CD34 orchestrates the formation and actions of microvilli for efficient E-selectin-mediated cell migration

CD34 orchestrates the formation and actions of microvilli for efficient E-selectin-mediated cell migration | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article CD34 orchestrates the formation and actions of microvilli for efficient E-selectin-mediated cell migration Jasmeen Merzaban, Mansour Aldehaiman, Abdullah Alghamdi, Shuho Nozue, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7280176/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Hematopoietic stem/progenitor cells (HSPCs) exit the blood stream and migrate to the bone marrow by extending their microvilli into tethers, in response to shear-resistant binding between ligands on HSPCs and E- and/or P-selectin, on the endothelial cell layer. Several selectin ligands have been identified; however, their mechanisms in microvilli formation and extension into tethers remain elusive. Using several super-resolution imaging techniques and functional assays, we showed that CD34 orchestrates these mechanisms. CD34 is indispensable for microvilli formation, enhancing tether formation by increasing its clustering and adopting a unique ring-like localization pattern at the microvillus tip, which can encapsulate other ligands, forming a dense tethering site for E-selectin. CD34 transduces E-selectin binding by phosphorylating the ERM protein ezrin, enhancing microvilli and tether formation in vitro and cell migration in vivo . Thus, CD34 plays a key role in the mechanical and signaling mechanisms of the microvilli, during cellular tethering and rolling. Biological sciences/Cell biology/Cell migration/RHO signalling Biological sciences/Cell biology/Cell adhesion Biological sciences/Biochemistry/Glycobiology Biological sciences/Cell biology/Cellular imaging/Super-resolution microscopy Biological sciences/Cancer/Haematological cancer/Leukaemia/Acute myeloid leukaemia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Cell migration is fundamental to several processes, including development, immune defense, and tissue regeneration 1 . It is also a key process for tumor-cell invasion and metastasis 2 , 3 . Therefore, understanding its underlying mechanisms may provide key tools for cancer diagnoses and treatment. In leukemia treatment, for example, intravenous transplantation of hematopoietic stem/progenitor cells (HSPCs) relies on the ability of stem cells to migrate from the blood to bone marrow 4 , 5 . Decades of research have established a multistep paradigm for cell migration: cell tethering and rolling on the endothelial cell-lining tissues and organs, activation and binding of integrins, cell arrest, and cell transmigration 4 , 6 , 7 , 8 . The prerequisite for cell tethering and rolling involves the establishment of shear-resistant binding between P- and/or E-selectin on the surface of the endothelium and several ligands on the cell in flow 8 , 9 , 10 , 11 , 12 , 13 . This leads to a gradual slowing of the cells in flow, allowing surface receptors to recognize chemokines in the vicinity and further progress toward cell migration 4 , 7 , 14 , 15 , 16 , 17 . Several ligands have been identified as E- and P-selectin ligands, which may vary depending on the cell type 18 . This raises the question as to whether these ligands play distinct, overlapping, or coordinated roles during cell tethering and rolling and if they are linked to the rest of the steps in cell migration. Super-resolution and single molecule imaging of cells before and after rolling have unraveled fundamental roles for microvilli in the cell tethering and rolling step. Several of the ligands were shown to cluster in these microvilli; upon their binding to E- or P-selectin, a shear force is built on the cell in flow, forcing its microvilli to extend into long tethers behind the cell 19 , 20 , 21 . The tethers may flip in front of the cell and act as slings to enhance cell rolling 22 . These findings highlight the importance of studying the roles of E- and P-selectin ligands within the context of microvilli, tethers, and slings to understand their mechanistic contribution to the cell tethering and rolling step. Microvilli are cellular extensions of the plasma membrane that are supported by polymerized actin filaments 23 , 24 , 25 . Microvilli are enriched with cross-linked actin bundles and the Ezrin, Radixin, Moesin (ERM) family proteins 26 , 27 , 28 . It has been proposed that during cell migration, ERM proteins link the actin cytoskeleton to several E-selectin ligands, including CD43 29, 30 , CD44 31 , and P-selectin glycoprotein ligand-1 (PSGL-1) 32 , 33 ; this link may contribute to ligand clustering 34 , 35 , 36 , 37 and the maintenance of the microvilli structure 38 . Two-color super-resolution imaging of fixed cells before rolling, confirms the nanoscale co-localization of the actin cytoskeleton and CD44 11 . However, after cellular rolling, although this co-localization is still observed on protrusions from the cell surface, it is absent on the tethers and slings formed. In fact, visualizing tethers and slings during live cell rolling using single molecule, microfluidics-based imaging 20 , confirms that tethers and slings detach from the actin cytoskeleton 20 , 39 , 40 . Remarkably, CD44 and PSGL-1 molecules continue to fully cover the surfaces of the tethers and slings 20 . Additionally, the tips of the tethers and slings that bind selectins, called tethering points, sometimes express a higher clustering of PSGL-1 molecules compared with the rest of the tethers and slings 20 , suggesting a specific mechanism of tethering point formation that may require such clustering to occur on the surface of cells as they roll. Collectively, these findings indicate dynamic structural changes in the microvilli, tethers, and slings, and in the spatial and temporal clustering of selectin ligands within these nanoscale structures. The findings also indicate potential linkages between selectin ligands and microvilli formation via ERM protein signaling. To decipher the role of the various selectin ligands in the formation of microvilli, tethers, and slings, and that of internal signal transduction via the ERM proteins, we focused on CD34 and the ERM protein Ezrin, as CD34 has been linked to microvilli formation and E-selectin binding 5 . We compared the findings from CD34 analysis to that from E-selectin ligands CD43, CD44, and PSGL-1. Our results unravel a complex spatial and temporal networking among the E-selectin ligands within the microvilli and establish CD34 as the orchestrator of this networking, leading to the enhancement of the mechanical and signaling mechanisms that form and support the microvilli function during cell tethering and rolling. These findings argue that some E-selectin ligands might be playing more pronounced role in the cell tethering and rolling step and indicate that the mechanical and signaling changes induced by CD34 and their differential effect on the clustering of E-selectin ligands might link this step to the subsequent multistep paradigm for cell migration. Results CD34 exhibits a “ring-like” nanoscale localization pattern on the microvilli To understand the role of CD34 in cellular homing, we characterized the nanoscale localization of CD34 prior to or post, cellular rolling, comparing it with the well-known E-selectin ligand CD44 41 . For pre-rolling, KG1a cells were attached onto the surface of a microfluidics channel slide that was pre-treated with silane; for post-rolling the cells were flown at a biologically relevant shear stress of 2 dyne.cm − 2 over recombinant E-selectin human IgG chimeric protein (rE-selectin) deposited on a Protein A-coated microfluidic chamber ( Fig. 1A ). The cells were then fixed and stained specifically for CD34 (Alexa Fluor 647; red) or CD44 (Alexa Fluor 488; green). Super-resolution images of either CD34 or CD44 at the slide’s surface were obtained using super-resolution localization microscopy that was configured with a highly inclined and laminated optical sheet (HILO), as described previously 11 . Consistent with our previous study 11 , significant CD44 clustering reorganization was observed, from a patchy spot-like clustering during pre-rolling, to elongated network-like structures post-rolling ( Fig. 1B ). We also observed similar reorganization of the CD34 clusters from diffusive to elongated network-like structures upon cellular rolling ( Fig. 1B ). Interestingly, CD34 clusters formed a unique hollow “ring-like” structure prior to and post- rolling ( Fig. 1B ). Co-localization analysis of CD34 and CD44 using the localizer extension, in the IGOR Pro Software 42 showed that in the majority of the cells (n = 15 cells), ring-like structure encircled the CD44 clusters which appeared not to encompass them prior to rolling but encompassed them post-rolling ( Fig. 1B ). We next compared the clustering dynamics between CD34 and CD44 in the tethers and slings that occasionally formed and fixed during rolling ( Fig. 1C ). Intriguingly, distal to the cell body near the microfluidics surface, where the tethering point of the tethers and slings was located, we observed a distinct patch of CD44 detached from the rest of the CD44 clusters and a dense clustering of CD34. Closer examination of the superimposed CD34 and CD44 signals revealed a CD34 ring-like structure that encircled and encompassed the detached CD44 cluster at the tethering point. Next, we zoomed in on the CD34 ring-like structure at the tips of the microvilli using a novel imaging technique that simultaneously combines 3D-total internal reflection fluorescence microscopy (TIRFM) and single molecule 3D- stochastic optical reconstruction microscopy (STORM) super-resolution imaging, to visualize the region near the tips of the microvilli, located near the tethering points within 120 nm of the surface of the glass coverslip. This enabled a 3D, higher resolution localization, of CD34 and CD44 prior to or post- rolling over rhE-selectin, at the single molecule level. Briefly, the microvilli topography near the tethering point was visualized by uniformly staining the cell surface using MemBrite Fix 640 and imaging them using TIRFM. Single molecules of CD34 and CD44 were visualized by staining with CD34 (AF488) or CD44 (AF488), respectively, and imaging using STORM ( Fig. 1D ). To visualize the 3D-topography of the microvilli using TIRFM, the cell surface fluorescence intensity was segmented and converted to distance, where the highest intensity was assigned to the tip of the microvilli near the glass surface as indicated in grayscale, in Fig. 1D . Higher counter threshold was applied to distinguish the tip of the different microvilli. 3D-STORM was used to generate the localization map of single CD34 or CD44 molecules and their distance from the glass coverslip surface are indicated by the color scale, in Fig. 1D . The maps from the 3D-topography and 3D-STORM from the same focal plane from the surface of the glass coverslip were overlayed, allowing for 3D-visualization of localized single CD34 or CD44 molecules near the tips of the microvilli. The 3D-maps were then converted to 2D-maps, which represent the spatial dimension of the 3D volume. Figure 1D shows the images of a large region of interest prior to or post-rolling. In both cases, CD34 molecules were located within the tips of the microvilli in a ring-like structure. The single molecule localization showed that CD34 was located slightly away from the tip of the microvillus ( Fig. 1D ). However, CD44 was randomly distributed in the tip of the microvillus prior to and post-rolling; CD44 was also present closer to the tip of the microvillus compared to CD34 ( Fig. 1D ). By screening the field of view for extended tethers near the surface, we visualized the dense localization of CD34 and small clusters of CD44 at the tethering point ( Fig. 1E ). The high resolution of this new imaging technique enabled us to analyze clustering criteria such as the average number of clusters per microvillus and average percent of molecules per cluster. These analyses showed that both parameters increased after cellular rolling in the case of CD34 and were not significantly affected in case of CD44 ( Fig. 1D , bottom panels). Overall, these results indicate that CD34 localizes in the microvilli and adopts a ring-like structure with increased clustering, post-cellular rolling. The 3D super-resolution images revealed that CD34 localizes below the very tip of the microvillus. We hypothesize that the CD34 ring-like structure may act as a scaffold that concentrates selectin ligands, as shown in the case of CD44, enhancing the ability of the microvilli to form tethers and slings. CD34 is key for microvilli formation and supports cellular rolling We investigated the effect of removing CD34, on the formation of microvilli and tethers and slings. KG1a cells were treated with siRNAs targeting CD34 (CD34-KD) or a scrambled control siRNA (sCT). The absence of CD34 was confirmed using western blot ( Supplementary Fig. 1 ). Cells were then fluorescent-stained for CD44 (Alexa Fluor 647; yellow) or CD34 (Alexa fluor 488; blue), prior to being perfused over a microfluidics chamber coated with rE-selectin, at a biologically relevant sheer stress of 2 dyne.cm − 2 . As illustrated in Fig. 2A , sCT-KG1a cells formed tethers and slings during rolling and expressed both CD34 and CD44 on the cell surface and on the tethers and slings. A merged image of anti-CD34 (blue) and anti-CD44 (yellow) antibodies on wild type (WT) KG1a cells showed a more even distribution of both proteins on the cell body. CD34-KD KG1a cells expressed significantly lower total numbers of tethers and slings compared to sCT-KG1a cells ( Fig. 2B ); when tethers and slings were observed, they were visibly shorter and less developed than those observed on the sCT-KG1a cells ( Fig. 2A ). A merged image captured using anti-CD34 (blue) and anti-CD44 (yellow) antibodies showed the absence of CD34 signals on the cell surface ( Fig. 2A ), indicating the effective knockdown of CD34. These results suggest that the tether and sling formation relies on CD34 expression. To evaluate if the paucity of tethers and slings in CD34-KD KG1a cells may be attributed to a deficiency of microvilli, we used scanning electron microscopy (SEM) to image the surface of the sCT-KG1a and CD34-KD KG1a cells ( Fig. 2C ). The microvilli were mostly eliminated in the CD34-KD KG1a cells. In contrast, knockdown of other E-selectin ligands PSGL-1 43 and CD43 44 and the ERM protein Ezrin in KG1a cells resulted in various degrees of loss of microvilli structures; this was however, to a much lesser extent compared to that in the CD34-KD KG1a cells ( Fig. 2C ). The number of microvilli per µm 2 on the cell surface was quantified and the same results were observed as that in the SEM images ( Fig. 2C , right panel). In a control experiment, we confirmed efficient knockdown of CD34, CD43, PSGL-1, and Ezrin ( Supplementary Fig. 1 ). To further compare the effect of various selectin ligands on the formation of microvilli, we used HEK cells that did not express CD34 and created HEK cells expressing CD34, CD44, or PSGL-1, and quantified the number of microvilli/µm 2 of HEK knock-ins. The CD34 knock-in had a substantially larger increase in the number of microvilli/µm 2 compared to other ligands, further supporting the conclusion that CD34 is key for the formation of microvilli, tethers, and slings ( Fig. 2D ). As microvilli are crucial for cellular rolling 5 , 45 , we also assessed the number of rolling CD34-, CD44-, PSGL-1-, and Ezrin-KD KG1a cells and their rolling velocity when perfused over rE-selectin at different shear stress forces, as described previously 11 , 46 , 47 . As illustrated in Figs. 2E and 2F , the removal of CD34 and PSGL-1 resulted in a consistent reduction in the number of rolling cells and an increase in their velocity under all tested shear stress forces. However, the magnitude of the change in CD34 knockdown was larger and more significant when compared to that for PSGL-1 KD. Surprisingly, the knockdown of CD43 and Ezrin resulted in the opposite trend of increasing the number of rolling cells and decreasing their rolling velocity, but the magnitudes of the changes were not significant in most cases, excluding the reduced rolling velocity in the knockdown of Ezrin at 5 and 8 dyne.cm − 2 and in CD43 at 5 dyne.cm − 2 ( Figs. 2E and 2F ), although the knockdowns decreased the number of microvilli ( Fig. 2C ). The SEM images showed that the cell morphologies were not affected by the knockdowns. This indicates that a balance in the density of microvilli may be required for effective cellular rolling. Collectively, these results demonstrate that CD34 had a more pronounced impact on the formation of microvilli, tethers, and slings and in achieving slow rolling compared to the other ligands, which is consistent with our previous work 5 . The results highlight the requirement for a balanced density of the microvilli for efficient rolling and tethering. CD34 affects the clustering of E-selectin ligands differentially in the microvillus Given that eliminating CD34 had the greatest effect on microvilli and rolling parameters compared to other E-selectin ligands, we determined whether CD34 knockdown changed the nanoscale dynamic organization of selectin ligands within the microvillus prior to or post-rolling over rE-selectin. To this end, we used our new imaging technique that combines 3D-TIRFM and 3D-SMLM super-resolution imaging to quantify the number of clusters per microvillus. The high resolution and topographical mapping of the cell surface near the tethering points in this technique enabled us to identify specific regions on the KG1a cell surface where microvilli are still present. CD34 knockdown affected the number of clusters per microvillus of CD44, CD43, PSGL-1, and Ezrin in a differential manner (Fig. 3 ). CD44 was the most affected ligand under both pre- and post-rolling conditions. CD43 and Ezrin were affected in the post-rolling condition, whereas PSGL-1 was affected in the pre-rolling condition. However, post-rolling, the localization of PSGL-1 at the tip of the microvillus was reduced to a greater extent than its distribution at the lower region of the microvillus tip. In a control experiment, using the HILO super-resolution imaging setup outlined in Fig. 1A , KG1a CD34-KD showed substantially dissociated CD44 clusters and an absence of nanoscale organization of the membrane surface (i.e., eliminated protrusions) ( Supplementary Fig. 2A ), and none of the KG1a knockdown in CD43, PSGL-1, and Ezrin had an effect on the nanoscale clustering of CD34 or CD44 ( Supplementary Fig. 2B ); CD44 knockdown was not possible. Collectively, these results highlight the critical role of CD34 in the nanoscale dynamic organization of selectin ligands within the microvillus prior to and post-rolling and indicate that this coordination varies among these ligands, with CD44 being the ligand most-linked to CD34. This differential spatial arrangement of the ligands within the microvilli indicates potential differences in their contributions along the multistep paradigm for cell migration via spatial distribution-driven mechanisms. E-selectin binding to CD34 promotes Ezrin phosphorylation and microvilli formation The effect of CD34 on the clustering dynamics of Ezrin in the microvilli (Fig. 3 ) prompted us to investigate the role of the CD34 intracellular mechanisms versus E-selectin binding in microvilli. We investigated the dynamics of Ezrin during cell rolling in relation to CD34. In microvilli biogenesis, it is essential that Ezrin is phosphorylated at the regulatory threonine 567 (T567) site (termed p-Ezrin) and recruited to the apical surface of epithelial cells 48 , 49 , 50 , 51 , 52 ; however, its role in tether and sling formation has not been explored. The relationship between full-length CD34 and p-Ezrin remains poorly understood as it pertains to microvilli and tether and sling formation prior to and upon binding to E-selectin; therefore, we used HEK cells that do not express CD34 and instead, created HEK cells expressing either full-length CD34 (KEK-CD34) or CD34 missing the E-selectin extra cellular binding domain (HEK-∆EC) ( Fig. 4A ); CD34 variants were stably expressed in HEK293ft cells using a lentiviral approach as described in the Methods section. These constructs also enabled us to identify which domain of CD34 was responsible for the formation of microvilli, tethers, and slings. Efficient and stable expression of the different forms of CD34 were assessed through western blotting ( Supplementary Fig. 3 ). Interestingly, analysis of p-Ezrin revealed an increased level of phosphorylation and activation of Ezrin in the HEK-∆EC cells compared to that in HEK and HEK-CD34 cells ( Fig. 4B ). In a control experiment, we showed that Ezrin and B-actin had a consistent expression in HEK, HEK-CD34, and HEK-∆EC cells ( Fig. 4B ). Next, we treated HEK-CD34 cells with fucosyltransferase 6 (FUT6) to decorate CD34 with sLe x sugar for proper interaction with E-selectin ( Supplementary Fig. 4 ) 53 and incubated them with rE-selectin for 1 h prior to western blotting. A significant increase was observed in p-Ezrin levels in the FUT6-treated HEK-CD34 cells compared to that in the HEK cell control, without altering the expression level of Ezrin ( Fig. 4C ). These results indicate that binding of E-selectin to the sLe x -decorated extracellular domain of CD34 or the expression of the truncated version of CD34 in the HEK-∆EC cells resulted in increased phosphorylation and activation of Ezrin. SEM analysis of microvilli formation in HEK, HEK-∆EC, and HEK-CD34 cells that were pretreated with FUT6 showed increased numbers of microvilli in HEK-∆EC and HEK-CD34 cells ( Fig. 4D ). Similarly, the number of tethers and slings upon cellular rolling showed that both, HEK-∆EC and HEK-CD34 cells displayed significantly more tethers compared to HEK cells post-rolling on E-selectin (Fig. 4E) . The finding that HEK-∆EC cells produced tethers without the presence of the external region of CD34 suggests the involvement of other E-selectin ligands. However, HEK-∆EC was not as efficient as HEK-CD34 in forming slings, indicating the weak binding to E-selectin at the tethering point. These results imply, for the first time, that CD34 can transduce signaling to downstream targets via Ezrin phosphorylation that may also be linked to E-selectin binding to promote the formation of microvilli, tethers, and slings. CD34 and Ezrin phosphorylation are required for cellular accumulation in vivo Control HEK, HEK-∆EC, and HEK-CD34 cells were stained with DiR membrane dye, treated with FUT6, and then injected intravenously into NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice as illustrated in Fig. 5A. The cells were then detected using an in vivo imaging system (IVIS) imaging system at various time points (1, 8, 24, and 48 h) following injection. Detection at these time points allowed the newly altered glycans on the surfaces of these cells to be maintained as they were shown to have a life of approximately 48–72 h after FUT6 treatment 53 . In vivo , whole-body IVIS imaging showed that HEK-CD34 cells expressed a significantly higher signal accumulation compared to HEK-∆EC cells as well as control HEK cells (Fig. 5B) . After observation at the last time point, the mice were euthanized. Ex vivo imaging of organs revealed that NSG mice receiving HEK-CD34 cells showed the highest average radiant efficiency compared to both, HEK-∆EC and control HEK cells. A significantly heightened radiant signal was observed in the spine, bones, and spleen (Fig. 5C) . These results suggest that while phosphorylated Ezrin can aid in the production of tethers and slings in vitro , proper accumulation and migration are not observed in vivo unless the cells possess both, the full-length CD34 and phosphorylated Ezrin. This finding may be attributed to the fact that although phosphorylated Ezrin can induce tether formation, it may be difficult for the cells to withstand the sheer stress in vivo without proper attachment of CD34 with E-selectin to aid the rolling process; this is consistent with the observation that HEK-∆EC was not efficient in forming slings in vitro ( Fig. 4E ). These findings suggest that the phosphorylation of Ezrin and presence of full-length CD34 are crucial for cellular rolling. Discussion Despite decades of investigations, the role of different selectin ligands in forming microvilli, tethers, and slings, and their spatiotemporal dynamics that facilitate stable rolling and tethering is only beginning to be understood. Recently, the clinical marker of hematopoietic progenitor cells, CD34 24, 54 , has been identified as an E-selectin ligand 5 ; however, its role in tethering and rolling remains poorly understood. Using a variety of microfluidics-based single-molecule super-resolution imaging techniques, we characterized the formation of microvilli, tethers, and slings, and unraveled the clustering and dynamics of CD34 alone and with other E-selectin ligands, under conditions mimicking pre- and post-rolling. CD34 knockdown severely compromised microvilli formation and their ability to extend to tethers and slings for slow rolling. This effect was more pronounced compared to that with the knockdown of the other E-selectin ligands CD43 and PSGL-1 ( Fig. 2C ). In fact, the knock-in of various E-selectin ligands in HEK cells that did not express CD34 showed that CD34 was most effective in increasing the number of microvilli/µm 2 ( Fig. 2D) . In addition, CD34 knockdown abolished CD44 clustering prior to and post rolling; however, its effect on the clustering of the other E-selectin ligands, CD43 and PSGL-1, was less and differential, indicating stronger link between CD34 and CD44 and a global effect on other selectin ligands in the microvillus (Fig. 3 ). Furthermore, for the first time through super-resolution imaging, we observed an interesting reorganization in CD34 clustering, in tethers and slings, compared to that in microvilli. CD34 exhibited denser clustering at the tethering points of the tethers and slings compared to the extended remains on the microvilli (Fig. 1C). As tethers and slings are believed to originate from singular microvilli 46 , CD34 may play a more crucial role than other ligands, in the extension of microvilli into tethers and slings due to its dense clustering around the tethering points. This is further supported by the fact that silencing CD34 mediated faster rolling than the knockdown of PSGL-1 (Fig. 2F) , an E-selectin ligand that has already been shown to cluster more toward the tips of the microvilli 55 , tethers, and slings 46 . It is possible, however, that a more pronounced role of CD34 at the tip of the microvilli results from the cumulative effect of forming a ring-like structure encompassing CD44 and other ligands, such as, PSGL-1. In fact, the CD34 ring-like structure is not located at the tip of the microvillus ( Fig. 1D ), indicating that it may also act by projecting other ligands within the tip of the microvillus. The clustering of ligands, such as, CD34 and CD44, may facilitate the formation of a signaling platform to facilitate the binding of different proteins and their targets. This phenomenon has been studied extensively in lipid rafts, which are microdomains consisting of cholesterol and glycosphingolipids 36 , 56 , 57 . Interestingly, CD44 clustering was disrupted by CD34 knockdown in a manner that is similar to that seen in treatment with MβCD, a cholesterol extractor that leads to the disruption of lipid raft domains and spatial clustering of selectin ligands 11 , 36 , 46 , 58 . Furthermore, MβCD treatment severely compromised microvilli formation and resulted in faster rolling kinetics as observed in the case of CD34 knockdown 46 . This suggests that CD34 may function similarly to the lipid rafts by facilitating the localization of other proteins into the lipid rafts. Clustering into a ring-like structure has been shown to play a role in T-cell receptor binding to antigen-presenting cells. Micro-adhesion rings made up of integrin and focal adhesion molecules that surround the T cell receptor micro-clusters are essential for T-cell receptor activation and clustering 59 . Impairment of these micro-adhesion rings resulted in the disruption of the T cell receptor micro-cluster formation and subsequent hindrance to cellular signaling and cell functions 59 . Thus, it is plausible that a similar effect can be played by the CD34 ring-like clustering where it can support the functions of other molecules and ligands by mediating better clustering and recruitment of other important players. This is evident by the encompassing of CD44 within the CD34 ring-like structure. ERM proteins are important players in linking the cytoskeletal cortex with the plasma membrane proteins 60 , 61 , 62 , 63 , 64 , 65 and play a crucial role in microvilli formation and activation 66 , 67 , 68 , and the motility of cancer cells 62 , 69 , 70 . Moreover, podocalyxin, a CD34-related family member, induces microvilli formation via recruitment of an adaptor protein NHERF-1 that links podocalyxin to actin via the ERM proteins 71 . These prior findings prompted us to elucidate the roles of different CD34 variants when expressed in HEK293ft cells. Interestingly, we observed a significant increase in Ezrin T567 phosphorylation in HEK-∆EC cells (Fig. 4B) but not in HEK-CD34 cells. This observation suggests a potential feedback inhibition by the CD34 extracellular domain on the functions of its intracellular domain. Interestingly, the binding of full length CD34 with E-selectin mimicked the effects on Ezrin phosphorylation seen in HEK-∆EC. These results imply that the intracellular tail of CD34 can directly or indirectly facilitate phosphorylation of Ezrin only when the extracellular domain of CD34 is occupied/inhibited with a ligand or removed completely. When these cells were treated with FUT6 to decorate CD34 with sLe x sugar for proper interaction with E-selectin, followed by injection into mice, HEK-CD34 cells showed remarkably improved accumulation compared to HEK-∆EC and control HEK cells (Fig. 5B) . These results imply that while T567-phosphorylated Ezrin impacts tether production, rolling and cellular migration still require intact CD34. In addition, analysis of cell migration into individual organs showed significantly more HEK-CD34 cells in all the tested organs compared to HEK-∆EC and control HEK cells (Fig. 5C) . This is consistent with previous findings that the CD34 family can aid in cell migration 72 . Collectively, while T567 Ezrin plays a role in tether formation, proper rolling and migration still require an intact and functional CD34 protein. The knockdown of CD34, CD43, PSGL-1, and Ezrin resulted in reduced number of microvilli ( Fig. 2C ). In contrast to CD34 and PSGL-1, the knockdown of CD43 and Ezrin showed consistent trend of improvement in the tethering and rolling kinetics measured by an increase in the number of rolling cells and decrease in their rolling velocity ( Figs. 2E and 2F ). However, these differences were not significant in most of the tested sheer stress forces and occasionally became significant with Ezrin reducing the rolling velocity at 5 and 8 dyne.cm − 2 and with CD43 at 5 dyne.cm − 2 ( Figs. 2E and 2F ). This enhancement despite the reduction in number of microvilli and tethers indicates that fine tuning the number of microvilli/µm 2 may be needed for efficient tethering and rolling. This proposition is strongly supported in the case of the Ezrin knockdown as E-selectin ligands were still present. Contrastingly, CD43 is a mucin with a high density of sialylated O-glycans that are negatively charged 73 , 74 and may provide a mechanism for regulating the density of the microvilli on the cell surface. In summary, our findings demonstrate t important role for CD34 in mediating slow rolling by maintaining microvilli and their extension into tethers and slings and coordinating the clustering, and potentially the activity, of other E-selectin ligands during the tethering and rolling steps. When CD34 was overexpressed, cells can roll appropriately and produce tethers that aid in the rolling process. These cells also showed enhanced migration in the tested mice. Our study highlights the novel role of CD34 as a signal transducer in the Ezrin signaling pathway, delineating its role in the clustering of ligands and production and maintenance of microvilli, tethers, and slings. Methods Statistical analysis Statistical analyses were performed using one-way ANOVA, two-way ANOVA, or T-tests of the relevant data set using GraphPad Prism version 10.2.1 for Windows, GraphPad Software, Boston, Massachusetts USA, www.graphpad.com . For the specific test used, please refer to the respective figure legends. Cell culture KG1a cells (human CD34 + leukemic progenitor cell line) and HEK293ft cells purchased from ATCC, were maintained in Roswell Park Memorial Institute (RPMI) 1640 media (10% fetal bovine serum (FBS); Gibco) or Dulbecco's modified Eagle medium (DMEM) media with streptomycin (100 µg/ml) at 37°C with 5% CO 2 , in a humidified atmosphere. Protein knockdown Cells were collected and transfected with 250 or 500 pmol of either a negative “scrambled” small interfering RNA (siRNA) (Ambion) or a specific SiRNA of choice (Table 1 ) using the Lonza 4D nucleofector protocol (Lonza). After transfection, cells were suspended in 2 mL of warm, antibiotic free RPMI 1640 media (10% FBS; Gibco) and incubated for 48–96 h at 37°C with 5% CO 2 . In some instances, pretreatment with 250 µg/mL (Sigma) of bromelain for 30 min at 37°C, followed by phosphate-buffered saline (PBS) washes, was used prior to transfection for effective knockdowns. Table 1 SiRNA and antibodies used for knockdown Name of siRNA Company and catalog # “Scramble” control siRNA Thermo Fisher Scientific (4390843) CD34 siRNA Thermo Fisher Scientific (s2644) PSGL-1 siRNA Thermo Fisher Scientific (s12688) CD43 siRNA Thermo Fisher Scientific (s13368) Ezrin siRNA Thermo Fisher Scientific (s14795) Thermo Fisher Scientific (s14797) Antibody/clone Company and catalog # CD34 antibody QBEND10 BIO-RAD (MCA547G) CD34 antibody EP373Y ABCAM (ab81289) CD44 antibody IM7 BioLegend (10302) CD44 antibody 515 BD Biosciences (550990) CD44 antibody Hermes 3 produced in-house PSGL1 antibody (KPL-1) BioLegend (328802) CD43 antibody (SP55) Thermo Fisher Scientific (MA5-16339) Ezrin Antibody 3C12 Thermo Fisher (357300) Phospho-Ezrin antibody (Thr567) Thermo Fisher (PA537763) Anti-Beta-Actin antibody (AC-15) Ambion (AM4302) Anti-rabbit IgG, HRP-linked Antibody Cell Signaling (7074S) Anti-mouse IgG, HRP-linked Antibody Cell Signaling (7076S) Anti mouse IgG (H + L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) Cell Signaling (4410S) Anti rabbit IgG (H + L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) Cell Signaling (4412S) Anti-mouse IgG (H + L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) Cell Signaling (4408S) Anti-rabbit IgG (H + L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) Cell Signaling (4414S) PE Rat Anti-Human Cutaneous Lymphocyte Antigen Clone HECA-452 BD Biosciences (563962) E-selectin produced in-house Western blot analysis A cell lysis buffer containing 1% phosphatase inhibitor (HaltTM, Thermo Scientific), 88% NP40 (InvitrogenTM NovexTM, Fisher Scientific), 1% PMSF, and 10% protease inhibitor (PierceTM, Thermo Scientific) was used to lyse the cells for 1 h at 4°C, with constant shaking. The lysate was incubated in a reducing mixture of 10% β-mercaptoethanol in NuPAGE LDS sample buffer (Invitrogen) for 10 min at 70°C. Reduced samples were moved onto a PVDF membrane following an SDS-PAGE gel run. Using 5% non-fat skim milk powder or 5% bovine serum albumin (BSA) in Tris-buffered saline with Tween-20 (TBST, Cell Signaling Technology), the membrane was blocked overnight at 4°C, on a tilting platform. Following TBST washes, the membrane was incubated with a primary antibody of interest (Table 1 ), then washed thrice with TBST and immunoblotted with HRP-conjugated secondary antibodies and prepared for imaging. Flow cytometry Cells (2 × 10 5 cells/mL) were washed and placed in a 96-well FACS plate in 50 µl of Hanks' balanced salt solution (HBSS; Gibco). Primary antibodies (10 µg/mL) were added for 25 min at 4°C. Cells were then incubated with a fluorophore-conjugated secondary antibody against the species used for the primary antibody, at a concentration of 5 µg/mL in HBSS, for 20 min at 4°C. Antibody information is presented in Table 1 . E-selectin deposition For super-resolution imaging, potassium hydroxide and ethanol were used to clean glass coverslips (No. 1.5, ibidi GmbH) by ultrasonication (P60H, Elma Schmidbauer GmbH). Using adhesives, the coverslip was attached to a sticky-Slide VI 0.4 microfluidic chamber (channel width, 3.8 mm; channel height, 0.4 mm; ibidi GmbH). Following firm attachment, protein A (10 µg/mL, Invitrogen) was incubated in the chamber overnight, at 4°C. After HBSS washing, rE-selectin (0.2 µg/mL) (Sino Biological) was incubated in the microfluidic chamber for 1 h at 4°C. The microfluidic chamber was then washed and blocked using 1% casein in PBS (Thermo) at room temperature (22°C) for 0.5-1 h. The chamber was then promptly used for super-resolution imaging. For live cell fluorescence rolling imaging, a µ-Slide VI 0.1 microfluidic chamber (channel width, 1 mm; channel height, 0.1 mm; ibidi GmbH) was incubated with rhE-selectin (1 µg/mL) (Sino Biological) overnight, at 4°C. Next, the microfluidic chamber was washed and blocked using 1% BSA in HBSS for 40 min at 4°C. The chamber was then promptly used for live cell rolling. Scanning Electron Microscopy (SEM) : Cells (5 × 10 5 ) were collected, washed twice with PBS, and then incubated in a buffer with a 1:10 ratio of 25% glutaraldehyde to 0.1 M cacodylate, for 12–24 h at room temperature. After incubation, cells were washed thrice with 0.1 M cacodylate buffer. Cells were resuspended in 200 µL of 0.1 M cacodylate buffer and deposited into a prepared coverslip lined with Poly-L-Lysine and left overnight at room temperature. Osmium tetroxide (1%) was diluted in 0.1 M cacodylate buffer and the slides were incubated in it for 1 h, covered with foil. The slides were washed thrice with distilled water for 10 min each, and then covered again in foil. This process was repeated with 30%, 50%, 70%, 90%, and 100% of ethanol diluted in distilled water. The slides were transferred to a critical point apparatus and dried. Coverslips were then mounted on a metal stub with a double-sided carbon metal adhesive and coated with platinum. Images were captured by SEM. Cell rolling and analysis of the cell-rolling behavior Cell rolling was performed at room temperature using either a sticky-Slide VI 0.4 microfluidics chamber that was already adhered to the rhE-selectin (0.2 µg/mL)-deposited coverslip (for super-resolution imaging) or a µ-Slide VI 0.1 microfluidics chamber incubated with rhE-selectin (1 µg/mL) (for live cell rolling). Male Luer connectors (ibidi GmbH) were used to connect the inlet and outlet of the chamber to a 0.8-mm silicon tubing (ibidi GmbH). The inlet silicon tubing was placed into HBSS rolling buffer with 1% BSA (Sigma) and 1 mM CaCl 2 (Sigma). The outlet silicon tubing was attached to a programmable syringe pump (PHD ULTRA, Harvard Apparatus) by adding a female Luer Lock connector (ibidi GmbH). The rolling buffer was allowed to flow into the chamber for 90 s before KG1a cell rolling to equilibrate the flow path. KG1a cells (10 6 ) were then suspended in the rolling buffer and perfused into the chamber at a sheer stress of 2 dyne.cm − 2 for 20 s, before stopping the flow for 30 s, to allow the cells to interact with the rE-selectin-incubated chamber. Flow was then resumed using the rolling buffer at a sheer stress of 2 dyne.cm − 2 to roll the cells for the required amount of time. Cell-rolling was visualized and performed by mounting the microfluidic chamber on a LCAch N 20X objective (Olympus)-equipped CXK41 inverted optical microscope (Olympus). An XC10 CCD camera (Olympus) and a CellSens software (Olympus) were used to record transmitted light images at video rate. An Olympus IX71 inverted optical microscope equipped with a UAPON 100XOTIRF high numerical aperture (NA) objective (Olympus) and an iXon3 897 EMCCD camera (Andor Technology) was also used to capture transmitted optical microscopy images. Cell rolling videos were analyzed using TrackMate Fiji, an ImageJ plugin. Using the Linear Assignment Problem (LAP) tracker, we localized and tracked each cell as it rolled. Mean rolling velocity was calculated by dividing the total displacement of the rolled cell by the duration of rolling per cell. To consider only cells that rolled properly, any cell that rolled for < 2.5 s or for < 30 µM was excluded from calculations. Fluorescence labeling of cells For live, two-color cell rolling, KG1a cells were washed with HBSS and incubated in 1.5 mL of FC blocker (Accurate Chemical & Scientific) for 30 min at 4°C. Cells were then incubated in 5 µg/mL of AF647-conjugated CD44 antibody (515-BD Bioscience) diluted in 2% BSA (Sigma) in HBSS for 45 min at 4°C. Cells were then incubated in 5 µg/mL of AF488-conjugated CD34 antibody (Qbend10-Biolegend) diluted with 2% BSA in HBSS, for 45 min at 4°C. Cells were resuspended in perfusion buffer (1X DMEM flour bright (Gibco) and 1 mM of CaCl 2 ) and rolled at a constant rate of 2 dyne.cm − 2 . For two-color super-resolution of cells in suspension, a fixing solution composed of 0.2% (w/v) glutaraldehyde (Electron Microscopy Sciences) and 3% (w/v) paraformaldehyde (Electron Microscopy Sciences) in HBSS was used to fix the cells for 20 min at room temperature. Goat serum (10%) (Sigma) was then used to block the cells at 37°C for 40 min. Next, the fixed/blocked cells were incubated with 15 µg/mL of a primary antibody against one of the proteins of interest that was diluted in 2% BSA (Sigma) in HBSS at room temperature for 30 min; this was followed by treatment with 5 µg/mL of AF488 conjugated goat secondary antibody (Invitrogen) diluted in 2% BSA in HBSS at room temperature for 25 min. Cells were then fixed using 0.2% (w/v) glutaraldehyde and 3% (w/v) paraformaldehyde in HBSS at room temperature for 10 min. Next, the cells were incubated with another primary antibody corresponding to another protein of interest diluted in 2% BSA in HBSS at room temperature for 30 min, followed by 5 µg/mL of AF647 conjugated secondary antibody (Invitrogen) diluted in 2% BSA in HBSS at room temperature for 30 min. The cells were again fixed using 3% (w/v) paraformaldehyde and 0.2% (w/v) glutaraldehyde in HBSS at room temperature for 10 min. The cells were placed in a sticky-Slide VI 0.4 microfluidic chamber that already adhered to a clean coverslip and coated with Silane (Sigma A3648) overnight, at 4°C. The cells were incubated in the chamber for 30 min at room temperature. For rhE-selectin rolled fluorescence labeling of KG1a cells, cells were perfused into a sticky-Slide VI 0.4 microfluidic chamber that was previously coated with E-selectin, as described earlier, at a constant shear stress of 2 dyne.cm − 2 . After the cells rolled for 90 s, a fixing solution made up of 0.2% (w/v) glutaraldehyde and 3% (w/v) paraformaldehyde in HBSS was auto-perfused at 2 dyne.cm − 2 for approximately 2–3 min to fix the cells to the chamber while still rolling. Cells were incubated in the fixing solution for another 17 min to ensure proper and complete fixing of the cells. Fluorescence labeling of cell proteins in the chamber was performed as outlined in the previous step. Fixed and immunolabeled Control KG1a cells were suspended in switching buffer before perfusing them into a clean silane-coated microfluidic chamber. Super-resolution “STORM” microscopy and analysis of super-resolution (SR) images To perform two-color super-resolution imaging on immunolabeled KG1a cells, a switching buffer composed of catalase (40 µg/mL; Sigma), 10 mM 2-aminoethanethiol (MEA; Fluka), TN buffer [50 mM tris (pH 8.0) and 10 mM NaCl], oxygen scavenging system [glucose oxidase (0.5 mg/mL; Sigma), and 10% (w/v) glucose] was added to the cells just prior to imaging. MEA solution was prepared in 1 M aqueous KOH, with pH adjusted to 8. The switching buffer was prepared just prior to imaging. A custom-built, widefield illumination fluorescence microscope on an inverted IX71 optical microscope platform (Olympus) was used for the super-resolution imaging 11 , 46 , 75 , 76 . Beam lines with wavelengths 640 nm and 488 nm (60 mW; MLD, Cobolt) were introduced coaxially into the inverted IX71 optical microscope. Using the HILO configuration, the samples were illuminated through a 100× objective (NA = 1.49; UAPON 100XOTIRF, Olympus) with illumination powers of 3.8 mW cm − 2 or 5.9 mW cm − 2 for the 640 nm or 488 nm beams, respectively. Sample fluorescence signals were captured using the same objective lens which was separated from the illumination light by a multiband dichroic mirror (Di03-405/488/561/635-t1-25x36, Semrock), and passed through a TuCam dual-camera adaptor (Andor Technology) equipped with a filter cassette containing a dichroic image splitter (FF580-FDi01-25 × 36, Semrock) to separate the fluorescence into two channels. Two EMCCD cameras (iXon3 897, Andor Technology) detected the split fluorescence from the samples through emission bandpass filters (FF01-550/88 − 25 and FF01-697/58 − 25, Semrock). To reconstruct SR localization microscopy images, 5,000 frames of the fluorescence image sequences were recorded using the Andor iQ3 software 11 . Reconstruction of the super-resolution images was done using the Localizer software 42 . AF647 and AF488 molecule positions were determined by 2D Gaussian fitting of the point spread function (PSF) images. For two-color super-resolution analysis, calibrating the shift between the two channels was conducted by the use of TetraSpeck microspheres (100 nm in diameter) that were deposited on a cleaned coverslip. Once images of the TetraSpeck beads were taken using the two laser beams, we produced a registration map that allowed us to correct the shift between the two detected images and applied these corrections to the obtained localized images. TIRFM and 3D-SR image acquisition To achieve a nanoscale spatial correlation between the cell surface morphology captured by TIRFM imaging and the 3D-SR coordinates of adhesion molecules characterized by the astigmatism-based 3D-single molecule localization microscopy (SMLM) imaging 77 , we collected TIRFM and 3D-SMLM data sequentially from the same KG1a cell under two different illumination conditions (i.e., TIR and HILO for TIRFM and 3D-SMLM imaging, respectively). Each image was recorded through an individual detection pathway using two EMCCD cameras (iXon3 897, Andor Technology). The image was split into different wavelength ranges using a dichroic mirror. This configuration does not require mechanical switching of the detection pathway for each measurement, allowing each image to be compatible with nano-scale correlation analysis. The microfluidic-based 3D-TIRFM imaging was utilized to map the cell surface morphology. We recorded TIRF images of membrane-stained KG1a cells (MemBrite-FX640) at a single angle of incidence 66.6 ° (θ), corresponding to a 120 nm evanescent field penetration depth, under weak illumination of 640-nm laser power (equivalent to 30 W cm − 2 at Epi configuration). Per a sample field of view, 10 frames were captured to average the fluorescence intensity before processing the TIRF image for topographical map construction. The pixel with maximum intensity was assigned as the closest one to the glass surface. Fluorescence signals were separated from the illumination light by a multiband dichroic mirror (Di03-405/488/561/635-t1-25x36, Semrock). Then the fluorescence signal was separated into two images using a dichroic image splitter (Semrock, FF580-FDi01-25 × 36) by different wavelengths, with each assigned to a different measurement mode. An emission bandpass filter was used for each separated image of TIRFM imaging (FF01-697/58 − 25) and 3D-SMLM imaging (FF01-550/88 − 25). A cylindrical lens with a focal length of 1000mm was used to create the astigmatism-based PSF for 3D-SMLM imaging. Details of the method will be published elsewhere. TIRFM image processing Using a custom-written MATLAB script, the initial membrane topography analysis was performed from TIRFM images, and included filtering, visualization, and data extraction to generate a topographical 3D map of a microvilli. Laplacian of Gaussian (LoG) filter (Gaussian σ = 0.5; kernel size = 10 × 10) and additional Gaussian smoothing was applied to enhance and resolve microvilli features and exclude background noise from the cell body, as it lay outside the edge of detection. To find contours representing microvilli boundaries, thresholding (min-intensity = 200) was applied to obtain a binary mask, and then edge detection was applied. The mask was also used to remove irrelevant localization outside the microvilli boundaries. After that, the intensity values of the image were normalized, and fluorescence intensity was converted to a height profile using the TIRF theory, generating corresponding contour maps of the individual microvilli. The topography map of individual microvilli was segmented into several contour levels by dividing the maximum distance by interval distance (10 nm) 78 . Reconstruction of 3D topographical map and single molecule coordinates A custom-written Python script was developed for 3D spatial overlaying of the cell surface topography map and obtaining 3D coordinates of the adhesion molecules. TIRFM data were analyzed and compared with single-molecule data to identify the structural similarity index (SSI) between microvilli and single-molecule data. After extracting the topographical map and single-molecule data within a 3D-bounding box of a region (microvilli), 3D surface fitting was applied to the single molecules. Next, the 3D topography surface of TIRF data and the fitted surface within a bounding box to an 8-bit grayscale image were normalized to calculate the SSI between the 2D projections. Microvilli and their corresponding single-molecule coordinates that exceeded the SSI threshold (x ≤ 0.8) were selected for downstream analysis. Density-based cluster analysis The SM localization maps of different proteins were analyzed using a custom-written Python code based on the algorithm density-based spatial clustering of applications with noise (DBSCAN). We determined minpts = 7 and epsilon = 45 nm 79 . In this way, the code defined and separated SM into groups and clusters in 3D. CD34 lentivirus variant design : CD34 lentivirus variants were designed and purchased from GenScript. Three variants were designed: 1) Empty vector lentivirus particles encoding puromycin resistance, 2) ‘HEK-CD34’ lentivirus particles encoding the full human CD34 amino acid sequence including the signal peptide and a puromycin resistance. Amino acid sequence of the insert was as follows: MLVRRGARAGPRMPRGWTALCLLSLLPSGFMSLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITETTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKTLIALVTSGALLAVLGITGYFLMNRRSWSPTGERLGEDPYYTENGGGQGYSSGPGTSPEAQGKASVNRGAQENGTGQATSRNGHSARQHVVADTEL*. ‘HEK-∆EC’ lentivirus particles encoding a CD34 variant with a missing extracellular domain excluding the first four amino acids (SQKT). Puromycin resistance was also included. Amino acid sequence of the insert was as follows: MLVRRGARAGPRMPRGWTALCLLSLLPSGFMSQKTLIALVTSGALLAVLGITGYFLMNRRSWSPTGERLGEDPYYTENGGGQGYSSGPGTSPEAQGKASVNRGAQENGTGQATSRNGHSARQHVVADTEL*. Lentivirus transduction into HEK293ft cells HEK293ft cells (6 × 10 5 ) were collected and plated in a 6-well plate overnight, in 2 mL of supplemented DMEM media (10% FBS and 100 µg/mL of streptomycin) at 37°C with 5% CO 2 . Media was removed and new supplemented DMEM media with 10 multiplicity of infection (MOI) of Lentivirus particles and 2 µg/mL of polybrene was added to the cells and incubated at 37°C with 5% CO 2 , for 48 h. Media was removed again and new supplemented media containing 3 µg/mL of puromycin was added until all un-transduced cells were killed. Fucosyltransferase 6 (FUT6) Treatment HEK293ft cells (4 × 10 5 ) were collected and incubated for FUT6 treatment (0.5 mM of GDP-fucose (Sigma), 25 mM Hepes (pH 7.5) (Gibco Invitrogen), 5 mM MnCl 2 , 0.1% human serum albumin (HAS), and 1 µg purified rhFTVI enzyme in HBSS) for 30 min, at 37°C with 5% CO 2 . Cells were washed twice with HBSS containing 10 mM of EDTA, and once with un-supplemented HBSS. E-selectin Treatment HEK293ft cells (1 × 10 6 ) were collected and incubated in 1 mL of supplemented DMEM media, 5 µL of Dimer E-selectin (0.23 mg/mL) for 1 h at 37°C, with 5% CO 2 . Cells were then washed twice with PBS. CD34 variant in vivo analysis : NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) Mice were procured from Charles River Laboratories (Lodi, Italy) and housed in the Animal Research Core Lab at the King Abdullah University of Science and Technology. The NSG mice were randomly allocated into four experimental groups: (i) an untreated control group injected with 100 µL of PBS, intravenously (IV) via the tail vein (n = 3), (ii) a group designated as 'F' that received 100 µL of 3 × 10 5 DIR pre-stained HEK-CD34 cells in PBS (n = 4) IV, (iii) an 'EC' group that was administered 100 µL of 3 × 10 8 DIR pre-stained HEK-∆EC cells in PBS (n = 4) IV, and (iv) a 'Negative' group that received 100 µL of 3 × 10 5 DIR pre-stained HEK cells in PBS (n = 4) IV. The biodistribution of HEK cells in the mice was longitudinally imaged at 1, 8, 24, and 48 h using the IVIS Spectrum system (PerkinElmer Inc., MA, USA), after which the mice were euthanized, and various organs including the spleen, liver, spine, heart, lung, femur, and tibia were excised and imaged using the IVIS imaging system. Images were captured by a charge-coupled device (CCD) camera with the following settings: binning set to medium and f/stop at 2. The filter sets were calibrated at 710 nm for excitation and 800 nm for emission wavelengths. Fluorescence intensity was quantitatively assessed with the Living Image software suite (Caliper Life Sciences, MA, USA). The accumulation of DIR-labeled HEK cells in the whole body, as well as in specific organs, was quantified by averaging out radiant efficiency ([p/s/cm²/sr] / [µW/cm²]). Data availability Data supporting the findings of this work are available within the paper and supplementary material. Declarations acknowledgements The authors would like to thank Umme Habiba for her efforts in reagent preparation and her role in the thawing and freezing of HEK293ft and KG1a cells. In addition, the authors would also like to thank the Imaging and Characterization core facility at King Abdullah University of Science and Technology (KAUST) and especially Maya Ayach for her efforts in providing some of the SEM data. Finally, the authors would like to thank Professor Samir M Hamdan for his helpful discussion. The research reported in this publication was supported by the King Abdullah University of Science and Technology (KAUST). author contributions M.M.A. designed, performed, and analyzed experiments and wrote the manuscript; A.A. designed, performed, and analyzed super-resolution experiments and wrote the manuscript; S.N. managed the SR imaging setup; A.I.A., K.A., performed super-resolution experiments; Y.L. performed and analyzed in vivo experiments in mice; A.T. performed and analyzed part of the SEM experiments; A.S.A. provided and aided in the use of the FUT6 enzyme during HEK293ft functionality experiments; I.I. aided in final figure production as well as in the use of E-selectin, during E-selectin treatments; S.H. conceived and provided all of the SR imaging platforms as well as reviewed the manuscript; and J.S.M. conceived, designed, and analyzed experiments and wrote the manuscript. 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Chem. 10–2022 , (2022) Ghosh, S., et al.: ERM-Dependent Assembly of T Cell Receptor Signaling and Co-stimulatory Molecules on Microvilli prior to Activation. Cell. Rep. 30 , 3434–3447e3436 (2020) Verzelli, P., Nold, A., Sun, C., Heilemann, M., Schuman, E.M., Tchumatchenko, T.: Unbiased choice of global clustering parameters for single-molecule localization microscopy. Sci. Rep. 12 , 22561 (2022) Additional Declarations There is NO Competing Interest. Supplementary Files Supplementaryfiles.docx Supplementary Figures Cite Share Download PDF Status: Under Review Version 1 posted 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. 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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-7280176","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":510303135,"identity":"84f3037d-46c9-4a94-900f-812aa6a1a8ba","order_by":0,"name":"Jasmeen 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Technology","correspondingAuthor":false,"prefix":"","firstName":"Satoshi","middleName":"","lastName":"Habuchi","suffix":""}],"badges":[],"createdAt":"2025-08-02 20:05:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7280176/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7280176/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91189075,"identity":"4d0c4753-43b3-41b1-ab2f-97e44b11eda6","added_by":"auto","created_at":"2025-09-12 14:26:08","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":320429,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSuperimposed SR images show nanoscale localization patterns of CD34 and CD44 on fixed KG1a cells before and after E-selectin rolling.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A\u003c/strong\u003e) Schematic depicting microfluidics approach for examining \u003cstrong\u003e(B) \u003c/strong\u003efixed or rolled and fixed KG1a cells or \u003cstrong\u003e(C) \u003c/strong\u003erolled and fixed KG1a cells after visualizing tether expression. CD44 (right panel) and CD34 (left panel) molecules were immunolabeled with Alexa Fluor 488 (AF488) or Alexa Fluor 647 (AF647) secondary antibodies, respectively. The cells were imaged via a super-resolution imaging platform. A superimposed image of CD34 and CD44 is shown in the middle panel. The rectangular insets show enlarged views of the yellow regions. Representative Super-resolution (SR) images out of 22 images captured in n=3 independent experiments are shown. \u003cstrong\u003e(D) \u003c/strong\u003eLocalization map, top view of a 3D projection of segmented microvilli to many contour levels based on the height profile (grayscale) and the corresponding overlaid CD34 and CD44 molecules (colored points), before and after rolling (Color scale; height in nm). Quantification of the average percentage of molecules per cluster and average numbers of clusters per microvillus is shown in the bottom graphs. Superimposed images of single CD44 and CD34 molecules co-localizing with respect to reconstructed microvilli or (\u003cstrong\u003eE)\u003c/strong\u003e tether surface within a region of interest (ROI). (Scale bar: 100 nm). Each\u003cstrong\u003e \u003c/strong\u003epixel measures 83 nm\u003cstrong\u003e.\u003c/strong\u003eStatistical analysis for the data in this figure was performed using two-way ANOVA using the GraphPad Prism software using at least three independent experiments (****P \u0026lt; 0.0001) (***P \u0026lt; 0.001) (**P \u0026lt; 0.01) (*P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7280176/v1/471d7d41efeebc3a86dfc824.jpeg"},{"id":91189060,"identity":"1d52df5f-a1a7-46ad-83da-90cf7c80457e","added_by":"auto","created_at":"2025-09-12 14:26:08","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":413575,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCD34 knockdown in KG1a cells reduces the cell ability to produce tethers and slings. (A) \u003c/strong\u003esCT-KG1a (transfected with 250 pmol of Scramble control siRNA) and CD34-KD KG1a (transfected with 250 pmol of CD34 siRNA) cells were immunolabeled with AF647-conjugated anti-CD44 antibody or AF488-conjugated anti-CD34 antibody and then perfused over a microfluidics chamber coated with recombinant E-selectin human IgG chimeric protein (rE-selectin) (concentration of 0.2 µg/mL). Tethers and slings were imaged in real-time using a single protein, microfluidics-based imaging platform. \u003cstrong\u003e(B)\u003c/strong\u003e The number of tethers and slings was quantified using GraphPad Prism software. \u003cstrong\u003e(C)\u003c/strong\u003e After treatment with bromelain for 30 min, WT KG1a cells were transfected with 250–500 pmol of CD34, CD43, PSGL-1, Ezrin, or Scramble siRNA. Cells were then fixed and imaged using SEM, as described in the methods section. Representation of n=3 independent experiments are shown. Bar graph shows the number of microvilli per 2 µm\u003csup\u003e2 \u003c/sup\u003eof surface area, quantified using GraphPad Prism. The values plotted were obtained from a representative number of cells (3) out of 20 cells from each sample taken from n=3 independent experiments. \u003cstrong\u003e(D) \u003c/strong\u003eHEK293ft cell lines transfected with either PSGL-1, CD44, or CD34 molecules using a lentivirus transduction system as described in the methods, were assessed for the expression of microvilli\u003cstrong\u003e \u003c/strong\u003eusing values from three different cells.\u003cstrong\u003e (E)\u003c/strong\u003e After treatment with bromelain for 30 min, WT KG1a cells were transfected with 250–500 pmol of either CD34, PSGL1, CD43, Ezrin, or Scramble siRNA. A modified parallel plate flow microfluidic chamber apparatus was used where rE-selectin was deposited on a microfluidic chamber at a controlled concentration (0.2 µg/mL). Cells were then rolled on the E-selectin-deposited microfluidics chamber at 2, 5, and 8 dyne.cm\u003csup\u003e−2\u003c/sup\u003e in continuous succession and tested for their ability to bind E-selectin in motion as well as for their \u003cstrong\u003e(F) \u003c/strong\u003evelocity. Ten or more rolled cells were tested from n=5 independent experiments. Statistical analysis for the data in this figure was performed using two-way ANOVA using the GraphPad Prism software, using at least three independent experiments (****P \u0026lt; 0.0001) (***P \u0026lt; 0.001) (**P \u0026lt; 0.01) (*P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7280176/v1/119cbf7b779601cf28353d47.jpeg"},{"id":91189061,"identity":"f608587d-e794-4e4a-819f-bc49b9f05946","added_by":"auto","created_at":"2025-09-12 14:26:08","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":232532,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCluster analysis of sCT-KG1a and CD34-KD KG1a cells. \u003c/strong\u003eLocalization map, top view of a 3D projection of segmented microvilli onto many contour levels based on the height profile (grayscale) and corresponding overlaid molecules (CD44, CD43, CD34, PSGL-1, and Ezrin) (colored points) before and after CD34 knockdown, as well as before and after rolling. (Color scale; height in nm). Superimposed images of respective single molecules co-localizing with respect to the reconstructed microvilli surface within a region of interest (ROI). (Scale bar: 100 nm). Each\u003cstrong\u003e \u003c/strong\u003epixel measures 83 nm. The column graphs show the calculated average number of clusters found per microvilli. Statistical analysis for the data in this figure was performed using Multiple unpaired t-tests using the GraphPad Prism software, using at least three independent experiments, where a “*” indicates a discovery / P \u0026lt; 0.05. “nd” indicates no discovery / P \u0026gt; 0.05. Results are from n=3 independent experiments.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7280176/v1/3e8d82e94086ae7cfc8f9e79.jpeg"},{"id":91189067,"identity":"ad847c05-3c2c-4896-98ad-0b09927eb26b","added_by":"auto","created_at":"2025-09-12 14:26:08","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":259116,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eP-ezrin (@T567) phosphorylation is increased due to E-selectin treatment of full-length CD34 or owing to ∆EC CD34 expression. (A)\u003c/strong\u003e Third-generation lentiviruses encoding two different variants of CD34 or an empty vector were purchased from GeneScript. “Empty Vector” encodes an empty lentivirus with only puromycin resistance. “Full CD34” encoded full-length CD34, and “∆EC” encoded a CD34 variant missing its extracellular domain, except the first four amino acids (SQKT). \u003cstrong\u003e(B) \u003c/strong\u003eWestern blot analysis of “HEK (Vector Control)”, “HEK-CD34,” and “HEK-∆EC” cells reveal that HEK-∆EC cells show higher levels of P-Ezrin (Thr567). The blot was derived after incubation with a P-Ezrin (T567) antibody. \u003cstrong\u003e(C)\u003c/strong\u003e Western blot analysis of HEK (control) and “HEK-CD34” cells shows that HEK-CD34 cells show higher levels of P-Ezrin (Thr567) after treatment with E-selectin. “HEK” and “HEK-CD34” cells were treated with fucosyltransferase 6 (FUT6) , both FUT6 and E-selectin, or left blank. The blot on top was obtained after incubation with Ezrin + CD34 antibodies. The blot on the bottom was obtained after incubation with the P-Ezrin (T567) antibody. \u003cstrong\u003e(D) \u003c/strong\u003eHEK, HEK-∆EC, and HEK-CD34 cells were prepared for SEM imaging as described in the methods section. In this figure, representative SEM images from n=3 independent experiments are shown. Measurements of microvilli quantity was done through a manual count of a 2 µm\u003csup\u003e2 \u003c/sup\u003earea on the cell surface of three different cells, for each sample and plotted using GraphPad Prism software. \u003cstrong\u003e(E) \u003c/strong\u003eControl, HEK-∆EC, and HEK-CD34 cells were dyed using DID membrane dye and perfused over an E-selectin-deposited chamber. The number of tethers and slings produced by cells upon rolling during live cell fluorescence imaging was evaluated. Data were collected after measurement of all tethers and slings present per 3,300 frames of video obtained of rolling cells. Statistical analysis of tethers and slings was performed using unpaired T-test using GraphPad Prism software (***P \u0026lt; 0.001) (**P \u0026lt; 0.01) (*P \u0026lt; 0.05). Statistical analysis of microvilli quantity was performed using one-way ANOVA using GraphPad Prism software (***P \u0026lt; 0.001) (**P \u0026lt; 0.01) (*P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7280176/v1/e72677a4b8c1b9c9e24c899d.jpeg"},{"id":91189065,"identity":"f7932aa1-2882-40c6-88a7-ee460af30d58","added_by":"auto","created_at":"2025-09-12 14:26:08","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":451788,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHEK-∆EC and HEK-CD34 cells accumulate better under \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eex vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e conditions, compared to control cells. \u003c/strong\u003eControl, HEK-∆EC, and HEK-CD34 cells were stained with DiR membrane dye, treated with fucosyltransferase 6 (FUT6) , and then injected intravenously into NOD.Cg-Prkdc\u003csup\u003escid\u003c/sup\u003e Il2rg\u003csup\u003etm1Wjl\u003c/sup\u003e/SzJ (NSG) mice and imaged using the in vivo imaging system (IVIS) at 1, 8, 24, and 48 h. \u003cstrong\u003e(A)\u003c/strong\u003e Illustrative overview of the IVIS imaging system and procedure. \u003cstrong\u003e(B)\u003c/strong\u003e Images of whole mice superimposed with captured IVIS signaling (left panel) reveal a substantial accumulation of cells in mice injected with FUT6-treated HEK-CD34 cells compared to that in other mice. The right panel shows the same data plotted per hour as total radiant efficiency. Two mice from a total of four mice per group are shown \u003cstrong\u003e(C)\u003c/strong\u003e Mice were then euthanized, and individual organs/tissues were prepared for \u003cem\u003eex vivo\u003c/em\u003e imaging. \u003cem\u003eEx vivo\u003c/em\u003e imaging of individual tissues/organs (spine, legs, heart, liver, lungs, and spleen) showed a remarkable accumulation of tissues and organs in mice injected with FUT6-treated HEK-CD34 cells compared to that in other mice. Each group comprised four mice. Statistical analysis in this figure was performed using T-test (*P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7280176/v1/3dd9709458e63dc641871ee6.jpeg"},{"id":91194085,"identity":"9c2ccd73-8c18-43c0-8948-f4019f1c8c1d","added_by":"auto","created_at":"2025-09-12 14:50:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3235597,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7280176/v1/15014173-2782-43da-865b-1b21d6836b98.pdf"},{"id":91192372,"identity":"4128bc7f-c377-41ae-813a-903548d5ccb9","added_by":"auto","created_at":"2025-09-12 14:42:08","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":893862,"visible":true,"origin":"","legend":"Supplementary Figures","description":"","filename":"Supplementaryfiles.docx","url":"https://assets-eu.researchsquare.com/files/rs-7280176/v1/d1c64af3ba33d950036fae38.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"CD34 orchestrates the formation and actions of microvilli for efficient E-selectin-mediated cell migration","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCell migration is fundamental to several processes, including development, immune defense, and tissue regeneration \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. It is also a key process for tumor-cell invasion and metastasis \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Therefore, understanding its underlying mechanisms may provide key tools for cancer diagnoses and treatment. In leukemia treatment, for example, intravenous transplantation of hematopoietic stem/progenitor cells (HSPCs) relies on the ability of stem cells to migrate from the blood to bone marrow \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Decades of research have established a multistep paradigm for cell migration: cell tethering and rolling on the endothelial cell-lining tissues and organs, activation and binding of integrins, cell arrest, and cell transmigration \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The prerequisite for cell tethering and rolling involves the establishment of shear-resistant binding between P- and/or E-selectin on the surface of the endothelium and several ligands on the cell in flow \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. This leads to a gradual slowing of the cells in flow, allowing surface receptors to recognize chemokines in the vicinity and further progress toward cell migration \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSeveral ligands have been identified as E- and P-selectin ligands, which may vary depending on the cell type \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. This raises the question as to whether these ligands play distinct, overlapping, or coordinated roles during cell tethering and rolling and if they are linked to the rest of the steps in cell migration. Super-resolution and single molecule imaging of cells before and after rolling have unraveled fundamental roles for microvilli in the cell tethering and rolling step. Several of the ligands were shown to cluster in these microvilli; upon their binding to E- or P-selectin, a shear force is built on the cell in flow, forcing its microvilli to extend into long tethers behind the cell \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The tethers may flip in front of the cell and act as slings to enhance cell rolling \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. These findings highlight the importance of studying the roles of E- and P-selectin ligands within the context of microvilli, tethers, and slings to understand their mechanistic contribution to the cell tethering and rolling step.\u003c/p\u003e\u003cp\u003eMicrovilli are cellular extensions of the plasma membrane that are supported by polymerized actin filaments \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Microvilli are enriched with cross-linked actin bundles and the Ezrin, Radixin, Moesin (ERM) family proteins \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. It has been proposed that during cell migration, ERM proteins link the actin cytoskeleton to several E-selectin ligands, including CD43 \u003csup\u003e29, 30\u003c/sup\u003e, CD44 \u003csup\u003e31\u003c/sup\u003e, and P-selectin glycoprotein ligand-1 (PSGL-1) \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e; this link may contribute to ligand clustering \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e and the maintenance of the microvilli structure \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Two-color super-resolution imaging of fixed cells before rolling, confirms the nanoscale co-localization of the actin cytoskeleton and CD44 \u003csup\u003e11\u003c/sup\u003e. However, after cellular rolling, although this co-localization is still observed on protrusions from the cell surface, it is absent on the tethers and slings formed. In fact, visualizing tethers and slings during live cell rolling using single molecule, microfluidics-based imaging \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, confirms that tethers and slings detach from the actin cytoskeleton \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Remarkably, CD44 and PSGL-1 molecules continue to fully cover the surfaces of the tethers and slings \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Additionally, the tips of the tethers and slings that bind selectins, called tethering points, sometimes express a higher clustering of PSGL-1 molecules compared with the rest of the tethers and slings \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, suggesting a specific mechanism of tethering point formation that may require such clustering to occur on the surface of cells as they roll. Collectively, these findings indicate dynamic structural changes in the microvilli, tethers, and slings, and in the spatial and temporal clustering of selectin ligands within these nanoscale structures. The findings also indicate potential linkages between selectin ligands and microvilli formation via ERM protein signaling.\u003c/p\u003e\u003cp\u003eTo decipher the role of the various selectin ligands in the formation of microvilli, tethers, and slings, and that of internal signal transduction via the ERM proteins, we focused on CD34 and the ERM protein Ezrin, as CD34 has been linked to microvilli formation and E-selectin binding \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. We compared the findings from CD34 analysis to that from E-selectin ligands CD43, CD44, and PSGL-1. Our results unravel a complex spatial and temporal networking among the E-selectin ligands within the microvilli and establish CD34 as the orchestrator of this networking, leading to the enhancement of the mechanical and signaling mechanisms that form and support the microvilli function during cell tethering and rolling. These findings argue that some E-selectin ligands might be playing more pronounced role in the cell tethering and rolling step and indicate that the mechanical and signaling changes induced by CD34 and their differential effect on the clustering of E-selectin ligands might link this step to the subsequent multistep paradigm for cell migration.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCD34 exhibits a \u0026ldquo;ring-like\u0026rdquo; nanoscale localization pattern on the microvilli\u003c/h2\u003e\u003cp\u003eTo understand the role of CD34 in cellular homing, we characterized the nanoscale localization of CD34 prior to or post, cellular rolling, comparing it with the well-known E-selectin ligand CD44 \u003csup\u003e41\u003c/sup\u003e. For pre-rolling, KG1a cells were attached onto the surface of a microfluidics channel slide that was pre-treated with silane; for post-rolling the cells were flown at a biologically relevant shear stress of 2 dyne.cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e over recombinant E-selectin human IgG chimeric protein (rE-selectin) deposited on a Protein A-coated microfluidic chamber (\u003cb\u003eFig.\u0026nbsp;1A\u003c/b\u003e). The cells were then fixed and stained specifically for CD34 (Alexa Fluor 647; red) or CD44 (Alexa Fluor 488; green). Super-resolution images of either CD34 or CD44 at the slide\u0026rsquo;s surface were obtained using super-resolution localization microscopy that was configured with a highly inclined and laminated optical sheet (HILO), as described previously \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Consistent with our previous study \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, significant CD44 clustering reorganization was observed, from a patchy spot-like clustering during pre-rolling, to elongated network-like structures post-rolling (\u003cb\u003eFig.\u0026nbsp;1B\u003c/b\u003e). We also observed similar reorganization of the CD34 clusters from diffusive to elongated network-like structures upon cellular rolling (\u003cb\u003eFig.\u0026nbsp;1B\u003c/b\u003e). Interestingly, CD34 clusters formed a unique hollow \u0026ldquo;ring-like\u0026rdquo; structure prior to and post- rolling (\u003cb\u003eFig.\u0026nbsp;1B\u003c/b\u003e). Co-localization analysis of CD34 and CD44 using the localizer extension, in the IGOR Pro Software \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e showed that in the majority of the cells (n\u0026thinsp;=\u0026thinsp;15 cells), ring-like structure encircled the CD44 clusters which appeared not to encompass them prior to rolling but encompassed them post-rolling (\u003cb\u003eFig.\u0026nbsp;1B\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eWe next compared the clustering dynamics between CD34 and CD44 in the tethers and slings that occasionally formed and fixed during rolling (\u003cb\u003eFig.\u0026nbsp;1C\u003c/b\u003e). Intriguingly, distal to the cell body near the microfluidics surface, where the tethering point of the tethers and slings was located, we observed a distinct patch of CD44 detached from the rest of the CD44 clusters and a dense clustering of CD34. Closer examination of the superimposed CD34 and CD44 signals revealed a CD34 ring-like structure that encircled and encompassed the detached CD44 cluster at the tethering point.\u003c/p\u003e\u003cp\u003eNext, we zoomed in on the CD34 ring-like structure at the tips of the microvilli using a novel imaging technique that simultaneously combines 3D-total internal reflection fluorescence microscopy (TIRFM) and single molecule 3D- stochastic optical reconstruction microscopy (STORM) super-resolution imaging, to visualize the region near the tips of the microvilli, located near the tethering points within 120 nm of the surface of the glass coverslip. This enabled a 3D, higher resolution localization, of CD34 and CD44 prior to or post- rolling over rhE-selectin, at the single molecule level. Briefly, the microvilli topography near the tethering point was visualized by uniformly staining the cell surface using MemBrite Fix 640 and imaging them using TIRFM. Single molecules of CD34 and CD44 were visualized by staining with CD34 (AF488) or CD44 (AF488), respectively, and imaging using STORM (\u003cb\u003eFig.\u0026nbsp;1D\u003c/b\u003e). To visualize the 3D-topography of the microvilli using TIRFM, the cell surface fluorescence intensity was segmented and converted to distance, where the highest intensity was assigned to the tip of the microvilli near the glass surface as indicated in grayscale, in \u003cb\u003eFig.\u0026nbsp;1D\u003c/b\u003e. Higher counter threshold was applied to distinguish the tip of the different microvilli. 3D-STORM was used to generate the localization map of single CD34 or CD44 molecules and their distance from the glass coverslip surface are indicated by the color scale, in \u003cb\u003eFig.\u0026nbsp;1D\u003c/b\u003e. The maps from the 3D-topography and 3D-STORM from the same focal plane from the surface of the glass coverslip were overlayed, allowing for 3D-visualization of localized single CD34 or CD44 molecules near the tips of the microvilli. The 3D-maps were then converted to 2D-maps, which represent the spatial dimension of the 3D volume. Figure\u0026nbsp;1D shows the images of a large region of interest prior to or post-rolling. In both cases, CD34 molecules were located within the tips of the microvilli in a ring-like structure. The single molecule localization showed that CD34 was located slightly away from the tip of the microvillus (\u003cb\u003eFig.\u0026nbsp;1D\u003c/b\u003e). However, CD44 was randomly distributed in the tip of the microvillus prior to and post-rolling; CD44 was also present closer to the tip of the microvillus compared to CD34 (\u003cb\u003eFig.\u0026nbsp;1D\u003c/b\u003e). By screening the field of view for extended tethers near the surface, we visualized the dense localization of CD34 and small clusters of CD44 at the tethering point (\u003cb\u003eFig.\u0026nbsp;1E\u003c/b\u003e). The high resolution of this new imaging technique enabled us to analyze clustering criteria such as the average number of clusters per microvillus and average percent of molecules per cluster. These analyses showed that both parameters increased after cellular rolling in the case of CD34 and were not significantly affected in case of CD44 (\u003cb\u003eFig.\u0026nbsp;1D\u003c/b\u003e, bottom panels).\u003c/p\u003e\u003cp\u003eOverall, these results indicate that CD34 localizes in the microvilli and adopts a ring-like structure with increased clustering, post-cellular rolling. The 3D super-resolution images revealed that CD34 localizes below the very tip of the microvillus. We hypothesize that the CD34 ring-like structure may act as a scaffold that concentrates selectin ligands, as shown in the case of CD44, enhancing the ability of the microvilli to form tethers and slings.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCD34 is key for microvilli formation and supports cellular rolling\u003c/h3\u003e\n\u003cp\u003eWe investigated the effect of removing CD34, on the formation of microvilli and tethers and slings. KG1a cells were treated with siRNAs targeting CD34 (CD34-KD) or a scrambled control siRNA (sCT). The absence of CD34 was confirmed using western blot (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e). Cells were then fluorescent-stained for CD44 (Alexa Fluor 647; yellow) or CD34 (Alexa fluor 488; blue), prior to being perfused over a microfluidics chamber coated with rE-selectin, at a biologically relevant sheer stress of 2 dyne.cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e. As illustrated in \u003cb\u003eFig.\u0026nbsp;2A\u003c/b\u003e, sCT-KG1a cells formed tethers and slings during rolling and expressed both CD34 and CD44 on the cell surface and on the tethers and slings. A merged image of anti-CD34 (blue) and anti-CD44 (yellow) antibodies on wild type (WT) KG1a cells showed a more even distribution of both proteins on the cell body. CD34-KD KG1a cells expressed significantly lower total numbers of tethers and slings compared to sCT-KG1a cells (\u003cb\u003eFig.\u0026nbsp;2B\u003c/b\u003e); when tethers and slings were observed, they were visibly shorter and less developed than those observed on the sCT-KG1a cells (\u003cb\u003eFig.\u0026nbsp;2A\u003c/b\u003e). A merged image captured using anti-CD34 (blue) and anti-CD44 (yellow) antibodies showed the absence of CD34 signals on the cell surface (\u003cb\u003eFig.\u0026nbsp;2A\u003c/b\u003e), indicating the effective knockdown of CD34. These results suggest that the tether and sling formation relies on CD34 expression.\u003c/p\u003e\u003cp\u003eTo evaluate if the paucity of tethers and slings in CD34-KD KG1a cells may be attributed to a deficiency of microvilli, we used scanning electron microscopy (SEM) to image the surface of the sCT-KG1a and CD34-KD KG1a cells (\u003cb\u003eFig.\u0026nbsp;2C\u003c/b\u003e). The microvilli were mostly eliminated in the CD34-KD KG1a cells. In contrast, knockdown of other E-selectin ligands PSGL-1 \u003csup\u003e43\u003c/sup\u003e and CD43 \u003csup\u003e44\u003c/sup\u003e and the ERM protein Ezrin in KG1a cells resulted in various degrees of loss of microvilli structures; this was however, to a much lesser extent compared to that in the CD34-KD KG1a cells (\u003cb\u003eFig.\u0026nbsp;2C\u003c/b\u003e). The number of microvilli per \u0026micro;m\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e on the cell surface was quantified and the same results were observed as that in the SEM images (\u003cb\u003eFig.\u0026nbsp;2C\u003c/b\u003e, right panel). In a control experiment, we confirmed efficient knockdown of CD34, CD43, PSGL-1, and Ezrin (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eTo further compare the effect of various selectin ligands on the formation of microvilli, we used HEK cells that did not express CD34 and created HEK cells expressing CD34, CD44, or PSGL-1, and quantified the number of microvilli/\u0026micro;m\u003csup\u003e2\u003c/sup\u003e of HEK knock-ins. The CD34 knock-in had a substantially larger increase in the number of microvilli/\u0026micro;m\u003csup\u003e2\u003c/sup\u003e compared to other ligands, further supporting the conclusion that CD34 is key for the formation of microvilli, tethers, and slings (\u003cb\u003eFig.\u0026nbsp;2D\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eAs microvilli are crucial for cellular rolling \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, we also assessed the number of rolling CD34-, CD44-, PSGL-1-, and Ezrin-KD KG1a cells and their rolling velocity when perfused over rE-selectin at different shear stress forces, as described previously \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. As illustrated in \u003cb\u003eFigs.\u0026nbsp;2E and 2F\u003c/b\u003e, the removal of CD34 and PSGL-1 resulted in a consistent reduction in the number of rolling cells and an increase in their velocity under all tested shear stress forces. However, the magnitude of the change in CD34 knockdown was larger and more significant when compared to that for PSGL-1 KD. Surprisingly, the knockdown of CD43 and Ezrin resulted in the opposite trend of increasing the number of rolling cells and decreasing their rolling velocity, but the magnitudes of the changes were not significant in most cases, excluding the reduced rolling velocity in the knockdown of Ezrin at 5 and 8 dyne.cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e and in CD43 at 5 dyne.cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e (\u003cb\u003eFigs.\u0026nbsp;2E and 2F\u003c/b\u003e), although the knockdowns decreased the number of microvilli (\u003cb\u003eFig.\u0026nbsp;2C\u003c/b\u003e). The SEM images showed that the cell morphologies were not affected by the knockdowns. This indicates that a balance in the density of microvilli may be required for effective cellular rolling.\u003c/p\u003e\u003cp\u003eCollectively, these results demonstrate that CD34 had a more pronounced impact on the formation of microvilli, tethers, and slings and in achieving slow rolling compared to the other ligands, which is consistent with our previous work \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The results highlight the requirement for a balanced density of the microvilli for efficient rolling and tethering.\u003c/p\u003e\n\u003ch3\u003eCD34 affects the clustering of E-selectin ligands differentially in the microvillus\u003c/h3\u003e\n\u003cp\u003eGiven that eliminating CD34 had the greatest effect on microvilli and rolling parameters compared to other E-selectin ligands, we determined whether CD34 knockdown changed the nanoscale dynamic organization of selectin ligands within the microvillus prior to or post-rolling over rE-selectin. To this end, we used our new imaging technique that combines 3D-TIRFM and 3D-SMLM super-resolution imaging to quantify the number of clusters per microvillus. The high resolution and topographical mapping of the cell surface near the tethering points in this technique enabled us to identify specific regions on the KG1a cell surface where microvilli are still present. CD34 knockdown affected the number of clusters per microvillus of CD44, CD43, PSGL-1, and Ezrin in a differential manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003e). CD44 was the most affected ligand under both pre- and post-rolling conditions. CD43 and Ezrin were affected in the post-rolling condition, whereas PSGL-1 was affected in the pre-rolling condition. However, post-rolling, the localization of PSGL-1 at the tip of the microvillus was reduced to a greater extent than its distribution at the lower region of the microvillus tip. In a control experiment, using the HILO super-resolution imaging setup outlined in \u003cb\u003eFig.\u0026nbsp;1A\u003c/b\u003e, KG1a CD34-KD showed substantially dissociated CD44 clusters and an absence of nanoscale organization of the membrane surface (i.e., eliminated protrusions) (\u003cb\u003eSupplementary Fig.\u0026nbsp;2A\u003c/b\u003e), and none of the KG1a knockdown in CD43, PSGL-1, and Ezrin had an effect on the nanoscale clustering of CD34 or CD44 (\u003cb\u003eSupplementary Fig.\u0026nbsp;2B\u003c/b\u003e); CD44 knockdown was not possible.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCollectively, these results highlight the critical role of CD34 in the nanoscale dynamic organization of selectin ligands within the microvillus prior to and post-rolling and indicate that this coordination varies among these ligands, with CD44 being the ligand most-linked to CD34. This differential spatial arrangement of the ligands within the microvilli indicates potential differences in their contributions along the multistep paradigm for cell migration via spatial distribution-driven mechanisms.\u003c/p\u003e\n\u003ch3\u003eE-selectin binding to CD34 promotes Ezrin phosphorylation and microvilli formation\u003c/h3\u003e\n\u003cp\u003eThe effect of CD34 on the clustering dynamics of Ezrin in the microvilli (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003e) prompted us to investigate the role of the CD34 intracellular mechanisms versus E-selectin binding in microvilli. We investigated the dynamics of Ezrin during cell rolling in relation to CD34. In microvilli biogenesis, it is essential that Ezrin is phosphorylated at the regulatory threonine 567 (T567) site (termed p-Ezrin) and recruited to the apical surface of epithelial cells \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e; however, its role in tether and sling formation has not been explored. The relationship between full-length CD34 and p-Ezrin remains poorly understood as it pertains to microvilli and tether and sling formation prior to and upon binding to E-selectin; therefore, we used HEK cells that do not express CD34 and instead, created HEK cells expressing either full-length CD34 (KEK-CD34) or CD34 missing the E-selectin extra cellular binding domain (HEK-∆EC) (\u003cb\u003eFig.\u0026nbsp;4A\u003c/b\u003e); CD34 variants were stably expressed in HEK293ft cells using a lentiviral approach as described in the Methods section. These constructs also enabled us to identify which domain of CD34 was responsible for the formation of microvilli, tethers, and slings. Efficient and stable expression of the different forms of CD34 were assessed through western blotting (\u003cb\u003eSupplementary Fig.\u0026nbsp;3\u003c/b\u003e). Interestingly, analysis of p-Ezrin revealed an increased level of phosphorylation and activation of Ezrin in the HEK-∆EC cells compared to that in HEK and HEK-CD34 cells (\u003cb\u003eFig.\u0026nbsp;4B\u003c/b\u003e). In a control experiment, we showed that Ezrin and B-actin had a consistent expression in HEK, HEK-CD34, and HEK-∆EC cells (\u003cb\u003eFig.\u0026nbsp;4B\u003c/b\u003e). Next, we treated HEK-CD34 cells with fucosyltransferase 6 (FUT6) to decorate CD34 with sLe\u003csup\u003ex\u003c/sup\u003e sugar for proper interaction with E-selectin (\u003cb\u003eSupplementary Fig.\u0026nbsp;4\u003c/b\u003e) \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e and incubated them with rE-selectin for 1 h prior to western blotting. A significant increase was observed in p-Ezrin levels in the FUT6-treated HEK-CD34 cells compared to that in the HEK cell control, without altering the expression level of Ezrin (\u003cb\u003eFig.\u0026nbsp;4C\u003c/b\u003e). These results indicate that binding of E-selectin to the sLe\u003csup\u003ex\u003c/sup\u003e-decorated extracellular domain of CD34 or the expression of the truncated version of CD34 in the HEK-∆EC cells resulted in increased phosphorylation and activation of Ezrin.\u003c/p\u003e\u003cp\u003eSEM analysis of microvilli formation in HEK, HEK-∆EC, and HEK-CD34 cells that were pretreated with FUT6 showed increased numbers of microvilli in HEK-∆EC and HEK-CD34 cells (\u003cb\u003eFig.\u0026nbsp;4D\u003c/b\u003e). Similarly, the number of tethers and slings upon cellular rolling showed that both, HEK-∆EC and HEK-CD34 cells displayed significantly more tethers compared to HEK cells post-rolling on E-selectin \u003cb\u003e(Fig.\u0026nbsp;4E)\u003c/b\u003e. The finding that HEK-∆EC cells produced tethers without the presence of the external region of CD34 suggests the involvement of other E-selectin ligands. However, HEK-∆EC was not as efficient as HEK-CD34 in forming slings, indicating the weak binding to E-selectin at the tethering point.\u003c/p\u003e\u003cp\u003eThese results imply, for the first time, that CD34 can transduce signaling to downstream targets via Ezrin phosphorylation that may also be linked to E-selectin binding to promote the formation of microvilli, tethers, and slings.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCD34 and Ezrin phosphorylation are required for cellular accumulation\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e\u003cp\u003eControl HEK, HEK-∆EC, and HEK-CD34 cells were stained with DiR membrane dye, treated with FUT6, and then injected intravenously into NOD.Cg-Prkdc\u003csup\u003escid\u003c/sup\u003e Il2rg\u003csup\u003etm1Wjl\u003c/sup\u003e/SzJ (NSG) mice as illustrated in \u003cb\u003eFig.\u0026nbsp;5A.\u003c/b\u003e The cells were then detected using an in vivo imaging system (IVIS) imaging system at various time points (1, 8, 24, and 48 h) following injection. Detection at these time points allowed the newly altered glycans on the surfaces of these cells to be maintained as they were shown to have a life of approximately 48\u0026ndash;72 h after FUT6 treatment \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eIn vivo\u003c/em\u003e, whole-body IVIS imaging showed that HEK-CD34 cells expressed a significantly higher signal accumulation compared to HEK-∆EC cells as well as control HEK cells \u003cb\u003e(Fig.\u0026nbsp;5B)\u003c/b\u003e. After observation at the last time point, the mice were euthanized. \u003cem\u003eEx vivo\u003c/em\u003e imaging of organs revealed that NSG mice receiving HEK-CD34 cells showed the highest average radiant efficiency compared to both, HEK-∆EC and control HEK cells. A significantly heightened radiant signal was observed in the spine, bones, and spleen \u003cb\u003e(Fig.\u0026nbsp;5C)\u003c/b\u003e. These results suggest that while phosphorylated Ezrin can aid in the production of tethers and slings \u003cem\u003ein vitro\u003c/em\u003e, proper accumulation and migration are not observed \u003cem\u003ein vivo\u003c/em\u003e unless the cells possess both, the full-length CD34 and phosphorylated Ezrin. This finding may be attributed to the fact that although phosphorylated Ezrin can induce tether formation, it may be difficult for the cells to withstand the sheer stress \u003cem\u003ein vivo\u003c/em\u003e without proper attachment of CD34 with E-selectin to aid the rolling process; this is consistent with the observation that HEK-∆EC was not efficient in forming slings \u003cem\u003ein vitro\u003c/em\u003e (\u003cb\u003eFig.\u0026nbsp;4E\u003c/b\u003e). These findings suggest that the phosphorylation of Ezrin and presence of full-length CD34 are crucial for cellular rolling.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDespite decades of investigations, the role of different selectin ligands in forming microvilli, tethers, and slings, and their spatiotemporal dynamics that facilitate stable rolling and tethering is only beginning to be understood. Recently, the clinical marker of hematopoietic progenitor cells, CD34 \u003csup\u003e24, 54\u003c/sup\u003e, has been identified as an E-selectin ligand \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e; however, its role in tethering and rolling remains poorly understood. Using a variety of microfluidics-based single-molecule super-resolution imaging techniques, we characterized the formation of microvilli, tethers, and slings, and unraveled the clustering and dynamics of CD34 alone and with other E-selectin ligands, under conditions mimicking pre- and post-rolling. CD34 knockdown severely compromised microvilli formation and their ability to extend to tethers and slings for slow rolling. This effect was more pronounced compared to that with the knockdown of the other E-selectin ligands CD43 and PSGL-1 (\u003cb\u003eFig.\u0026nbsp;2C\u003c/b\u003e). In fact, the knock-in of various E-selectin ligands in HEK cells that did not express CD34 showed that CD34 was most effective in increasing the number of microvilli/\u0026micro;m\u003csup\u003e2\u003c/sup\u003e (\u003cb\u003eFig.\u0026nbsp;2D)\u003c/b\u003e. In addition, CD34 knockdown abolished CD44 clustering prior to and post rolling; however, its effect on the clustering of the other E-selectin ligands, CD43 and PSGL-1, was less and differential, indicating stronger link between CD34 and CD44 and a global effect on other selectin ligands in the microvillus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFurthermore, for the first time through super-resolution imaging, we observed an interesting reorganization in CD34 clustering, in tethers and slings, compared to that in microvilli. CD34 exhibited denser clustering at the tethering points of the tethers and slings compared to the extended remains on the microvilli \u003cb\u003e(Fig.\u0026nbsp;1C).\u003c/b\u003e As tethers and slings are believed to originate from singular microvilli \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, CD34 may play a more crucial role than other ligands, in the extension of microvilli into tethers and slings due to its dense clustering around the tethering points. This is further supported by the fact that silencing CD34 mediated faster rolling than the knockdown of PSGL-1 \u003cb\u003e(Fig.\u0026nbsp;2F)\u003c/b\u003e, an E-selectin ligand that has already been shown to cluster more toward the tips of the microvilli \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, tethers, and slings \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. It is possible, however, that a more pronounced role of CD34 at the tip of the microvilli results from the cumulative effect of forming a ring-like structure encompassing CD44 and other ligands, such as, PSGL-1. In fact, the CD34 ring-like structure is not located at the tip of the microvillus (\u003cb\u003eFig.\u0026nbsp;1D\u003c/b\u003e), indicating that it may also act by projecting other ligands within the tip of the microvillus.\u003c/p\u003e\u003cp\u003eThe clustering of ligands, such as, CD34 and CD44, may facilitate the formation of a signaling platform to facilitate the binding of different proteins and their targets. This phenomenon has been studied extensively in lipid rafts, which are microdomains consisting of cholesterol and glycosphingolipids \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Interestingly, CD44 clustering was disrupted by CD34 knockdown in a manner that is similar to that seen in treatment with MβCD, a cholesterol extractor that leads to the disruption of lipid raft domains and spatial clustering of selectin ligands \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Furthermore, MβCD treatment severely compromised microvilli formation and resulted in faster rolling kinetics as observed in the case of CD34 knockdown \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. This suggests that CD34 may function similarly to the lipid rafts by facilitating the localization of other proteins into the lipid rafts.\u003c/p\u003e\u003cp\u003eClustering into a ring-like structure has been shown to play a role in T-cell receptor binding to antigen-presenting cells. Micro-adhesion rings made up of integrin and focal adhesion molecules that surround the T cell receptor micro-clusters are essential for T-cell receptor activation and clustering \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Impairment of these micro-adhesion rings resulted in the disruption of the T cell receptor micro-cluster formation and subsequent hindrance to cellular signaling and cell functions \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Thus, it is plausible that a similar effect can be played by the CD34 ring-like clustering where it can support the functions of other molecules and ligands by mediating better clustering and recruitment of other important players. This is evident by the encompassing of CD44 within the CD34 ring-like structure.\u003c/p\u003e\u003cp\u003eERM proteins are important players in linking the cytoskeletal cortex with the plasma membrane proteins \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e and play a crucial role in microvilli formation and activation \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e, and the motility of cancer cells \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Moreover, podocalyxin, a CD34-related family member, induces microvilli formation via recruitment of an adaptor protein NHERF-1 that links podocalyxin to actin via the ERM proteins \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. These prior findings prompted us to elucidate the roles of different CD34 variants when expressed in HEK293ft cells. Interestingly, we observed a significant increase in Ezrin T567 phosphorylation in HEK-∆EC cells \u003cb\u003e(Fig.\u0026nbsp;4B)\u003c/b\u003e but not in HEK-CD34 cells. This observation suggests a potential feedback inhibition by the CD34 extracellular domain on the functions of its intracellular domain. Interestingly, the binding of full length CD34 with E-selectin mimicked the effects on Ezrin phosphorylation seen in HEK-∆EC. These results imply that the intracellular tail of CD34 can directly or indirectly facilitate phosphorylation of Ezrin only when the extracellular domain of CD34 is occupied/inhibited with a ligand or removed completely. When these cells were treated with FUT6 to decorate CD34 with sLe\u003csup\u003ex\u003c/sup\u003e sugar for proper interaction with E-selectin, followed by injection into mice, HEK-CD34 cells showed remarkably improved accumulation compared to HEK-∆EC and control HEK cells \u003cb\u003e(Fig.\u0026nbsp;5B)\u003c/b\u003e. These results imply that while T567-phosphorylated Ezrin impacts tether production, rolling and cellular migration still require intact CD34. In addition, analysis of cell migration into individual organs showed significantly more HEK-CD34 cells in all the tested organs compared to HEK-∆EC and control HEK cells \u003cb\u003e(Fig.\u0026nbsp;5C)\u003c/b\u003e. This is consistent with previous findings that the CD34 family can aid in cell migration \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. Collectively, while T567 Ezrin plays a role in tether formation, proper rolling and migration still require an intact and functional CD34 protein.\u003c/p\u003e\u003cp\u003eThe knockdown of CD34, CD43, PSGL-1, and Ezrin resulted in reduced number of microvilli (\u003cb\u003eFig.\u0026nbsp;2C\u003c/b\u003e). In contrast to CD34 and PSGL-1, the knockdown of CD43 and Ezrin showed consistent trend of improvement in the tethering and rolling kinetics measured by an increase in the number of rolling cells and decrease in their rolling velocity (\u003cb\u003eFigs.\u0026nbsp;2E and 2F\u003c/b\u003e). However, these differences were not significant in most of the tested sheer stress forces and occasionally became significant with Ezrin reducing the rolling velocity at 5 and 8 dyne.cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e and with CD43 at 5 dyne.cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e (\u003cb\u003eFigs.\u0026nbsp;2E and 2F\u003c/b\u003e). This enhancement despite the reduction in number of microvilli and tethers indicates that fine tuning the number of microvilli/\u0026micro;m\u003csup\u003e2\u003c/sup\u003e may be needed for efficient tethering and rolling. This proposition is strongly supported in the case of the Ezrin knockdown as E-selectin ligands were still present. Contrastingly, CD43 is a mucin with a high density of sialylated O-glycans that are negatively charged \u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e and may provide a mechanism for regulating the density of the microvilli on the cell surface.\u003c/p\u003e\u003cp\u003eIn summary, our findings demonstrate t important role for CD34 in mediating slow rolling by maintaining microvilli and their extension into tethers and slings and coordinating the clustering, and potentially the activity, of other E-selectin ligands during the tethering and rolling steps. When CD34 was overexpressed, cells can roll appropriately and produce tethers that aid in the rolling process. These cells also showed enhanced migration in the tested mice. Our study highlights the novel role of CD34 as a signal transducer in the Ezrin signaling pathway, delineating its role in the clustering of ligands and production and maintenance of microvilli, tethers, and slings.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003cp\u003eStatistical analyses were performed using one-way ANOVA, two-way ANOVA, or T-tests of the relevant data set using GraphPad Prism version 10.2.1 for Windows, GraphPad Software, Boston, Massachusetts USA, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.graphpad.com\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.graphpad.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. For the specific test used, please refer to the respective figure legends.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003cp\u003eKG1a cells (human CD34\u0026thinsp;+\u0026thinsp;leukemic progenitor cell line) and HEK293ft cells purchased from ATCC, were maintained in Roswell Park Memorial Institute (RPMI) 1640 media (10% fetal bovine serum (FBS); Gibco) or Dulbecco's modified Eagle medium (DMEM) media with streptomycin (100 \u0026micro;g/ml) at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e, in a humidified atmosphere.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eProtein knockdown\u003c/strong\u003e\u003cp\u003eCells were collected and transfected with 250 or 500 pmol of either a negative \u0026ldquo;scrambled\u0026rdquo; small interfering RNA (siRNA) (Ambion) or a specific SiRNA of choice (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) using the Lonza 4D nucleofector protocol (Lonza). After transfection, cells were suspended in 2 mL of warm, antibiotic free RPMI 1640 media (10% FBS; Gibco) and incubated for 48\u0026ndash;96 h at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. In some instances, pretreatment with 250 \u0026micro;g/mL (Sigma) of bromelain for 30 min at 37\u0026deg;C, followed by phosphate-buffered saline (PBS) washes, was used prior to transfection for effective knockdowns.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSiRNA and antibodies used for knockdown\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eName of siRNA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCompany and catalog #\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026ldquo;Scramble\u0026rdquo; control siRNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eThermo Fisher Scientific (4390843)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD34 siRNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eThermo Fisher Scientific (s2644)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePSGL-1 siRNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eThermo Fisher Scientific (s12688)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD43 siRNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eThermo Fisher Scientific (s13368)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEzrin siRNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eThermo Fisher Scientific (s14795)\u003c/p\u003e\u003cp\u003eThermo Fisher Scientific (s14797)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAntibody/clone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCompany and catalog #\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD34 antibody QBEND10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBIO-RAD (MCA547G)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD34 antibody EP373Y\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eABCAM (ab81289)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD44 antibody IM7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBioLegend (10302)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD44 antibody 515\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBD Biosciences (550990)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD44 antibody Hermes 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eproduced in-house\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePSGL1 antibody (KPL-1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBioLegend (328802)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD43 antibody (SP55)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eThermo Fisher Scientific (MA5-16339)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEzrin Antibody 3C12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eThermo Fisher (357300)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhospho-Ezrin antibody (Thr567)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eThermo Fisher (PA537763)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-Beta-Actin antibody (AC-15)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAmbion (AM4302)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-rabbit IgG, HRP-linked Antibody\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCell Signaling (7074S)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-mouse IgG, HRP-linked Antibody\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCell Signaling (7076S)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti mouse IgG (H\u0026thinsp;+\u0026thinsp;L), F(ab')2 Fragment (Alexa Fluor\u0026reg; 647 Conjugate)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCell Signaling (4410S)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti rabbit IgG (H\u0026thinsp;+\u0026thinsp;L), F(ab')2 Fragment (Alexa Fluor\u0026reg; 488 Conjugate)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCell Signaling (4412S)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L), F(ab')2 Fragment (Alexa Fluor\u0026reg; 488 Conjugate)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCell Signaling (4408S)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L), F(ab')2 Fragment (Alexa Fluor\u0026reg; 647 Conjugate)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCell Signaling (4414S)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePE Rat Anti-Human Cutaneous Lymphocyte Antigen Clone HECA-452\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBD Biosciences\u003c/p\u003e\u003cp\u003e(563962)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eE-selectin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eproduced in-house\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/p\u003e\u003cp\u003e\u003cstrong\u003eWestern blot analysis\u003c/strong\u003e\u003cp\u003eA cell lysis buffer containing 1% phosphatase inhibitor (HaltTM, Thermo Scientific), 88% NP40 (InvitrogenTM NovexTM, Fisher Scientific), 1% PMSF, and 10% protease inhibitor (PierceTM, Thermo Scientific) was used to lyse the cells for 1 h at 4\u0026deg;C, with constant shaking. The lysate was incubated in a reducing mixture of 10% β-mercaptoethanol in NuPAGE LDS sample buffer (Invitrogen) for 10 min at 70\u0026deg;C. Reduced samples were moved onto a PVDF membrane following an SDS-PAGE gel run. Using 5% non-fat skim milk powder or 5% bovine serum albumin (BSA) in Tris-buffered saline with Tween-20 (TBST, Cell Signaling Technology), the membrane was blocked overnight at 4\u0026deg;C, on a tilting platform. Following TBST washes, the membrane was incubated with a primary antibody of interest (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), then washed thrice with TBST and immunoblotted with HRP-conjugated secondary antibodies and prepared for imaging.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003cp\u003eCells (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL) were washed and placed in a 96-well FACS plate in 50 \u0026micro;l of Hanks' balanced salt solution (HBSS; Gibco). Primary antibodies (10 \u0026micro;g/mL) were added for 25 min at 4\u0026deg;C. Cells were then incubated with a fluorophore-conjugated secondary antibody against the species used for the primary antibody, at a concentration of 5 \u0026micro;g/mL in HBSS, for 20 min at 4\u0026deg;C. Antibody information is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eE-selectin deposition\u003c/strong\u003e\u003cp\u003eFor super-resolution imaging, potassium hydroxide and ethanol were used to clean glass coverslips (No. 1.5, ibidi GmbH) by ultrasonication (P60H, Elma Schmidbauer GmbH). Using adhesives, the coverslip was attached to a sticky-Slide VI 0.4 microfluidic chamber (channel width, 3.8 mm; channel height, 0.4 mm; ibidi GmbH). Following firm attachment, protein A (10 \u0026micro;g/mL, Invitrogen) was incubated in the chamber overnight, at 4\u0026deg;C. After HBSS washing, rE-selectin (0.2 \u0026micro;g/mL) (Sino Biological) was incubated in the microfluidic chamber for 1 h at 4\u0026deg;C. The microfluidic chamber was then washed and blocked using 1% casein in PBS (Thermo) at room temperature (22\u0026deg;C) for 0.5-1 h. The chamber was then promptly used for super-resolution imaging.\u003c/p\u003e\u003c/p\u003e\u003cp\u003eFor live cell fluorescence rolling imaging, a \u0026micro;-Slide VI 0.1 microfluidic chamber (channel width, 1 mm; channel height, 0.1 mm; ibidi GmbH) was incubated with rhE-selectin (1 \u0026micro;g/mL) (Sino Biological) overnight, at 4\u0026deg;C. Next, the microfluidic chamber was washed and blocked using 1% BSA in HBSS for 40 min at 4\u0026deg;C. The chamber was then promptly used for live cell rolling.\u003c/p\u003e\u003cp\u003e\u003cb\u003eScanning Electron Microscopy (SEM)\u003c/b\u003e: Cells (5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) were collected, washed twice with PBS, and then incubated in a buffer with a 1:10 ratio of 25% glutaraldehyde to 0.1 M cacodylate, for 12\u0026ndash;24 h at room temperature. After incubation, cells were washed thrice with 0.1 M cacodylate buffer. Cells were resuspended in 200 \u0026micro;L of 0.1 M cacodylate buffer and deposited into a prepared coverslip lined with Poly-L-Lysine and left overnight at room temperature. Osmium tetroxide (1%) was diluted in 0.1 M cacodylate buffer and the slides were incubated in it for 1 h, covered with foil. The slides were washed thrice with distilled water for 10 min each, and then covered again in foil. This process was repeated with 30%, 50%, 70%, 90%, and 100% of ethanol diluted in distilled water. The slides were transferred to a critical point apparatus and dried. Coverslips were then mounted on a metal stub with a double-sided carbon metal adhesive and coated with platinum. Images were captured by SEM.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCell rolling and analysis of the cell-rolling behavior\u003c/strong\u003e\u003cp\u003eCell rolling was performed at room temperature using either a sticky-Slide VI 0.4 microfluidics chamber that was already adhered to the rhE-selectin (0.2 \u0026micro;g/mL)-deposited coverslip (for super-resolution imaging) or a \u0026micro;-Slide VI 0.1 microfluidics chamber incubated with rhE-selectin (1 \u0026micro;g/mL) (for live cell rolling). Male Luer connectors (ibidi GmbH) were used to connect the inlet and outlet of the chamber to a 0.8-mm silicon tubing (ibidi GmbH). The inlet silicon tubing was placed into HBSS rolling buffer with 1% BSA (Sigma) and 1 mM CaCl\u003csub\u003e2\u003c/sub\u003e (Sigma). The outlet silicon tubing was attached to a programmable syringe pump (PHD ULTRA, Harvard Apparatus) by adding a female Luer Lock connector (ibidi GmbH). The rolling buffer was allowed to flow into the chamber for 90 s before KG1a cell rolling to equilibrate the flow path. KG1a cells (10\u003csup\u003e6\u003c/sup\u003e) were then suspended in the rolling buffer and perfused into the chamber at a sheer stress of 2 dyne.cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e for 20 s, before stopping the flow for 30 s, to allow the cells to interact with the rE-selectin-incubated chamber. Flow was then resumed using the rolling buffer at a sheer stress of 2 dyne.cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e to roll the cells for the required amount of time. Cell-rolling was visualized and performed by mounting the microfluidic chamber on a LCAch N 20X objective (Olympus)-equipped CXK41 inverted optical microscope (Olympus). An XC10 CCD camera (Olympus) and a CellSens software (Olympus) were used to record transmitted light images at video rate. An Olympus IX71 inverted optical microscope equipped with a UAPON 100XOTIRF high numerical aperture (NA) objective (Olympus) and an iXon3 897 EMCCD camera (Andor Technology) was also used to capture transmitted optical microscopy images.\u003c/p\u003e\u003c/p\u003e\u003cp\u003eCell rolling videos were analyzed using TrackMate Fiji, an ImageJ plugin. Using the Linear Assignment Problem (LAP) tracker, we localized and tracked each cell as it rolled. Mean rolling velocity was calculated by dividing the total displacement of the rolled cell by the duration of rolling per cell. To consider only cells that rolled properly, any cell that rolled for \u0026lt;\u0026thinsp;2.5 s or for \u0026lt;\u0026thinsp;30 \u0026micro;M was excluded from calculations.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFluorescence labeling of cells\u003c/strong\u003e\u003cp\u003eFor live, two-color cell rolling, KG1a cells were washed with HBSS and incubated in 1.5 mL of FC blocker (Accurate Chemical \u0026amp; Scientific) for 30 min at 4\u0026deg;C. Cells were then incubated in 5 \u0026micro;g/mL of AF647-conjugated CD44 antibody (515-BD Bioscience) diluted in 2% BSA (Sigma) in HBSS for 45 min at 4\u0026deg;C. Cells were then incubated in 5 \u0026micro;g/mL of AF488-conjugated CD34 antibody (Qbend10-Biolegend) diluted with 2% BSA in HBSS, for 45 min at 4\u0026deg;C. Cells were resuspended in perfusion buffer (1X DMEM flour bright (Gibco) and 1 mM of CaCl\u003csub\u003e2\u003c/sub\u003e) and rolled at a constant rate of 2 dyne.cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e.\u003c/p\u003e\u003c/p\u003e\u003cp\u003eFor two-color super-resolution of cells in suspension, a fixing solution composed of 0.2% (w/v) glutaraldehyde (Electron Microscopy Sciences) and 3% (w/v) paraformaldehyde (Electron Microscopy Sciences) in HBSS was used to fix the cells for 20 min at room temperature. Goat serum (10%) (Sigma) was then used to block the cells at 37\u0026deg;C for 40 min. Next, the fixed/blocked cells were incubated with 15 \u0026micro;g/mL of a primary antibody against one of the proteins of interest that was diluted in 2% BSA (Sigma) in HBSS at room temperature for 30 min; this was followed by treatment with 5 \u0026micro;g/mL of AF488 conjugated goat secondary antibody (Invitrogen) diluted in 2% BSA in HBSS at room temperature for 25 min. Cells were then fixed using 0.2% (w/v) glutaraldehyde and 3% (w/v) paraformaldehyde in HBSS at room temperature for 10 min. Next, the cells were incubated with another primary antibody corresponding to another protein of interest diluted in 2% BSA in HBSS at room temperature for 30 min, followed by 5 \u0026micro;g/mL of AF647 conjugated secondary antibody (Invitrogen) diluted in 2% BSA in HBSS at room temperature for 30 min. The cells were again fixed using 3% (w/v) paraformaldehyde and 0.2% (w/v) glutaraldehyde in HBSS at room temperature for 10 min. The cells were placed in a sticky-Slide VI 0.4 microfluidic chamber that already adhered to a clean coverslip and coated with Silane (Sigma A3648) overnight, at 4\u0026deg;C. The cells were incubated in the chamber for 30 min at room temperature.\u003c/p\u003e\u003cp\u003eFor rhE-selectin rolled fluorescence labeling of KG1a cells, cells were perfused into a sticky-Slide VI 0.4 microfluidic chamber that was previously coated with E-selectin, as described earlier, at a constant shear stress of 2 dyne.cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e. After the cells rolled for 90 s, a fixing solution made up of 0.2% (w/v) glutaraldehyde and 3% (w/v) paraformaldehyde in HBSS was auto-perfused at 2 dyne.cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e for approximately 2\u0026ndash;3 min to fix the cells to the chamber while still rolling. Cells were incubated in the fixing solution for another 17 min to ensure proper and complete fixing of the cells. Fluorescence labeling of cell proteins in the chamber was performed as outlined in the previous step. Fixed and immunolabeled Control KG1a cells were suspended in switching buffer before perfusing them into a clean silane-coated microfluidic chamber.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eSuper-resolution \u0026ldquo;STORM\u0026rdquo; microscopy and analysis of super-resolution (SR) images\u003c/strong\u003e\u003cp\u003eTo perform two-color super-resolution imaging on immunolabeled KG1a cells, a switching buffer composed of catalase (40 \u0026micro;g/mL; Sigma), 10 mM 2-aminoethanethiol (MEA; Fluka), TN buffer [50 mM tris (pH 8.0) and 10 mM NaCl], oxygen scavenging system [glucose oxidase (0.5 mg/mL; Sigma), and 10% (w/v) glucose] was added to the cells just prior to imaging. MEA solution was prepared in 1 M aqueous KOH, with pH adjusted to 8. The switching buffer was prepared just prior to imaging. A custom-built, widefield illumination fluorescence microscope on an inverted IX71 optical microscope platform (Olympus) was used for the super-resolution imaging \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. Beam lines with wavelengths 640 nm and 488 nm (60 mW; MLD, Cobolt) were introduced coaxially into the inverted IX71 optical microscope. Using the HILO configuration, the samples were illuminated through a 100\u0026times; objective (NA\u0026thinsp;=\u0026thinsp;1.49; UAPON 100XOTIRF, Olympus) with illumination powers of 3.8 mW cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e or 5.9 mW cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e for the 640 nm or 488 nm beams, respectively. Sample fluorescence signals were captured using the same objective lens which was separated from the illumination light by a multiband dichroic mirror (Di03-405/488/561/635-t1-25x36, Semrock), and passed through a TuCam dual-camera adaptor (Andor Technology) equipped with a filter cassette containing a dichroic image splitter (FF580-FDi01-25 \u0026times; 36, Semrock) to separate the fluorescence into two channels. Two EMCCD cameras (iXon3 897, Andor Technology) detected the split fluorescence from the samples through emission bandpass filters (FF01-550/88\u0026thinsp;\u0026minus;\u0026thinsp;25 and FF01-697/58\u0026thinsp;\u0026minus;\u0026thinsp;25, Semrock). To reconstruct SR localization microscopy images, 5,000 frames of the fluorescence image sequences were recorded using the Andor iQ3 software \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/p\u003e\u003cp\u003eReconstruction of the super-resolution images was done using the Localizer software \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. AF647 and AF488 molecule positions were determined by 2D Gaussian fitting of the point spread function (PSF) images. For two-color super-resolution analysis, calibrating the shift between the two channels was conducted by the use of TetraSpeck microspheres (100 nm in diameter) that were deposited on a cleaned coverslip. Once images of the TetraSpeck beads were taken using the two laser beams, we produced a registration map that allowed us to correct the shift between the two detected images and applied these corrections to the obtained localized images.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eTIRFM and 3D-SR image acquisition\u003c/strong\u003e\u003cp\u003eTo achieve a nanoscale spatial correlation between the cell surface morphology captured by TIRFM imaging and the 3D-SR coordinates of adhesion molecules characterized by the astigmatism-based 3D-single molecule localization microscopy (SMLM) imaging\u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e, we collected TIRFM and 3D-SMLM data sequentially from the same KG1a cell under two different illumination conditions (i.e., TIR and HILO for TIRFM and 3D-SMLM imaging, respectively). Each image was recorded through an individual detection pathway using two EMCCD cameras (iXon3 897, Andor Technology). The image was split into different wavelength ranges using a dichroic mirror. This configuration does not require mechanical switching of the detection pathway for each measurement, allowing each image to be compatible with nano-scale correlation analysis. The microfluidic-based 3D-TIRFM imaging was utilized to map the cell surface morphology. We recorded TIRF images of membrane-stained KG1a cells (MemBrite-FX640) at a single angle of incidence 66.6\u003csup\u003e\u0026deg;\u003c/sup\u003e (θ), corresponding to a 120 nm evanescent field penetration depth, under weak illumination of 640-nm laser power (equivalent to 30 W cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e at Epi configuration). Per a sample field of view, 10 frames were captured to average the fluorescence intensity before processing the TIRF image for topographical map construction. The pixel with maximum intensity was assigned as the closest one to the glass surface.\u003c/p\u003e\u003c/p\u003e\u003cp\u003eFluorescence signals were separated from the illumination light by a multiband dichroic mirror (Di03-405/488/561/635-t1-25x36, Semrock). Then the fluorescence signal was separated into two images using a dichroic image splitter (Semrock, FF580-FDi01-25 \u0026times; 36) by different wavelengths, with each assigned to a different measurement mode. An emission bandpass filter was used for each separated image of TIRFM imaging (FF01-697/58\u0026thinsp;\u0026minus;\u0026thinsp;25) and 3D-SMLM imaging (FF01-550/88\u0026thinsp;\u0026minus;\u0026thinsp;25). A cylindrical lens with a focal length of 1000mm was used to create the astigmatism-based PSF for 3D-SMLM imaging. Details of the method will be published elsewhere.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eTIRFM image processing\u003c/strong\u003e\u003cp\u003eUsing a custom-written MATLAB script, the initial membrane topography analysis was performed from TIRFM images, and included filtering, visualization, and data extraction to generate a topographical 3D map of a microvilli. Laplacian of Gaussian (LoG) filter (Gaussian σ\u0026thinsp;=\u0026thinsp;0.5; kernel size\u0026thinsp;=\u0026thinsp;10 \u0026times; 10) and additional Gaussian smoothing was applied to enhance and resolve microvilli features and exclude background noise from the cell body, as it lay outside the edge of detection. To find contours representing microvilli boundaries, thresholding (min-intensity\u0026thinsp;=\u0026thinsp;200) was applied to obtain a binary mask, and then edge detection was applied. The mask was also used to remove irrelevant localization outside the microvilli boundaries. After that, the intensity values of the image were normalized, and fluorescence intensity was converted to a height profile using the TIRF theory, generating corresponding contour maps of the individual microvilli. The topography map of individual microvilli was segmented into several contour levels by dividing the maximum distance by interval distance (10 nm) \u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eReconstruction of 3D topographical map and single molecule coordinates\u003c/strong\u003e\u003cp\u003eA custom-written Python script was developed for 3D spatial overlaying of the cell surface topography map and obtaining 3D coordinates of the adhesion molecules. TIRFM data were analyzed and compared with single-molecule data to identify the structural similarity index (SSI) between microvilli and single-molecule data. After extracting the topographical map and single-molecule data within a 3D-bounding box of a region (microvilli), 3D surface fitting was applied to the single molecules. Next, the 3D topography surface of TIRF data and the fitted surface within a bounding box to an 8-bit grayscale image were normalized to calculate the SSI between the 2D projections. Microvilli and their corresponding single-molecule coordinates that exceeded the SSI threshold (x\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026le;\u003c/span\u003e\u0026thinsp;0.8) were selected for downstream analysis.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eDensity-based cluster analysis\u003c/strong\u003e\u003cp\u003eThe SM localization maps of different proteins were analyzed using a custom-written Python code based on the algorithm density-based spatial clustering of applications with noise (DBSCAN). We determined minpts\u0026thinsp;=\u0026thinsp;7 and epsilon\u0026thinsp;=\u0026thinsp;45 nm \u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e. In this way, the code defined and separated SM into groups and clusters in 3D.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCD34 lentivirus variant design\u003c/b\u003e: CD34 lentivirus variants were designed and purchased from GenScript. Three variants were designed: 1) Empty vector lentivirus particles encoding puromycin resistance, 2) \u0026lsquo;HEK-CD34\u0026rsquo; lentivirus particles encoding the full human CD34 amino acid sequence including the signal peptide and a puromycin resistance. Amino acid sequence of the insert was as follows: MLVRRGARAGPRMPRGWTALCLLSLLPSGFMSLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITETTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKTLIALVTSGALLAVLGITGYFLMNRRSWSPTGERLGEDPYYTENGGGQGYSSGPGTSPEAQGKASVNRGAQENGTGQATSRNGHSARQHVVADTEL*. \u0026lsquo;HEK-∆EC\u0026rsquo; lentivirus particles encoding a CD34 variant with a missing extracellular domain excluding the first four amino acids (SQKT). Puromycin resistance was also included. Amino acid sequence of the insert was as follows: MLVRRGARAGPRMPRGWTALCLLSLLPSGFMSQKTLIALVTSGALLAVLGITGYFLMNRRSWSPTGERLGEDPYYTENGGGQGYSSGPGTSPEAQGKASVNRGAQENGTGQATSRNGHSARQHVVADTEL*.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eLentivirus transduction into HEK293ft cells\u003c/strong\u003e\u003cp\u003eHEK293ft cells (6 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) were collected and plated in a 6-well plate overnight, in 2 mL of supplemented DMEM media (10% FBS and 100 \u0026micro;g/mL of streptomycin) at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Media was removed and new supplemented DMEM media with 10 multiplicity of infection (MOI) of Lentivirus particles and 2 \u0026micro;g/mL of polybrene was added to the cells and incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e, for 48 h. Media was removed again and new supplemented media containing 3 \u0026micro;g/mL of puromycin was added until all un-transduced cells were killed.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFucosyltransferase 6 (FUT6) Treatment\u003c/strong\u003e\u003cp\u003eHEK293ft cells (4 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) were collected and incubated for FUT6 treatment (0.5 mM of GDP-fucose (Sigma), 25 mM Hepes (pH 7.5) (Gibco Invitrogen), 5 mM MnCl\u003csub\u003e2\u003c/sub\u003e, 0.1% human serum albumin (HAS), and 1 \u0026micro;g purified rhFTVI enzyme in HBSS) for 30 min, at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were washed twice with HBSS containing 10 mM of EDTA, and once with un-supplemented HBSS.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eE-selectin Treatment\u003c/strong\u003e\u003cp\u003eHEK293ft cells (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) were collected and incubated in 1 mL of supplemented DMEM media, 5 \u0026micro;L of Dimer E-selectin (0.23 mg/mL) for 1 h at 37\u0026deg;C, with 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were then washed twice with PBS.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCD34 variant in vivo analysis\u003c/b\u003e: NOD.Cg-Prkdc\u003csup\u003escid\u003c/sup\u003e Il2rg\u003csup\u003etm1Wjl\u003c/sup\u003e/SzJ (NSG) Mice were procured from Charles River Laboratories (Lodi, Italy) and housed in the Animal Research Core Lab at the King Abdullah University of Science and Technology. The NSG mice were randomly allocated into four experimental groups: (i) an untreated control group injected with 100 \u0026micro;L of PBS, intravenously (IV) via the tail vein (n\u0026thinsp;=\u0026thinsp;3), (ii) a group designated as 'F' that received 100 \u0026micro;L of 3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e DIR pre-stained HEK-CD34 cells in PBS (n\u0026thinsp;=\u0026thinsp;4) IV, (iii) an 'EC' group that was administered 100 \u0026micro;L of 3 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e DIR pre-stained HEK-∆EC cells in PBS (n\u0026thinsp;=\u0026thinsp;4) IV, and (iv) a 'Negative' group that received 100 \u0026micro;L of 3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e DIR pre-stained HEK cells in PBS (n\u0026thinsp;=\u0026thinsp;4) IV.\u003c/p\u003e\u003cp\u003eThe biodistribution of HEK cells in the mice was longitudinally imaged at 1, 8, 24, and 48 h using the IVIS Spectrum system (PerkinElmer Inc., MA, USA), after which the mice were euthanized, and various organs including the spleen, liver, spine, heart, lung, femur, and tibia were excised and imaged using the IVIS imaging system. Images were captured by a charge-coupled device (CCD) camera with the following settings: binning set to medium and f/stop at 2. The filter sets were calibrated at 710 nm for excitation and 800 nm for emission wavelengths. Fluorescence intensity was quantitatively assessed with the Living Image software suite (Caliper Life Sciences, MA, USA). The accumulation of DIR-labeled HEK cells in the whole body, as well as in specific organs, was quantified by averaging out radiant efficiency ([p/s/cm\u0026sup2;/sr] / [\u0026micro;W/cm\u0026sup2;]).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eData availability\u003c/h3\u003e\n\u003cp\u003eData supporting the findings of this work are available within the paper and supplementary material.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eacknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Umme Habiba for her efforts in reagent preparation and her role in the thawing and freezing of HEK293ft and KG1a cells. In addition, the authors would also like to thank the Imaging and Characterization core facility at King Abdullah University of Science and Technology (KAUST) and especially Maya Ayach for her efforts in providing some of the SEM data. Finally, the authors would like to thank Professor Samir M Hamdan for his helpful discussion. The research reported in this publication was supported by the King Abdullah University of Science and Technology (KAUST).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eauthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.M.A. designed, performed, and analyzed experiments and wrote the manuscript; A.A. designed, performed, and analyzed super-resolution experiments and wrote the manuscript;\u0026nbsp;S.N.\u0026nbsp;managed the SR imaging setup; A.I.A., K.A.,\u0026nbsp;performed super-resolution experiments; Y.L. performed and analyzed \u003cem\u003ein vivo\u003c/em\u003e experiments in mice; A.T. performed and analyzed part of the SEM experiments; A.S.A. provided and aided in the use of the FUT6 enzyme during HEK293ft functionality experiments; I.I. aided in final figure production as well as in the use of E-selectin, during E-selectin treatments; S.H. conceived and provided all of the SR imaging platforms as well as reviewed the manuscript; and J.S.M. conceived, designed, and analyzed experiments and wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e: The authors declare no competing financial interests.\u003c/p\u003e\n\u003cp\u003eCorrespondence: [email protected] or [email protected].\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLauffenburger, D.A., Horwitz, A.F.: Cell Migration: A Physically Integrated Molecular Process. 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Rep. \u003cb\u003e12\u003c/b\u003e, 22561 (2022)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7280176/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7280176/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHematopoietic stem/progenitor cells (HSPCs) exit the blood stream and migrate to the bone marrow by extending their microvilli into tethers, in response to shear-resistant binding between ligands on HSPCs and E- and/or P-selectin, on the endothelial cell layer. Several selectin ligands have been identified; however, their mechanisms in microvilli formation and extension into tethers remain elusive. Using several super-resolution imaging techniques and functional assays, we showed that CD34 orchestrates these mechanisms. CD34 is indispensable for microvilli formation, enhancing tether formation by increasing its clustering and adopting a unique ring-like localization pattern at the microvillus tip, which can encapsulate other ligands, forming a dense tethering site for E-selectin. CD34 transduces E-selectin binding by phosphorylating the ERM protein ezrin, enhancing microvilli and tether formation \u003cem\u003ein vitro\u003c/em\u003e and cell migration \u003cem\u003ein vivo\u003c/em\u003e. Thus, CD34 plays a key role in the mechanical and signaling mechanisms of the microvilli, during cellular tethering and rolling.\u003c/p\u003e","manuscriptTitle":"CD34 orchestrates the formation and actions of microvilli for efficient E-selectin-mediated cell migration","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-12 14:26:03","doi":"10.21203/rs.3.rs-7280176/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f0e19ad0-91cc-4513-b302-2e6d65e949c2","owner":[],"postedDate":"September 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":54204377,"name":"Biological sciences/Cell biology/Cell migration/RHO signalling"},{"id":54204378,"name":"Biological sciences/Cell biology/Cell adhesion"},{"id":54204379,"name":"Biological sciences/Biochemistry/Glycobiology"},{"id":54204380,"name":"Biological sciences/Cell biology/Cellular imaging/Super-resolution microscopy"},{"id":54204381,"name":"Biological sciences/Cancer/Haematological cancer/Leukaemia/Acute myeloid leukaemia"}],"tags":[],"updatedAt":"2026-05-13T15:24:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-12 14:26:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7280176","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7280176","identity":"rs-7280176","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}