.\u003c/em\u003e Proceedings of the National Academy of Sciences of the United States of America-Biological Sciences, 1982. \u003cstrong\u003e79\u003c/strong\u003e(2): p. 451-455.\u003c/li\u003e\n \u003cli\u003eEggenschwiler, J.T., et al., \u003cem\u003eMouse Rab23 regulates hedgehog signaling from smoothened to Gli proteins.\u003c/em\u003e Dev Biol, 2006. \u003cstrong\u003e290\u003c/strong\u003e(1): p. 1-12.\u003c/li\u003e\n \u003cli\u003eTong, J., P. Taylor, and M.F. Moran, \u003cem\u003eProteomic analysis of the epidermal growth factor receptor (EGFR) interactome and post-translational modifications associated with receptor endocytosis in response to EGF and stress.\u003c/em\u003e Mol Cell Proteomics, 2014. \u003cstrong\u003e13\u003c/strong\u003e(7): p. 1644-58.\u003c/li\u003e\n \u003cli\u003eYao, D., et al., \u003cem\u003eTransforming growth factor-beta receptors interact with AP2 by direct binding to beta2 subunit.\u003c/em\u003e Mol Biol Cell, 2002. \u003cstrong\u003e13\u003c/strong\u003e(11): p. 4001-12.\u003c/li\u003e\n \u003cli\u003eNozawa, T., et al., \u003cem\u003eThe small GTPases Rab9A and Rab23 function at distinct steps in autophagy during Group A Streptococcus infection.\u003c/em\u003e Cellular Microbiology, 2012. \u003cstrong\u003e14\u003c/strong\u003e(8): p. 1149-1165.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePathways and gene list\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"31.57894736842105%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePathways\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"68.42105263157895%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"31.57894736842105%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eVesicle-mediated transport\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"68.42105263157895%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eAnk3, Ap2b1, Arfip2, Chmp4b, Copz2, Galnt1, Gosr2, Kdelr2, Kif1a, Kif23, Rab3a, Sec31a, Sparc, Syt1, Txndc5\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"31.57894736842105%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eGenes functions in the cell surface\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"68.42105263157895%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eAp2b1, Atp1b1, Cav1, Grb7, Pik3r1, Rab3a, Syt1, Tgfb1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"31.57894736842105%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eTgf-beta receptor signaling\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"68.42105263157895%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eAp2b1. Ccnd1, Cav1, Mef2c, Pik3r1, Sparc, Tgfb1, Vdr\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Adaptor protein, Coat protein, Endocytosis, RAB23, Vesicle, Signaling","lastPublishedDoi":"10.21203/rs.3.rs-4539384/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4539384/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRAB23 is known to regulate several growth factors signaling during organogenesis. RABs and other small GTPases function as molecular switches during cellular membrane trafficking. However, what has not been established is how RAB23 functions during cellular membrane trafficking and how this influences cell signaling. To address this, we characterized RAB23\u0026rsquo;s localization in the endocytic pathway and determined the route of endocytosis. We find that RAB23 interacts with β-adaptin (AP2β1) subunit of the clathrin adaptor protein 2 (AP-2) complex, suggesting RAB23\u0026rsquo;s involvement in clathrin-dependent endocytosis at the plasma membrane. Our results show that RAB23 might function at multiple steps during clathrin-coated nascent vesicle formation. We find that RAB23 interacts with clathrin assembly protein PICALM, vesicle curvature protein endophilin A2, and a protein linked with vesicle scission, cortactin. To understand the functionality of RAB23, we performed time-lapse live cell imaging of transferrin uptake, which showed that clathrin-dependent endocytosis is affected in RAB23 deficient osteoprogenitors with inefficient cargo internalization. Our results show that deficiency of RAB23 reduced the interaction between β-adaptin and clathrin. We demonstrate that vesicle formation upon BMP stimulation and subsequent signal transduction is aberrant in RAB23-deficient cells. We further show evidence by providing microarray data-driven hypergeometric test of differentially expressed genes in WT and RAB23-deficient samples which suggests RAB23\u0026rsquo;s participation in vesicle formation, endocytosis and cell signaling. Collectively, our data indicate a role for RAB23 in vesicle formation, membrane trafficking, and cell signaling.\u003c/p\u003e","manuscriptTitle":"RAB23 facilitates clathrin-coated nascent vesicle formation at the plasma membrane and modulates cell signaling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-26 15:20:09","doi":"10.21203/rs.3.rs-4539384/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-04-25T00:14:15+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-24T21:15:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-24T11:34:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Life Sciences","date":"2024-04-24T06:40:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"51a29614-6eac-4e3e-977b-96ea50520d9d","owner":[],"postedDate":"June 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-28T16:03:42+00:00","versionOfRecord":{"articleIdentity":"rs-4539384","link":"https://doi.org/10.1007/s00018-025-05694-w","journal":{"identity":"cellular-and-molecular-life-sciences","isVorOnly":false,"title":"Cellular and Molecular Life Sciences"},"publishedOn":"2025-04-22 15:58:07","publishedOnDateReadable":"April 22nd, 2025"},"versionCreatedAt":"2024-06-26 15:20:09","video":"","vorDoi":"10.1007/s00018-025-05694-w","vorDoiUrl":"https://doi.org/10.1007/s00018-025-05694-w","workflowStages":[]},"version":"v1","identity":"rs-4539384","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4539384","identity":"rs-4539384","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}