Caveolin-1 Modulates Notch Transcriptional Activity During Respiratory Multiciliated Cell Maturation

Caveolin-1 Modulates Notch Transcriptional Activity During Respiratory Multiciliated Cell Maturation | 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 Caveolin-1 Modulates Notch Transcriptional Activity During Respiratory Multiciliated Cell Maturation Jose Maria Carvajal-Gonzalez, Marcos Olivera-Gómez, Guadalupe Cumplido-Laso, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6189425/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Feb, 2026 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract The airway epithelium, a sophisticated multicellular layer lining the respiratory tract, serves as our first line of defense against pathogens, allergens, and environmental pollutants. Recent studies have illuminated the intricate interplay between basal cell self-renewal, differentiation, and epithelial homeostasis following injury. Notably, expression of caveolin-1 (Cav-1) has been linked to specific cell types within the airway epithelium, primarily basal stem cells (BSCs) and multiciliated cells (MCCs). Despite its specific expression, the precise function of Cav-1 in BSC differentiation remains largely enigmatic. In this study, we investigate Cav-1’s function within the airway epithelium, exploring the molecular mechanisms underlying BSC differentiation into MCCs and secretory cells (SCs). Our results reveal limited Cav-1 expression in mouse airway epithelial BSCs, with additional enrichment observed in MCCs. Notably, deficiency of Cav-1 accelerates MCC differentiation and maturation. Additionally, we found that Cav-1 downregulation dramatically affects Notch intracellular domain (NICD) transcriptional activity. That leads us to propose that Cav-1 participates indirectly in a transcriptional program orchestrated by NICD, thereby modulating both BSC differentiation and MCC maturation. Biological sciences/Stem cells Biological sciences/Stem cells/Adult stem cells Biological sciences/Stem cells/Stem cell differentiation airway epithelium caveolin 1 Notch signaling multiciliated cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The respiratory tract, lined by a single layer of epithelial cells, serves as a critical barrier separating our internal milieu from the external environment 1 . To ensure proper respiratory function and the ability to regenerate in response to environmental challenges, understanding the processes governing differentiation and self-renewal in adult stem cells within the epithelium is paramount 2 , 3 . Moreover, unraveling the intricate mechanisms driving epithelial development is essential for creating comprehensive and innovative experimental model systems, spanning both two-dimensional and three-dimensional culture settings 4 , 5 . At homeostasis, the airway epithelium comprises a diverse array of specialized cell types 6 . Among these, three major constituents dominate. Basal Stem Cells (BSCs), these multipotent stem cells play a pivotal role in tissue regeneration, demonstrating the remarkable ability to fully restore the epithelium. Secretory Cells (SCs), within this category, club cells (CCs) and goblet cells (GCs) stand out 7 – 10 . SCs are responsible for producing mucus—a crucial defense mechanism that traps pathogens and pollutants 11 . And Multiciliated Cells (MCCs), these cells continuously propel mucus toward the mouth, ensuring airway clearance and maintaining respiratory health 12 . But the airway epithelium’s complexity extends further. Less abundant populations—such as tuft cells (immune sentinels), pulmonary neuroendocrine cells (environmental sensors), and pulmonary ionocytes (recently unveiled through single-cell RNA-sequencing)—contribute to its multifaceted function 13 , 14 . Maintaining the delicate balance among these cell types is essential for the proper functioning of the pseudostratified epithelium. Dysregulation, characterized by imbalances in BSC abundance or differentiation, underpins various airway diseases—asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF) 15 , 16 . Our quest to understand the molecular pathways governing these processes remains an active area of research, with profound implications for regenerative medicine and therapeutic intervention. Differential gene expression analysis has served a dual purpose: classifying distinct cell types and unraveling the biology inherent to each cell population 13 , 14 , 17 – 19 . The caveolin family, comprising three members—caveolin-1, caveolin-2, and caveolin-3—plays a pivotal role in cellular dynamics 20 . Among them, caveolin-1 (Cav-1) emerges as a major structural protein of caveolae, specialized plasma membrane invaginations involved in diverse cell activities, including molecular transport, cell adhesion, and signal transduction 21 – 25 . In the context of airway epithelium, Cav-1 garners attention. It is membrane-associated and localized within BSCs and MCCs of the large airways in both rats and humans 26 . Recent investigations underscore its differential enrichment in basal stem cells (BC-2) within the mouse trachea, linking it to WNT, TGFβ, and Hedgehog signaling pathways 27 . Despite this intriguing expression pattern, a definitive role for Cav-1 in airway epithelium remains elusive. Reduced Cav-1 expression disrupts E-cadherin-mediated cell-cell adhesion in asthma patients’ airways, leading to deregulation of the respiratory epithelial barrier 28 . Furthermore, Cav-1 expression in lung tissues acts as a safeguard against fibrogenic effects induced by TGF-β1, preventing conditions like pulmonary fibrosis and allergen-induced airway remodeling 20 . Notably, Cav-1 deficiency exacerbates pulmonary fibrosis, emphasizing its protective function 20 . Beyond structural integrity, Cav-1’s impact extends further. Its deletion correlates with increased pro-inflammatory cytokines (IL-6 and TNF-α) and altered production of MUC-5AC in lung epithelial cells 24 , 29 . In this study, our primary aim is to analyze Cav-1’s function in airway epithelium and identify molecular mechanisms involved in the differentiation of BSCs to MCCs and SCs. By unraveling the intricate interplay between Cav-1, Notch signaling, and MCC maturation, we provide new insights into cellular fate determination within the airway epithelium. Results Caveolin-1 expression is heterogeneously express in the airway epithelium To investigate the localization of Cav-1 in the airway tract, we conducted immunofluorescence on whole-mount trachea using Cav-1 antibodies and acetylated tubulin staining, which labels the apical surface of the multiciliated cells of the luminal airway epithelium. Confocal imaging and subsequent analysis revealed heterogeneous Cav-1 expression across different epithelial strata. At the basal planes where BSCs reside, Cav-1 expression varied, with some cells exhibiting high levels at the plasma membrane and others having lower levels (Figure 1, panel a). In the pseudostratified epithelium at medial planes, this heterogeneity was more pronounced (Figure 1, panel a), accompanied by enlarged puncta at the membrane. Conversely, at the luminal side where differentiated cells are located, Cav-1 expression was restricted to a subset of cells with very low levels compared to basal planes (Figure 1, panel a). This localization pattern correlated with single-cell RNA expression data from differentiated cells in ALI culture. Our analysis of published data indicated that Cav-1 was predominantly expressed in basal cells, cycling basal cells, and a subset of basal cells predisposed to differentiation (Krt4/13) (Figure 1, panel b). Further examination of MCCs revealed Cav-1 expression at both the apical and basolateral plasma membranes (Figure 1, panel c). In comparison, Flotillin 1, a caveolae component, exhibited more uniform expression across different strata and cell types in the airway epithelium (Figure 1, panel b). Additionally, Cav-2 showed higher expression in other cell types like Brush cells (Figure 1, panel b). To assess the specificity of the expression pattern in Krt4/13 cells, we examined genes that exhibited significantly higher expression in Krt4/13 compared to all other cell types (Supplementary Figure 1, panel a). We identified 70 genes that were significantly enriched, showing more than double the expression in Krt4/13 cells compared to other cell types. This distinct expression pattern, characterized by high expression in basal cells and even higher levels in Krt4/13 cells, motivates further investigation into the potential role of Cav-1 in airway epithelium biology. To further characterize the localization and function of Cav-1 in BSCs, we investigated its localization pattern in air-liquid interface (ALI) cultures in vitro during the differentiation process. Our findings revealed that at ALI 2 (day 2 of differentiation), Cav-1 levels and localization were homogeneous in the epithelial monolayer (Supplementary Figure 1, panel b). However, in ALI 4, ALI 6, and ALI 14, as pseudostratification and differentiation occurred, Cav-1 expression became heterogeneous among luminal cells, with higher levels observed in basal cells—consistent with in vivo observations (Figure 1, panel d and Supplementary Figure 1, panel b). Furthermore, global RNA expression levels revealed a decline in Cav-1 expression along the differentiation protocol (Figure 1, panel f), which coincided with an increased expression of Foxj1, a marker for MCCs (Supplementary Figure 1, panel c). This reduction in Cav-1 expression at the epithelial level was also evident in ALI 14 at the protein level, as measured by Western blot (Figure 1, panel e and Supplementary Figure 1, panel d). Based on these findings, we infer that caveolin-1 is predominantly expressed in BSCs, and its expression is significantly reduced in ALI 14 (Figure 1, panel e and Figure 1, panel f), likely due to a decrease in the proportion of BSCs. Deficiency of caveolin-1 increase MCCs differentiation To investigate the role of Cav-1 in the differentiation process from BSCs to luminal differentiated cells, we conducted knockdown experiments targeting Cav1 (Cav1-KD) in BSCs (Figure 2, panel a). Using three distinct shRNA sequences against Cav1, we observed a significant reduction in expression compared to control conditions (Luc-KD, luciferase knockdown) (Figure 2, panel a). Still, no significant changes in the relative expression of Cav-2 and Cav-3 were detected in Cav1-KD (Supplementary Figure 2, panel a). This Cav1-KD was also evident at the protein level (Figure 2, panel b and Supplementary Figure 2, panel b). Subsequently, we assessed various cellular processes in control and Cav1-KD cells, including proliferation, self-renewal, cell attachment, migration, and cell cycle. Notably, Cav1-KD did not impact BSC proliferation, as indicated by doubling time (Figure 2, panel c), airway organoid size (Figure 2, panel d and Supplementary Figure 2, panels c and d), attachment (Supplementary Figure 2, panel e), or migration (Supplementary Figure 2, panel f). Furthermore, the distribution of cells across different cell cycle phases remained similar between control and Cav1-KD BSCs (Figure 2, panel e). Next, we assessed the impact of Cav1 knockdown (Cav1-KD) on the airway epithelium developed in vitro from BSCs (Supplementary Figure 2, panel g). Remarkably, the Cav1-KD epithelium formed a compacted epithelial monolayer with normal barrier function, comparable to control conditions (Luc-KD). Transepithelial resistance measurements confirmed this similarity between control and Cav1-KD epithelia (Figure 2, panel f). Furthermore, we investigated the cellular composition of the epithelium under both control and Cav1-KD conditions by assessing the expression of cell type markers for BSCs, MCCs, and SCs (club and goblet cells). Notably, there were no significant differences in BSC markers ( Trp63 and Krt5 ) or goblet cell markers ( Spdef and Muc5A/c ) (Figure 2, panel g and j). However, we observed significant differences in MCCs ( Foxj1 ) and secretory cells ( Scgb1a1 ) markers (Figure 2, panels h and i). Interestingly, other markers such as Mcidas or Scgb3a2 did not exhibit significant differences (Figure 2, panels h and i). Additionally, p63 and Scgb1a1 staining was comparable between Cav1-KD and control conditions (Figure 2, panel k and Supplementary Figure 2, panel h), while Foxj1 showed an increase in positive cells in Cav1-KD epithelia (Figure 2, panel l and Supplementary Figure 2, panel i). Additionally, in a parallel experiment where Cav-1 was overexpressed, we did not observe any significant changes in the tested genes compared to the control group (Supplementary Figure 3). Overall, our findings suggest that the absence of Cav-1 does not significantly alter BSC behavior compared to control cells. The Cav1-KD cells successfully formed a functional airway epithelial monolayer, characterized by an increased presence of MCCs. Lack of Caveolin-1 triggers early transcriptional programs related to MCCs To further elucidate the role of Cav-1 in airway epithelial composition, we utilized BSCs isolated from both wild-type (WT) and Caveolin-1 knock-out (Cav1-KO) mice 30 . These BSCs were seeded and differentiated in air-liquid interface (ALI) cultures, and we analyzed cell type markers. Notably, expression levels of BSC and secretory cell (SC) markers were comparable between Cav1-KO and WT epithelia, except for Muc5ac (Figure 3 panels a-c and Supplementary Figure 4, panel a). However, markers associated with MCCs, such as Foxj1 and Mcidas , were significantly upregulated in Cav1-KO epithelium (Figure 3, panel d). Immunofluorescence staining for Foxj1 further confirmed the increased abundance of MCCs in Cav1-KO conditions compared to WT (Figure 3, panel e and Supplementary Figure 4, panel b). We have previously found that early transcriptional activity during differentiation affect BSC differentiation 17,18 . Hence, to gain deeper insight into the molecular mechanisms linking Cav-1 and MCC differentiation, we investigated the epithelial status during early differentiation stages (ALI 6) using RNAseq. Initially, we examined known cell type markers for each described cell type. Remarkably, 8 out of 8 MCC markers were upregulated in Cav1-KO epithelium, except for Mcidas (Figure 3, panel f). In contrast, other cell type categories did not exhibit clear upregulation or downregulation in Cav1-KO epithelia compared to wild-type (WT) (Figure 3, panel f). A broader analysis of expression patterns revealed that a subset of 80 genes was downregulated, while 477 genes were upregulated in Cav1-KO epithelium at ALI 6 (Figure 3, panel g). Additionally, gene ontology (GO) analyses highlighted at least 6 cilia-related GO terms significantly enriched in Cav1-KO epithelium (Figure 3, panel h). These sets encompassed functional and structural aspects of cilia, including cell projection and motile cilium. Furthermore, when we analyzed GO terms related to other signaling pathways, including Wnt, Shh, HIPPO, and endocytosis, we did not observe significant changes in gene expression (Supplementary Figure 5, panels c and d). Overall, our findings suggest that downregulation or absence of Cav-1 consistently leads to an increased population of MCCs in airway epithelium and an activation of a transcriptional program associated with MCCs. Lack of caveolin-1 expression leads to early maturation of MCCs Based on the GO­ analyses, we undertook a direct evaluation of cilia in both wild-type (WT) and Caveolin-1 knock-out (Cav1-KO) airway epithelium. Initially, we performed staining and confocal imaging of acetylated tubulin in control and Cav1-KO cells at ALI 14. Surprisingly, we did not observe apparent changes in cilia between control and Cav1-KO MCCs (Figure 4, panel a). Subsequently, we prepared airway epithelial monolayers for scanning electron microscopy (SEM) in differentiated cells up to ALI14. Once again, no major structural differences were evident between control and Cav1-KO conditions (Figure 4, panel b), except for a significant increase in the length of the cilia in Cav1-KO MCCs (Supplementary Figure 4, panel c). Given our earlier finding that spermatogenesis and cell projection GO terms were enriched in Cav1-KO cells, we decided to explore early stages of MCC differentiation (from ALI 4 to ALI 7). Notably, differences in MCC numbers were already significant at ALI 6 and ALI 7 between WT and KO epithelium (Figure 4, panel c). Furthermore, staining with Centriolin allowed us to categorize MCCs into different types representing distinct stages of differentiation 31 . These stages encompassed basal body maturation (Stage II), migration and docking of basal bodies in the apical membrane (Stage III), and the generation of motile cilia from the basal bodies (Stage IV) 31 . Analyzing these categories from ALI 4 to ALI 7, we observed that the absence of Cav-1 accelerated MCC differentiation. Specifically, we found significantly more cells in the Stage IV/V category at ALI 4 and ALI 5 in Cav1-KO epithelium (Figure 4, panel d and Supplementary Figure 4, panel d). Finally, at the functional level, we discovered that ciliary beating was significantly higher in Cav1-KO compared to WT (Figure 4, panel e). Caveolin-1 promotes activation of Notch signaling at the onset of airway cell differentiation As previously described in the literature, Notch signaling exerts pivotal control over airway epithelial cell fate. Activation of Notch directs cells toward secretory lineages (club cells and goblet cells), while its suppression promotes the expansion of MCCs 32,33 . The Notch signaling pathway features transmembrane Notch receptors (Notch1-4) that interact with ligand proteins (such as Jag1, Jag2, and Dll). Upon ligand binding, proteolytic cleavage occurs by ADAMs (a metalloproteinase) at the extracellular side, followed by intracellular cleavage by g-secretase. This cleavage releases the intracellular domain (NICD), which subsequently associates with transcriptional regulators such as Mastermind (Mam) and CSL (CBF1/RBP-Jκ/Su(H)/LAG-1), thereby modulating gene expression within the cell nucleus (Figure 5 panel a) 34–36 . Building upon these insights, we conducted further investigations into MCC differentiation. We began by analyzing the expression levels of Notch intracellular domain (NICD) target genes—Hes1, Hes5, Hey1, and Hey2—in wild-type and Caveolin-1 knock-out (Cav1-KO) mouse tracheal epithelial cells (MTECs) at ALI 14 using RT-qPCR (Supplementary Figure 5, panel a). Surprisingly, their expression was comparable between the two groups. However, when examining these target genes at ALI 6 (the onset of differentiation events), we observed a decrease specifically in Hes5, Hey1, and Hey2 expression in Cav1-KO cells (Figure 5 panel b), while Hes1 expression remained unchanged (Figure 5 panel b). This finding indicated that Notch signaling was suppressed in Cav1-KO cells during early differentiation (ALI 6), but this effect was not sustained at ALI 14. Moreover, we explored the expression of Notch1 and Notch2 receptors—critical determinants of airway epithelial cell fate—as well as the expression of Notch ligands (Jag1, Jag2, and Dll1). Importantly, both receptors and all ligands exhibited comparable expression levels in wild-type and Caveolin-1 knock-out (Cav1-KO) cells (Supplementary Figure 5, panel b). To gain deeper insights, we assessed Notch transcriptional activity by examining its binding to chromatin at ALI 6 in both Luc-KD and Cav1-KD conditions. Strikingly, depletion of Caveolin dramatically reduced Notch intracellular domain (NICD) binding to chromatin. Specifically, we detected only 766 genes in Cav1-KD cells, in contrast to over 5000 genes observed in control conditions (Figure 5, panel c). Importantly, we did not observe a shift in NICD (Notch intracellular domain) binding to other genomic regions, as evidenced by Venn diagram and distribution analyses (Figure 5, panel c-e). The DNA motifs that were found to be enriched in both Luc-KD and Cav1-KD peaks were similar, including Pitx2 and Znf460, that maintain a subtle homology to CSL consensus sequence (Figure 5, panel f). When we combined NICD-bound genes in Luc-KD conditions with the results from Cav1-KO RNA-seq, we found that the genes that were over expressed in Cav1-KO conditions were slightly more probable to be bound by NICD (Figure 5, panels g-i), suggesting a repressive role of NICD in Luc-KD cells. These genes that were bound by NICD and upregulated in Cav1-KO conditions were functionally associated to cilium (Figure 5, panel j). Notch1 Expression and Subcellular Distribution in Control and Cav1-KD Cells Although caveolae and clathrin-dependent endocytosis are well-established mechanisms for receptor internalization, no studies have yet demonstrated Cav-1-dependent Notch internalization 37 . Nevertheless, our observations suggest a correlation between Cav-1 and Notch activity. Could Cav-1 depletion be affecting Notch localization or protein levels? We first assessed Cav-1 localization by performing confocal imaging, which revealed the localization of Notch1 in control and Cav1-KD cells at ALI 6 (Figure 6, panel a). No major changes in levels or localization were observed between genotypes. Next, we used Western blotting to evaluate the expression levels of full-length Notch1 (300 kDa) and its cleaved form (120 kDa) in Luc-KD and Cav1-KD cells (Figure 6, panel b). Quantification of relative protein expression levels indicated a significant reduction in both full-length Notch1 (Figure 6, panel c) and cleaved Notch1 (Figure 6, panel d) in Cav1-KD cells compared to controls. Additionally, we checked the levels of E-Cadherin, a membrane protein previously related to Caveolin. We found that E-Cadherin levels were significantly reduced in Cav1-KD cells compared to control cells without major changes in E-Cadherin localization (Figure 6, panel e and Supplementary Figure 6, panel a). Furthermore, to quantitatively explore the cellular distribution of Notch1, we performed subcellular fractionation. The subcellular distribution of full-length Notch1 and its processed forms (TMD-NICD and NICD) was analyzed in the cytosol, membrane, and chromatin fractions of control and Cav1-KD cells (Figure 6, panel f and Supplementary Figure 6, panels b and c). Quantification of relative protein expression levels revealed a slight, although significant, reduction of the TMD-NICD in Cav1-KD cells compared to control cells (Figure 6, panel g). Based on these findings, we propose a working model in which Caveolin-1 (Cav-1) regulates basal stem cell (BSC) differentiation by modulating the binding capacity of the Notch intracellular domain (NICD) to chromatin together with other transcriptional partners (Figure 6, panel h). This differential binding capacity may influence the transcriptional activity of Notch target genes, thereby affecting cell fate decisions. These results collectively suggest that Cav-1 plays a critical role in the regulation of Notch1 signaling and its subcellular localization, which may have significant implications for BSC differentiation and epithelial homeostasis. Discussion In our present study, we elucidate the intricate role of caveolin-1 (Cav-1) expression within the mouse airway epithelium, building upon previous findings in rat and human samples. Our investigation reveals specific associations between Cav-1 and distinct cell types, primarily BSCs and MCCs. Notably, while downregulation or absence of Cav-1 does not significantly impact BSC biology or epithelial barrier function, it emerges as a critical regulator of MCC differentiation and maturation. The precise mechanism underlying this function remains an intriguing puzzle. We explored two potential scenarios. The first scenario involves direct endocytosis modulation: Cav-1 may directly influence Notch signaling by participating in its subcellular distribution, mainly through endocytosis after activation at the plasma membrane. Although caveolae and clathrin-dependent endocytosis are well-established mechanisms for receptor internalization 37 , no studies have yet demonstrated Cav-1-dependent Notch internalization. Nevertheless, our observations suggest a correlation between Cav-1 and Notch processing. Alternatively, a connection between γ-secretase and Cav-1 could be at play. References suggest that γ-secretase, situated within lipid rafts, affects Notch cleavage when altered altered 38 , 39 . However, we did not find major significant differences in the subcellular distribution or Notch 1 processing. In the second scenario, we explored the possibility that Cav-1 could modulate the amount of Notch1 within the cells. In this case, we found that Cav-1 deficiency affects Notch1 protein levels. Although a significant decrease in Notch1 is observed, we do not believe this is the major mechanism regulating Notch1 activity. At this point, we consider that third-party mediation could also be occurring (Fig. 6 , panel h). Of course, this a less defined scenario. But, for instance, we note that chronic cigarette smoke exposure markedly increases Cav-1 in airway epithelial cells 21 . This upregulation correlates with p53 induction and airway mucus hypersecretion 40 . Given our previous findings on p53/Mdm2 regulation of BSC differentiation, a potential link between MCCs and Cav-1 via p53 protein levels warrants exploration 17 . Beyond MCC determination, we uncover a phenotype related to MCC maturation and length. Previous studies highlight Cav-1’s roles in regulating ciliary membrane composition, function, and primary cilium length 41 . Intriguingly, depletion of Cav-1 expression promotes primary cilia formation 42 . While our experimental model system focuses on airway MCCs, it’s essential to consider their broader roles. MCCs extend beyond the airway tract, contributing to ovule movement in the fallopian tubes and cerebrospinal fluid (CSF) flow within brain ventricles. Investigating whether Cav-1 affects reproduction and CSF dynamics in these contexts holds promise for understanding broader physiological implications. In summary, our study underscores the multifaceted impact of Cav-1 in airway epithelium, from MCC differentiation to ciliary function, and prompts further exploration of its intricate connections. Materials and Methods Isolation of mouse tracheal epithelial cells (MTECs) The isolation of MTECs in wild-type and Cav1 constitutive knockout (provided by Miguel Ángel del Pozo) C57BL/6J adult mice was carried out using a modified version of the procedure described by You et al (2002). All animal studies have been performed in accordance with the National and European legislation (Spanish Royal Decree RD53/2013 and EU Directive 86/609/CEE as modified by 2003/65/ CE, respectively) and in accordance with the Institute of Laboratory Animal Resources (ILAR) for the protection of animals used for research. Experimental protocols were approved by the Bioethics Committee for Animal Experimentation of the University of Extremadura (Registry July 7, 2017). Firstly, the mice were euthanized, and the tracheas were dissected from the bronchial main to the larynx and placed in cold Ham’s F-12 medium supplemented with 1% of penicillin and streptomycin. After resection, vascular, fatty tissues, and muscle were removed in cold media, the clean tracheas were longitudinally excised and incubated with 1.5 mg/ml of pronase (Roche Molecular Biochemicals) in Ham’s F-12 medium with penicillin–streptomycin for 16 hours at 4°C. Afterward, fetal bovine serum (FBS, Gibco) was added to a final concentration of 10%. The processed tracheas were discarded, and the isolated cells were collected by centrifugation at 500 g for 5 min at 4°C. Then, the cells were then incubated in F-12 medium containing 0.5 mg/ml pancreatic DNase I (Sigma-Aldrich) for 10 min and collected again by centrifugation. After this step, cells were seeded within PneumaCult-Ex Plus complete medium (StemCell) in primary tissue culture plates (Corning) for 4 h in 5% CO 2 at 37°C to remove fibroblasts. Finally, the supernatant was collected, and cells were seeded in 60-mm plates previously treated with type I rat tail collagen (Gibco) in 0.02 N acetic acid. We used a 60-mm plate for every three tracheas. Cell Culture and Differentiation of MTECs MTECs were expanded in PneumaCult-Ex Plus medium (StemCell) at 37°C in 5% CO 2 until 70–80% confluence (3-5 days after plating). Then, cells were detached from plates by two consecutive incubations, with 0.02% EDTA in PBS for 20 minutes at 37°C (to provide separation of cell-cell junction), and Accutase (Gibco) for 10 min at room temperature (to separate cells from plate). Following that, suspension cells were centrifugued at 1000 g for 5 min and pellet containing cells was resuspended in 1 mL of Pneumacult-Ex plus and counted. For MTECs differentiation, 9 × 10 4 cells/cm 2 were seeded in supported polyester porous membranes (Transwell 0.4 μm pores, Corning). The upper and lower chambers were filled with PneumaCult-Ex Plus medium, which was changed every 2 days. Cells proliferated during 4-6 days with media in the upper chamber and lower chamber with expansion media. Once the epithelial monolayer reached the medium was removed from the upper chamber and changed in the lower chamber to ALI medium (Air-Liquid Interface Medium, StemCell) to simulate the organization of respiratory tract tissue. ALI was replaced every 2 days and maintained until the end of the differentiation, 14 days (ALI 14). Lentivirus Production and Infection Short hairpin RNAs (shRNAs) against Cav1 were cloned in the pLKO.1 vector which drives the expression of shRNAs from the U6 human promoter and contains a puromycin-IRESmCherry selection cassette. The shRNA against luciferase was provided by Miguel Fidalgo’s laboratory. The shRNAs (Supplementary Table I) were cloned into the pLKO.1 using EcoRI and AgeI sites as described by Woo et al., (2019) 43 . Purified DNAs were transfected with the packaging and envelope vectors psPAX2 and PMD2.G into HEK-293T cells using polyethylenimine (PEI) (Sigma). Viruses were collected 48 and 72 h post-transfection and concentrated with Amicon Ultra-15 (Merck) by centrifugation at 3500 g for 30 min at 4°C. The approximate concentration of infectious virus particles was adjusted to 2 x 10 5 infectious virus particles/ml. We added a volume of 75 μl of concentrated virus in 3 ml of medium in the presence of 8 μg/ml polybrene (Sigma) in a 60-mm plate. Infected MTECs were selected with puromycin 3 μg/ml during 48 h. Finally, cells were expanded and plated for experiments. For caveolin-1 overexpression, we used pRRL-IRES-eGPF-hCav1 lentiviral vector (provided by Miguel A. Del Pozo's laboratory) and followed the same protocol for lentivirus generation and MTEC infection as was used for knockdown generation. However, this vector does not allow for cell selection using puromycin. Culture of mouse airway organoids (AOs) MTECs were expanded and prepared as described previously to culture mouse airway organoids 17–19 . When cells reached 60%-70% confluence, they were detached and prepared for organoid formation. For mouse organoids, 96-well plates were coated with 100 μL of 30% matrigel in PneumaCult-Ex Plus medium and incubated at 37°C for 20 minutes until gelation occurred. Then, 500 MTECs control or Cav1-KD were suspended in 100 μL of 2% matrigel in PneumaCult-Ex Plus and were seeded onto the matrigel layer. After 5 days, images of the airway organoids were captured using an EVOS Floid Cell Imaging Station (Invitrogen). Immunofluorescence (IF) MTECs cultures were fixed in 4% paraformaldehyde (PFA) (PolyScience) for 10 min at room temperature, permeabilized in PBS-Triton X-100 0.1% for 10 min and blocked with PBS-Triton X-100 with 2% bovine serum albumin (BSA, Roche) for 1h. Primary antibodies were incubated overnight at 4°C in the blocking buffer. The primary antibodies used for immunofluorescence (IF) were anti-p63 (Abcam #ab124762, 1:200), anti-Foxj1 (Invitrogen, #14-9965-82, 1:200), anti-Scgb1a1 (Abcam, ab213203, 1:500), anti-Acetylated tubulin (Sigma, #T6793,1:100), anti-Caveolin1 (Cell Signaling, #3238, 1:400), anti-Centriolin (Santa Cruz, SE-365521, 1:100), anti-Notch1 (CellSignalling, #3608, 1:200) and anti-E-Cadherin (BD Biosciences, #610182, 1:1000). After the incubation with primary antibodies, the samples were washed five times in 0.1% Triton and then incubated for 1 hour with fluorescent secondary antibodies diluted in the same buffer. The secondary antibodies used for IF were, Alexa Fluor 488 anti-Rabbit (Invitrogen, #A21206, 1:500), and Alexa Fluor 594 anti-Mouse (Invitrogen, #A11005, 1:500). Nuclei were stained with DAPI at a concentration of 0.5 μg/mL (Thermo Fisher Scientific, #62248, 1:1000) and actin were stained with Phalloidin (Invitrogen, A12381, 1:500). Five additional washes were performed in PBS-Triton 0.1%. Slides were mounted in Vectashield (Vector Labs), and images were captured using an Olympus FV 1000 confocal microscope. After acquisition, the images were processed using ImageJ (Fiji) and Adobe Photoshop CC 2023. For the quantitative assessment of cells expressing a specific cell-type marker, we counted the number of positive cells in a minimum of four fields of view captured using a confocal microscope and then projected them into a single image. To determine the percentage of positive cells, DAPI staining was employed to calculate the total number of cells in a given field of view. Gene expression analysis Total RNA was isolated using the illustra RNAspin Mini kit (GE Healthcare). Following elution, for quantitative PCR (qPCR) 200-400 ng of RNA was subjected to reverse transcription using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's instructions. qPCR was performed to analyze gene expression using the oligonucleotides listed in Supplementary Table II and the Power SYBR Green PCR Master Mix (Applied Biosystems) as per the provided instructions. Melt curve analyses were performed to confirm the specificity of the PCR reactions. For RNA-seq analysis, libraries from RNA samples were built following Illumina recommendations at Novogene UK and then sequenced. Raw reads were pseudoaligned to Mus musculus (mm10) genome using kallisto 44 . An ad hoc script transformed these outputs to the specific format required to be analyzed by Gene Set Enrichment Analysis (GSEA) 45 . Western blot (WB) Cells were lysed using ice-cold lysis buffer composed of 50 mM Tris-HCl (pH 7.5), 1 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 5 mM sodium pyrophosphate, 10 mM sodium fluoride, 0.27 M sucrose, 0.1 mM phenylmethylsulphonyl fluoride, 0.1% (v/v) 2-mercaptoethanol, 1% (v/v) Triton X-100, and complete protease inhibitor cocktail (Roche). The protein concentration was determined using the Bio-Rad protein assay, and 20 μg of protein was subjected to SDS-PAGE electrophoresis and transferred onto nitrocellulose membranes (Bio-Rad Laboratories). The membranes were then blocked with 5% dry milk in TBS-Tween (Tris-buffered saline containing 0.05% Tween-20) and incubated with primary antibodies overnight at 4°C. The primary antibodies for WB were diluted in blocking solution. The antibodies used anti-Caveolin1 (Cell Signaling, #3238, 1:1000), anti-GAPDH (Cell Singnaling, #5174, 1:1000), anti-Flotillin (Cell Signaling, #18634 1:1000), anti-Vinculin (Sigma, #V4505, 1:1000), anti-Notch1 (CellSignalling, #3608, 1:1000), anti-E-Cadherin (BD Biosciences, #610182, 1:1000) and anti-Actin (Sigma, #A2066, 1:1000) in blocking solution. The membranes were washed five times with TBS-Tween and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit-HRP, Cell Signaling, #7074; anti-mouse-HRP, Cell Signaling, #7076) in blocking solution (1:1000) for 1 h at room temperature. After additional washes with TBS-Tween, the proteins were visualized using a chemiluminescence detection system (SuperSignal West Dura, Thermo Fisher Scientific) and detected with iBright CL1000. Subcellular Protein Fractionation The subcellular fractionation kit (#78840, Thermo Fisher Scientific) was used for separation and preparation of cytoplasmic, membrane and chromatin-bound protein extracts. The procedure was performed according to the manufacturer’s instructions. Protein concentrations were determined, and equal amounts of protein (10 µg per fraction) were subjected to western blot analysis. For SDS-PAGE we have used 4-20% Mini-PROTEAN® TGX gels (Bio-Rad, #4561094) which allows for the separation of a wider range of protein size. The primary antibodies used were anti-Notch1 (CellSignalling, #3608, 1:1000), anti-GAPDH (CellSignalling, #2118, 1:2500) and Histone 3 (CellSignalling, #4499, 1:10000) incubated 1h at room temperature. The HRP-conjugated secondary antibodies that were used are anti-Rabbit-HRP (ThermoFisher, #31460, 1:10000) and anti-Mouse-HRP (ThermoFisher, #62-6520, 1:5000) incubated 1h at room temperature. Ponceau S staining was used to confirm homogeneous loading. Cell cycle assay For quantitative assessment of the cell cycle, MTECs were detached as described above and fixed with cold 70% ethanol. Then, the cells were treated with RNAsa and PI overnight. 1 x 10 5 cells were analyzed on the MACSQuant X flow cytometer (Miltenyi Biotec) and the data were processed in Flowlogic software (Miltenyi Biotec). Transepithelial resistance measurements (TEER) The permeability of tight junction along the differentiation process of MTECs was tested by TEER in ALI 2, ALI 4, ALI 6 and ALI 14. First, the upper chamber of Transwells was washed once in PBS. Then, DMEM (Dulbecco's Modified Eagle Medium ) at 37°C was added in the upper and lower chambers of Transwells and TEER was measured with an epithelial Ohm-voltmeter (EVOM3, WPI), positioning the electrode in the upper chamber. Resistance was recorded in kilohms (kW). For each time point, a fold change relative to control cells was calculated. Finally, graphs were obtained using Microsoft Excel and GraphPad Prism 8. Scanning electron microscopy (SEM) MTECs were differentiated up to ALI 14 and prepared for SEM by fixation with 2.5% glutaraldehyde for 1h and 30 min at 4°C, washed in 0.2 M cacodylate buffer and stained with 1% osmium tetroxide (Sigma) in 0.2 M cacodylate for 2 hours at 4°C. Cells were further dehydrated by incubation in increasing concentrations of ethanol (10%, 30%, 50%, 70%, 90% and 100%) for 20 minutes each. Finally, samples were dried using the technique of liquid carbon dioxide critical point, gold sputter coated, and visualized in a Quanta 3D FEG (ESEM-FIB; FEI Company) electron microscope. Cilia beating frequency measurement Cilia beating frequency was measured in ALI 14 MTECs wild-type and Cav1 knockdown. The upper chamber of 12-mm transwells was incubated with 5 μL of Dynabeads Streptavidin C1 (Invitrogen) in 300 μL of Ca 2+ /Mg 2+ -PBS for 10 min at 37°C. After, the medium from the upper chamber was removed and transwells were incubated for 1h and 30 min at 37°C. Before movie recording, 100 μL of Ca 2+ /Mg 2+ -PBS at 37°C was added on top of the transwell. Cilia beating movie was obtained using Motic AE20 microscope and an iPhone 14 at 240 frames per second (fps), and a custom MATLAB function was developed to assess the movement of the beads as well as to quantify the cilia beating frequencies using FFT. Cilia length measurement Cilia lengths were measured using Fiji software. Images were acquired at 8,000x magnification using SEM, providing sufficient resolution for detailed morphological analysis. To convert pixel measurements to micrometers (µm), a known scale embedded in the images was used. The Set Scale function in the Analyze menu of Fiji was employed, where the known scale length was entered, and the unit of measurement was set to micrometers. Measurements of the lengths of the structures were conducted using the Segmented Lines tool, which allowed tracing over morphological features in the images to record their lengths in micrometers. For each phenotype (wild type and Cav1-KO) analyzed, at least 50 independent measurements were performed. Chromatin immunoprecipitation sequencing (ChIP-seq) To analyze the binding of NICD to different promoters, ChIP was performed as described above 46,47 . After, libraries for ChIP-seq were built following Illumina recommendations at STAB VIDA. 20M reads were obtained per input and IP sample, respectively. They were aligned to the M. musculus (mm10) genome using BWA, and peaks were retrieved with MACS2 48 . ChIPseeker 49 and HOMER 50 were used to annotate the peaks, and custom MATLAB functions were developed for other analyses. Single cell RNA sequencing (scRNA-seq) We used the GSE102580 dataset 14 from NCBI GEO to analyze the cell-type expression of specific genes in mouse airway epithelia. A custom MATLAB function was developed in order to generate a dot plot showing average expression and cell percentage. Statistical analyses Different experimental groups (GraphPad Prism) were compared against control conditions by means of two-tailed t -tests. The n value in figure legends for WB and qPCR represents independent samples which are single transwells coming from at least four differentiation sets. In qPCR experiments, each independent sample is the result of 2-3 technical replicates. Relative expression was obtained using EIF1a as a housekeeping gene. Declarations Data availability Data from NGS experiments are publicly available at figshare server (10.6084/m9.figshare.26417137). Acknowledgements: Confocal microscopy and scanning electron microscopy were performed at the UEX microscopy core facilities. This work was supported by BFU2017-85547-P, TED2021-130560B-I00 and PID2021-126905NB-I00 grants from the Ministry of Economy, IB18014 from Junta de Extremadura to J.M. C-G and GR21140 from Junta de Extremadura to S.M-N. This study was also supported by grants to M.A.d.P. from the Spanish Ministry of Science and Innovation (MICIIN)/Agencia Estatal de Investigación (AEI)/European Regional Development Fund (ARDF/FEDER) “A way to make Europe” PID2020-118658RB-I00 and PDC2021-121572-I00, and Fundación Obra Social La Caixa (AtheroConvergence, HR20-00075. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation) and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). S.G-J. was a recipient of a Fellowship from the Universidad de Extremadura. S. D-Ch and C.M.N-Q were recipients of a Fellowship from Junta de Extremadura. All Spanish funding is co-sponsored by the European Union FEDER program. Author contributions M.O.-G., G. C.-L., J.F. B.-L., N. V.-P., A. D.-P., M. T.-D., and M. C.-M. performed all of the experiments. G. C.-L. and D.A.B. designed and performed the data analysis. S.M.-N., A.C. R., M. A.P. and J.M.C.-G. designed the experiments, analyzed data and wrote the manuscript. Disclosure and competing interest statement The authors declare no competing financial interests. References Hewitt, R. J. & Lloyd, C. M. Regulation of immune responses by the airway epithelial cell landscape. doi:10.1038/s41577-020-00477-9. Whitsett, J. A., Kalin, T. V., Xu, Y. & Kalinichenko, V. V. Building and regenerating the lung cell by cell. Physiological Reviews 99 , 513–554 (2019). Davis, J. D. & Wypych, T. P. Cellular and functional heterogeneity of the airway epithelium. Mucosal Immunology 14 , 978–990 (2021). Pampaloni, F., Reynaud, E. G. & Stelzer, E. H. K. 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Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Published Journal Publication published 15 Feb, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 24 Jul, 2025 Reviews received at journal 17 Jul, 2025 Reviewers agreed at journal 05 Jul, 2025 Reviews received at journal 17 Apr, 2025 Reviewers agreed at journal 13 Apr, 2025 Reviewers agreed at journal 26 Mar, 2025 Reviewers invited by journal 25 Mar, 2025 Editor assigned by journal 25 Mar, 2025 Editor invited by journal 19 Mar, 2025 Submission checks completed at journal 18 Mar, 2025 First submitted to journal 09 Mar, 2025 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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(a)\u003c/strong\u003e Maximal projection of confocal images\u003cstrong\u003e \u003c/strong\u003ein mouse whole-mount trachea for caveolin-1 in green, acetylated tubulin in red and nuclei in blue. \u003cstrong\u003e(b) \u003c/strong\u003eAnalysis of flotillin, caveolin-2 and caveolin-1 expression in MTECs differentiated \u003cem\u003ein vitro\u003c/em\u003e by scRNA-seq. The size of the circle represents the percentage of cells (%) with positive expression, while the color reflects the average expression level (reads counts). \u003cstrong\u003e(c) \u003c/strong\u003eCaveolin-1 expression in MCCs in ALI 14. Caveolin-1 is shown in green, acetylated tubulin in red, flotillin in green and nuclei in blue\u003cstrong\u003e. (d) \u003c/strong\u003eMaximal projection of confocal images of caveolin-1 expression from the apical plane to the base plane in MTECs in ALI 14\u003cstrong\u003e. (e) \u003c/strong\u003eRelative protein expression quantification for caveolin-1 protein assayed during the differentiation process at ALI 2, ALI 4, ALI 6, and ALI 14. \u003cstrong\u003e(f) \u003c/strong\u003eCaveolin-1 mRNA expression levels in ALI 2, ALI 4, ALI 6, and ALI 14. Scale bar in panels a and d represent 20 μm. Scale bar in panel c represent 10 μm.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6189425/v1/11feb0485d312f22dd121b31.png"},{"id":80034746,"identity":"fbe47063-0db3-41dd-aec3-89909faac42e","added_by":"auto","created_at":"2025-04-07 08:10:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":665733,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCav1-KD and its effect on the proliferation and self-renewal of BSCs. (a) \u003c/strong\u003emRNA expression levels of caveolin-1 assessed in differentiated MTECs infected with control viruses (Luc-KD) or Cav1 shRNAs (Cav1-KD). \u003cstrong\u003e(b)\u003c/strong\u003e Western blots images in Luc-KD and knockdown of Cav1 (Cav1-KD) cells in ALI 6. Flotilin and Gadph were assayed as a loading control. \u003cstrong\u003e(c) \u003c/strong\u003eDoubling time (hours) in Luc-KD and Cav1-KD cells. \u003cstrong\u003e(d)\u003c/strong\u003eAOs size quantification (A.U.) at 7 days in Luc-KD and Cav1-KD. \u003cstrong\u003e(e) \u003c/strong\u003eCell cycle analyses in Luc-KD and Cav1-KD cells. \u003cstrong\u003e(f)\u003c/strong\u003e Transepithelial electrical resistance (TEER) was used to test tight-junction permeability during the differentiation process of MTECs in Cav1-KD compared to Luc-KD cells. \u003cstrong\u003e(g-j)\u003c/strong\u003e mRNA expression levels in Luc-KD versus Cav1-KD. Trp63 and Krt5 for BSCs (a), Scgb1a1 and Scgb3a2 for CCs (b), Foxj1 and Mcidas for MCCs (c), and Spdef and Muc5ac for GCs (d). \u003cstrong\u003e(k, l) \u003c/strong\u003eMaximal projection of confocal images in Luc-KD and Cav1-KD differentiated cells for p63 (k) and Foxj1 (l) in green, phalloidin in red, and nucleus in blue. Scale bar in panels k and l represent 20 μm. p-values in all conditions were obtained using two-tailed t-test (**** represents p\u0026lt;0,0001 and n.s. means no significative differences).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6189425/v1/7d4674db40e5960a10c6b66e.png"},{"id":80034748,"identity":"6d79ba04-436e-4d76-9873-c21d380a4a9e","added_by":"auto","created_at":"2025-04-07 08:10:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":860547,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCaveolin-1 expression is required for MCCs differentiation. (a-d) \u003c/strong\u003emRNA expression levels in control (WT) versus Cav1-KO. Trp63 and Krt5 for BScs (a), Scgb1a1 and Scgb3a2 for CCs (b), Spdef and Muc5ac for GCs (c), and Foxj1 and Mcidas for MCCs (d). \u003cstrong\u003e(e)\u003c/strong\u003e Confocal images for WT and Cav1-KO cells processed for immunofluorescence to evaluate MCCs positive for Foxj1 in green. \u003cstrong\u003e(f)\u003c/strong\u003eHeat map of gene expression in WT and Cav1-KO cells in ALI 6 that shown an increase in the expression of MCCs markers. \u003cstrong\u003e(g)\u003c/strong\u003e Volcano plot for RNA-seq (WT vs Cav1-KO cells in ALI 6). \u003cstrong\u003e(h) \u003c/strong\u003eGene ontology (GO) analyses using DAVID (WT vs Cav1-KO cells in ALI 6). Scale bar in e represent 20 μm. p-values in all conditions were obtained using two-tailed t-test (**** represents p\u0026lt;0,0001, *** represents p\u0026lt;0,001, and n.s. means no significative differences).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6189425/v1/5c0e3849f7c591699f796065.png"},{"id":80035218,"identity":"0cb7a01a-f3b0-44f2-a245-87d03d0261ea","added_by":"auto","created_at":"2025-04-07 08:18:54","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":663727,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLack of caveolin-1 expression leads to early maturation of MCCs. (a) \u003c/strong\u003eMaximal projection of confocal images for acetylated tubulin (in green), phalloidin (in red) and nucleus (in blue) in control (WT) and Cav1-KO cells in ALI 14. \u003cstrong\u003e(b)\u003c/strong\u003eScanning electron microscopy images of airway epithelial in WT and Cav1-KO cells in ALI 14. \u0026nbsp;\u003cstrong\u003e(c)\u003c/strong\u003e Number of MCCs\u003cstrong\u003e \u003c/strong\u003eper field in WT and Cav1-KO cells in ALI 4, ALI 5, ALI 6 and ALI 7. \u003cstrong\u003e(d) \u003c/strong\u003eQuantification of deuterosomal/MCCs in different stages of differentiation (ALI 4, ALI 5, ALI 6 and ALI 7) in WT and Cav1-KO cells. Type II/III are those cells with Centriolin staining in aggregates while Type IV/V are those cells with Centriolin staining disperse at the apical membrane.\u003cstrong\u003e (e)\u003c/strong\u003eCilia beating frequency quantification as number of beats per second in WT and Cav1-KO cells in ALI 14. Scale bar in a represent 20 μm. p-values in all conditions were obtained using two-tailed t-test (**** represents p\u0026lt;0,0001, ** represents p\u0026lt;0,01 and n.s. means no significative differences).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6189425/v1/2f6f4572873d403c3c459524.jpeg"},{"id":80034749,"identity":"401bcc1d-1d0b-4750-91f3-80e1a0a5210f","added_by":"auto","created_at":"2025-04-07 08:10:54","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":670606,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCaveolin-1 promotes activation of Notch signaling at the onset of airway cell differentiation. (a)\u003c/strong\u003e Schematic representation of the Notch signaling pathway. \u003cstrong\u003e(b)\u003c/strong\u003e NICD target genes (Hes1, Hes5, Hey1 and Hey2) mRNA expression levels in WT versus Cav1-KO in and ALI 6. \u003cstrong\u003e(c)\u003c/strong\u003e Venn diagram shows intersection of genes in close proximity to NICD-bound regulatory elements in Luc-KD and Cav1-KD. \u003cstrong\u003e(d, e)\u003c/strong\u003eNICD peak distribution across the mouse genome in Luc-KD (d) and Cav1-KD (e). \u003cstrong\u003e(f)\u003c/strong\u003e Logo of the NICD (forming a complex with CSL) detected motif in the peak regions in Luc-KD and Cav1-KD cells in ALI 6. \u003cstrong\u003e(g-i)\u003c/strong\u003e Number of genes (%) up (g), down (h) and non-regulated (i) by RNA-seq that present NICD binding. \u003cstrong\u003e(j)\u003c/strong\u003e Gene ontology (GO) analyses using DAVID (WT vs Cav1-KO cells in ALI 6) of up-regulated and NICD binding genes. p-values in all conditions were obtained using two-tailed t-test (**** represents p\u0026lt;0,0001, ** represents p\u0026lt;0,01 and n.s. means no significative differences).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6189425/v1/1181c2669859cb05c58c5809.jpeg"},{"id":80035220,"identity":"1adc4958-5821-4bf9-acb2-7fd4f2c37984","added_by":"auto","created_at":"2025-04-07 08:18:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1203634,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of Notch1 Expression and Subcellular Distribution in Control and Cav1-KD Cells. (a)\u003c/strong\u003e Maximal projection of confocal images for Notch1 (in yellow), and nucleus (in blue) in control and Cav1-KD cells in ALI 6. \u003cstrong\u003eb)\u003c/strong\u003e Western blot images of Luc-KD and Cav1-KD cells showing Notch 1 (full-length at 300 kDa and cleaved at 120 kDa) and E-Cadherin expression. Each condition was tested in triplicate (L1, L2, and L3 for each genotype). β-actin was used as a loading control. \u003cstrong\u003e(c-e)\u003c/strong\u003e Relative protein expression levels quantification of Notch1 300kDa (c), Notch1 120kDa (d) and E-Cadherin (e). \u003cstrong\u003e(f) \u003c/strong\u003eAnalyses of Notch 1 full-length and processed forms (TMD+NICD and NICD) subcellular distribution in the cytosol (Cyt.), membrane (Mem.), and chromatin (Chr.) in control and Cav1-KD cells. Cell fractionation and gradient SDS-Gels were used for improved resolution. \u003cstrong\u003e(g) \u003c/strong\u003eRelative protein expression quantification of Notch 1 subcellular distribution in control (WT) and Cav1-KD cells.\u003cstrong\u003e (h) \u003c/strong\u003eProposed working model of the mechanism by which Cav-1 regulates BSC differentiation, involving differential NICD binding capacity to chromatin together with other partners. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ep-values in all conditions were obtained using two-tailed t-test (*** represents p\u0026lt;0,001, ** represents p\u0026lt;0,01 and n.s. means no significative differences).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6189425/v1/01d428844a477ae46611cd32.png"},{"id":102786722,"identity":"a03befb0-0215-4d82-9d75-f092a3812699","added_by":"auto","created_at":"2026-02-16 16:14:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5848254,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6189425/v1/1a92647c-3391-4b81-a678-f9d5ded7ef11.pdf"},{"id":80036163,"identity":"717be1c8-0385-472b-a99e-d801353f37bb","added_by":"auto","created_at":"2025-04-07 08:26:54","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4834438,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6189425/v1/b20490db5b12db0dc20261c4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Caveolin-1 Modulates Notch Transcriptional Activity During Respiratory Multiciliated Cell Maturation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe respiratory tract, lined by a single layer of epithelial cells, serves as a critical barrier separating our internal milieu from the external environment\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. To ensure proper respiratory function and the ability to regenerate in response to environmental challenges, understanding the processes governing differentiation and self-renewal in adult stem cells within the epithelium is paramount\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Moreover, unraveling the intricate mechanisms driving epithelial development is essential for creating comprehensive and innovative experimental model systems, spanning both two-dimensional and three-dimensional culture settings \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAt homeostasis, the airway epithelium comprises a diverse array of specialized cell types\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Among these, three major constituents dominate. Basal Stem Cells (BSCs), these multipotent stem cells play a pivotal role in tissue regeneration, demonstrating the remarkable ability to fully restore the epithelium. Secretory Cells (SCs), within this category, club cells (CCs) and goblet cells (GCs) stand out\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. SCs are responsible for producing mucus\u0026mdash;a crucial defense mechanism that traps pathogens and pollutants\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. And Multiciliated Cells (MCCs), these cells continuously propel mucus toward the mouth, ensuring airway clearance and maintaining respiratory health\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. But the airway epithelium\u0026rsquo;s complexity extends further. Less abundant populations\u0026mdash;such as tuft cells (immune sentinels), pulmonary neuroendocrine cells (environmental sensors), and pulmonary ionocytes (recently unveiled through single-cell RNA-sequencing)\u0026mdash;contribute to its multifaceted function\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Maintaining the delicate balance among these cell types is essential for the proper functioning of the pseudostratified epithelium. Dysregulation, characterized by imbalances in BSC abundance or differentiation, underpins various airway diseases\u0026mdash;asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Our quest to understand the molecular pathways governing these processes remains an active area of research, with profound implications for regenerative medicine and therapeutic intervention.\u003c/p\u003e \u003cp\u003eDifferential gene expression analysis has served a dual purpose: classifying distinct cell types and unraveling the biology inherent to each cell population\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The caveolin family, comprising three members\u0026mdash;caveolin-1, caveolin-2, and caveolin-3\u0026mdash;plays a pivotal role in cellular dynamics\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Among them, caveolin-1 (Cav-1) emerges as a major structural protein of caveolae, specialized plasma membrane invaginations involved in diverse cell activities, including molecular transport, cell adhesion, and signal transduction\u003csup\u003e\u003cspan additionalcitationids=\"CR22 CR23 CR24\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In the context of airway epithelium, Cav-1 garners attention. It is membrane-associated and localized within BSCs and MCCs of the large airways in both rats and humans\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Recent investigations underscore its differential enrichment in basal stem cells (BC-2) within the mouse trachea, linking it to WNT, TGFβ, and Hedgehog signaling pathways\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite this intriguing expression pattern, a definitive role for Cav-1 in airway epithelium remains elusive. Reduced Cav-1 expression disrupts E-cadherin-mediated cell-cell adhesion in asthma patients\u0026rsquo; airways, leading to deregulation of the respiratory epithelial barrier\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Furthermore, Cav-1 expression in lung tissues acts as a safeguard against fibrogenic effects induced by TGF-β1, preventing conditions like pulmonary fibrosis and allergen-induced airway remodeling\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Notably, Cav-1 deficiency exacerbates pulmonary fibrosis, emphasizing its protective function\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Beyond structural integrity, Cav-1\u0026rsquo;s impact extends further. Its deletion correlates with increased pro-inflammatory cytokines (IL-6 and TNF-α) and altered production of MUC-5AC in lung epithelial cells\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, our primary aim is to analyze Cav-1\u0026rsquo;s function in airway epithelium and identify molecular mechanisms involved in the differentiation of BSCs to MCCs and SCs. By unraveling the intricate interplay between Cav-1, Notch signaling, and MCC maturation, we provide new insights into cellular fate determination within the airway epithelium.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCaveolin-1 expression is heterogeneously express in the airway epithelium\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the localization of Cav-1 in the airway tract, we conducted immunofluorescence on whole-mount trachea using Cav-1 antibodies and acetylated tubulin staining, which labels the apical surface of the multiciliated cells of the luminal airway epithelium. Confocal imaging and subsequent analysis revealed heterogeneous Cav-1 expression across different epithelial strata. At the basal planes where BSCs reside, Cav-1 expression varied, with some cells exhibiting high levels at the plasma membrane and others having lower levels (Figure 1, panel a). In the pseudostratified epithelium at medial planes, this heterogeneity was more pronounced (Figure 1, panel a), accompanied by enlarged puncta at the membrane. Conversely, at the luminal side where differentiated cells are located, Cav-1 expression was restricted to a subset of cells with very low levels compared to basal planes (Figure 1, panel a). This localization pattern correlated with single-cell RNA expression data from differentiated cells in ALI culture. Our analysis of published data indicated that Cav-1 was predominantly expressed in basal cells, cycling basal cells, and a subset of basal cells predisposed to differentiation (Krt4/13) (Figure 1, panel b). Further examination of MCCs revealed Cav-1 expression at both the apical and basolateral plasma membranes (Figure 1, panel c). In comparison, Flotillin 1, a caveolae component, exhibited more uniform expression across different strata and cell types in the airway epithelium (Figure 1, panel b). Additionally, Cav-2 showed higher expression in other cell types like Brush cells (Figure 1, panel b).\u003c/p\u003e\n\u003cp\u003eTo assess the specificity of the expression pattern in Krt4/13 cells, we examined genes that exhibited significantly higher expression in Krt4/13 compared to all other cell types (Supplementary Figure 1, panel a). We identified 70 genes that were significantly enriched, showing more than double the expression in Krt4/13 cells compared to other cell types. This distinct expression pattern, characterized by high expression in basal cells and even higher levels in Krt4/13 cells, motivates further investigation into the potential role of Cav-1 in airway epithelium biology.\u003c/p\u003e\n\u003cp\u003eTo further characterize the localization and function of Cav-1 in BSCs, we investigated its localization pattern in air-liquid interface (ALI) cultures \u003cem\u003ein vitro\u003c/em\u003e during the differentiation process. Our findings revealed that at ALI 2 (day 2 of differentiation), Cav-1 levels and localization were homogeneous in the epithelial monolayer (Supplementary Figure 1, panel b). However, in ALI 4, ALI 6, and ALI 14, as pseudostratification and differentiation occurred, Cav-1 expression became heterogeneous among luminal cells, with higher levels observed in basal cells\u0026mdash;consistent with \u003cem\u003ein vivo\u003c/em\u003e observations (Figure 1, panel d and Supplementary Figure 1, panel b). Furthermore, global RNA expression levels revealed a decline in Cav-1 expression along the differentiation protocol (Figure 1, panel f), which coincided with an increased expression of Foxj1, a marker for MCCs (Supplementary Figure 1, panel c). This reduction in Cav-1 expression at the epithelial level was also evident in ALI 14 at the protein level, as measured by Western blot (Figure 1, panel e and Supplementary Figure 1, panel d).\u003c/p\u003e\n\u003cp\u003eBased on these findings, we infer that caveolin-1 is predominantly expressed in BSCs, and its expression is significantly reduced in ALI 14 (Figure 1, panel e and Figure 1, panel f), likely due to a decrease in the proportion of BSCs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeficiency of caveolin-1 increase MCCs differentiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the role of Cav-1 in the differentiation process from BSCs to luminal differentiated cells, we conducted knockdown experiments targeting Cav1 (Cav1-KD) in BSCs (Figure 2, panel a). Using three distinct shRNA sequences against Cav1, we observed a significant reduction in expression compared to control conditions (Luc-KD, luciferase knockdown) (Figure 2, panel a). Still, no significant changes in the relative expression of Cav-2 and Cav-3 were detected in Cav1-KD (Supplementary Figure 2, panel a). This Cav1-KD was also evident at the protein level (Figure 2, panel b and Supplementary Figure 2, panel b). Subsequently, we assessed various cellular processes in control and Cav1-KD cells, including proliferation, self-renewal, cell attachment, migration, and cell cycle. Notably, Cav1-KD did not impact BSC proliferation, as indicated by doubling time (Figure 2, panel c), airway organoid size (Figure 2, panel d and Supplementary Figure 2, panels c and d), attachment (Supplementary Figure 2, panel e), or migration (Supplementary Figure 2, panel f). Furthermore, the distribution of cells across different cell cycle phases remained similar between control and Cav1-KD BSCs (Figure 2, panel e).\u003c/p\u003e\n\u003cp\u003eNext, we assessed the impact of Cav1 knockdown (Cav1-KD) on the airway epithelium developed \u003cem\u003ein vitro\u003c/em\u003e from BSCs (Supplementary Figure 2, panel g). Remarkably, the Cav1-KD epithelium formed a compacted epithelial monolayer with normal barrier function, comparable to control conditions (Luc-KD). Transepithelial resistance measurements confirmed this similarity between control and Cav1-KD epithelia (Figure 2, panel f). Furthermore, we investigated the cellular composition of the epithelium under both control and Cav1-KD conditions by assessing the expression of cell type markers for BSCs, MCCs, and SCs (club and goblet cells). Notably, there were no significant differences in BSC markers (\u003cem\u003eTrp63\u003c/em\u003e and \u003cem\u003eKrt5\u003c/em\u003e) or goblet cell markers (\u003cem\u003eSpdef\u003c/em\u003e and \u003cem\u003eMuc5A/c\u003c/em\u003e) (Figure 2, panel g and j). However, we observed significant differences in MCCs (\u003cem\u003eFoxj1\u003c/em\u003e) and secretory cells (\u003cem\u003eScgb1a1\u003c/em\u003e) markers (Figure 2, panels h and i). Interestingly, other markers such as \u003cem\u003eMcidas\u003c/em\u003e or \u003cem\u003eScgb3a2\u003c/em\u003e did not exhibit significant differences (Figure 2, panels h and i). Additionally, p63 and Scgb1a1 staining was comparable between Cav1-KD and control conditions (Figure 2, panel k and Supplementary Figure 2, panel h), while Foxj1 showed an increase in positive cells in Cav1-KD epithelia (Figure 2, panel l and Supplementary Figure 2, panel i). Additionally, in a parallel experiment where Cav-1 was overexpressed, we did not observe any significant changes in the tested genes compared to the control group (Supplementary Figure 3).\u003c/p\u003e\n\u003cp\u003eOverall, our findings suggest that the absence of Cav-1 does not significantly alter BSC behavior compared to control cells. The Cav1-KD cells successfully formed a functional airway epithelial monolayer, characterized by an increased presence of MCCs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLack of Caveolin-1 triggers early transcriptional programs related to MCCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further elucidate the role of Cav-1 in airway epithelial composition, we utilized BSCs isolated from both wild-type (WT) and Caveolin-1 knock-out (Cav1-KO) mice\u003csup\u003e30\u003c/sup\u003e. These BSCs were seeded and differentiated in air-liquid interface (ALI) cultures, and we analyzed cell type markers. Notably, expression levels of BSC and secretory cell (SC) markers were comparable between Cav1-KO and WT epithelia, except for \u003cem\u003eMuc5ac\u003c/em\u003e (Figure 3 panels a-c and Supplementary Figure 4, panel a). However, markers associated with MCCs, such as \u003cem\u003eFoxj1\u003c/em\u003e and \u003cem\u003eMcidas\u003c/em\u003e, were significantly upregulated in Cav1-KO epithelium (Figure 3, panel d). Immunofluorescence staining for Foxj1 further confirmed the increased abundance of MCCs in Cav1-KO conditions compared to WT (Figure 3, panel e and Supplementary Figure 4, panel b).\u003c/p\u003e\n\u003cp\u003eWe have previously found that early transcriptional activity during differentiation affect BSC differentiation\u003csup\u003e17,18\u003c/sup\u003e. Hence, to gain deeper insight into the molecular mechanisms linking Cav-1 and MCC differentiation, we investigated the epithelial status during early differentiation stages (ALI 6) using RNAseq. Initially, we examined known cell type markers for each described cell type. Remarkably, 8 out of 8 MCC markers were upregulated in Cav1-KO epithelium, except for \u003cem\u003eMcidas\u003c/em\u003e (Figure 3, panel f). In contrast, other cell type categories did not exhibit clear upregulation or downregulation in Cav1-KO epithelia compared to wild-type (WT) (Figure 3, panel f). A broader analysis of expression patterns revealed that a subset of 80 genes was downregulated, while 477 genes were upregulated in Cav1-KO epithelium at ALI 6 (Figure 3, panel g). Additionally, gene ontology (GO) analyses highlighted at least 6 cilia-related GO terms significantly enriched in Cav1-KO epithelium (Figure 3, panel h). These sets encompassed functional and structural aspects of cilia, including cell projection and motile cilium. Furthermore, when we analyzed GO terms related to other signaling pathways, including Wnt, Shh, HIPPO, and endocytosis, we did not observe significant changes in gene expression (Supplementary Figure 5, panels c and d). Overall, our findings suggest that downregulation or absence of Cav-1 consistently leads to an increased population of MCCs in airway epithelium and an activation of a transcriptional program associated with MCCs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLack of caveolin-1 expression leads to early maturation of MCCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the GO\u0026shy; analyses, we undertook a direct evaluation of cilia in both wild-type (WT) and Caveolin-1 knock-out (Cav1-KO) airway epithelium. Initially, we performed staining and confocal imaging of acetylated tubulin in control and Cav1-KO cells at ALI 14. Surprisingly, we did not observe apparent changes in cilia between control and Cav1-KO MCCs (Figure 4, panel a). Subsequently, we prepared airway epithelial monolayers for scanning electron microscopy (SEM) in differentiated cells up to ALI14. Once again, no major structural differences were evident between control and Cav1-KO conditions (Figure 4, panel b), except for a significant increase in the length of the cilia in Cav1-KO MCCs (Supplementary Figure 4, panel c).\u003c/p\u003e\n\u003cp\u003eGiven our earlier finding that spermatogenesis and cell projection GO terms were enriched in Cav1-KO cells, we decided to explore early stages of MCC differentiation (from ALI 4 to ALI 7). Notably, differences in MCC numbers were already significant at ALI 6 and ALI 7 between WT and KO epithelium (Figure 4, panel c). Furthermore, staining with Centriolin allowed us to categorize MCCs into different types representing distinct stages of differentiation\u003csup\u003e31\u003c/sup\u003e. These stages encompassed basal body maturation (Stage II), migration and docking of basal bodies in the apical membrane (Stage III), and the generation of motile cilia from the basal bodies (Stage IV)\u003csup\u003e31\u003c/sup\u003e. Analyzing these categories from ALI 4 to ALI 7, we observed that the absence of Cav-1 accelerated MCC differentiation. Specifically, we found significantly more cells in the Stage IV/V category at ALI 4 and ALI 5 in Cav1-KO epithelium (Figure 4, panel d and Supplementary Figure 4, panel d). Finally, at the functional level, we discovered that ciliary beating was significantly higher in Cav1-KO compared to WT (Figure 4, panel e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCaveolin-1 promotes activation of Notch signaling at the onset of airway cell differentiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs previously described in the literature, Notch signaling exerts pivotal control over airway epithelial cell fate. Activation of Notch directs cells toward secretory lineages (club cells and goblet cells), while its suppression promotes the expansion of MCCs\u003csup\u003e32,33\u003c/sup\u003e. The Notch signaling pathway features transmembrane Notch receptors (Notch1-4) that interact with ligand proteins (such as Jag1, Jag2, and Dll). Upon ligand binding, proteolytic cleavage occurs by ADAMs (a metalloproteinase) at the extracellular side, followed by intracellular cleavage by g-secretase. This cleavage releases the intracellular domain (NICD), which subsequently associates with transcriptional regulators such as Mastermind (Mam) and CSL (CBF1/RBP-J\u0026kappa;/Su(H)/LAG-1), thereby modulating gene expression within the cell nucleus (Figure 5 panel a)\u003csup\u003e34\u0026ndash;36\u003c/sup\u003e. Building upon these insights, we conducted further investigations into MCC differentiation. We began by analyzing the expression levels of Notch intracellular domain (NICD) target genes\u0026mdash;Hes1, Hes5, Hey1, and Hey2\u0026mdash;in wild-type and Caveolin-1 knock-out (Cav1-KO) mouse tracheal epithelial cells (MTECs) at ALI 14 using RT-qPCR (Supplementary Figure 5, panel a). Surprisingly, their expression was comparable between the two groups. However, when examining these target genes at ALI 6 (the onset of differentiation events), we observed a decrease specifically in Hes5, Hey1, and Hey2 expression in Cav1-KO cells (Figure 5 panel b), while Hes1 expression remained unchanged (Figure 5 panel b). This finding indicated that Notch signaling was suppressed in Cav1-KO cells during early differentiation (ALI 6), but this effect was not sustained at ALI 14. Moreover, we explored the expression of Notch1 and Notch2 receptors\u0026mdash;critical determinants of airway epithelial cell fate\u0026mdash;as well as the expression of Notch ligands (Jag1, Jag2, and Dll1). Importantly, both receptors and all ligands exhibited comparable expression levels in wild-type and Caveolin-1 knock-out (Cav1-KO) cells (Supplementary Figure 5, panel b).\u003c/p\u003e\n\u003cp\u003eTo gain deeper insights, we assessed Notch transcriptional activity by examining its binding to chromatin at ALI 6 in both Luc-KD and Cav1-KD conditions. Strikingly, depletion of Caveolin dramatically reduced Notch intracellular domain (NICD) binding to chromatin. Specifically, we detected only 766 genes in Cav1-KD cells, in contrast to over 5000 genes observed in control conditions (Figure 5, panel c). Importantly, we did not observe a shift in NICD (Notch intracellular domain) binding to other genomic regions, as evidenced by Venn diagram and distribution analyses (Figure 5, panel c-e). The DNA motifs that were found to be enriched in both Luc-KD and Cav1-KD peaks were similar, including Pitx2 and Znf460, that maintain a subtle homology to CSL consensus sequence (Figure 5, panel f). When we combined NICD-bound genes in Luc-KD conditions with the results from Cav1-KO RNA-seq, we found that the genes that were over expressed in Cav1-KO conditions were slightly more probable to be bound by NICD (Figure 5, panels g-i), suggesting a repressive role of NICD in Luc-KD cells. These genes that were bound by NICD and upregulated in Cav1-KO conditions were functionally associated to cilium (Figure 5, panel j).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNotch1 Expression and Subcellular Distribution in Control and Cav1-KD Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlthough caveolae and clathrin-dependent endocytosis are well-established mechanisms for receptor internalization, no studies have yet demonstrated Cav-1-dependent Notch internalization\u003csup\u003e37\u003c/sup\u003e. Nevertheless, our observations suggest a correlation between Cav-1 and Notch activity. Could Cav-1 depletion be affecting Notch localization or protein levels? We first assessed Cav-1 localization by performing confocal imaging, which revealed the localization of Notch1 in control and Cav1-KD cells at ALI 6 (Figure 6, panel a). No major changes in levels or localization were observed between genotypes. Next, we used Western blotting to evaluate the expression levels of full-length Notch1 (300 kDa) and its cleaved form (120 kDa) in Luc-KD and Cav1-KD cells (Figure 6, panel b). Quantification of relative protein expression levels indicated a significant reduction in both full-length Notch1 (Figure 6, panel c) and cleaved Notch1 (Figure 6, panel d) in Cav1-KD cells compared to controls. Additionally, we checked the levels of E-Cadherin, a membrane protein previously related to Caveolin. We found that E-Cadherin levels were significantly reduced in Cav1-KD cells compared to control cells without major changes in E-Cadherin localization (Figure 6, panel e and Supplementary Figure 6, panel a).\u003c/p\u003e\n\u003cp\u003eFurthermore, to quantitatively explore the cellular distribution of Notch1, we performed subcellular fractionation. The subcellular distribution of full-length Notch1 and its processed forms (TMD-NICD and NICD) was analyzed in the cytosol, membrane, and chromatin fractions of control and Cav1-KD cells (Figure 6, panel f and Supplementary Figure 6, panels b and c). Quantification of relative protein expression levels revealed a slight, although significant, reduction of the TMD-NICD in Cav1-KD cells compared to control cells (Figure 6, panel g).\u003c/p\u003e\n\u003cp\u003eBased on these findings, we propose a working model in which Caveolin-1 (Cav-1) regulates basal stem cell (BSC) differentiation by modulating the binding capacity of the Notch intracellular domain (NICD) to chromatin together with other transcriptional partners (Figure 6, panel h). This differential binding capacity may influence the transcriptional activity of Notch target genes, thereby affecting cell fate decisions. These results collectively suggest that Cav-1 plays a critical role in the regulation of Notch1 signaling and its subcellular localization, which may have significant implications for BSC differentiation and epithelial homeostasis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn our present study, we elucidate the intricate role of caveolin-1 (Cav-1) expression within the mouse airway epithelium, building upon previous findings in rat and human samples. Our investigation reveals specific associations between Cav-1 and distinct cell types, primarily BSCs and MCCs. Notably, while downregulation or absence of Cav-1 does not significantly impact BSC biology or epithelial barrier function, it emerges as a critical regulator of MCC differentiation and maturation. The precise mechanism underlying this function remains an intriguing puzzle.\u003c/p\u003e \u003cp\u003eWe explored two potential scenarios. The first scenario involves direct endocytosis modulation: Cav-1 may directly influence Notch signaling by participating in its subcellular distribution, mainly through endocytosis after activation at the plasma membrane. Although caveolae and clathrin-dependent endocytosis are well-established mechanisms for receptor internalization\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, no studies have yet demonstrated Cav-1-dependent Notch internalization. Nevertheless, our observations suggest a correlation between Cav-1 and Notch processing. Alternatively, a connection between γ-secretase and Cav-1 could be at play. References suggest that γ-secretase, situated within lipid rafts, affects Notch cleavage when altered altered\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. However, we did not find major significant differences in the subcellular distribution or Notch 1 processing.\u003c/p\u003e \u003cp\u003eIn the second scenario, we explored the possibility that Cav-1 could modulate the amount of Notch1 within the cells. In this case, we found that Cav-1 deficiency affects Notch1 protein levels. Although a significant decrease in Notch1 is observed, we do not believe this is the major mechanism regulating Notch1 activity. At this point, we consider that third-party mediation could also be occurring (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, panel h). Of course, this a less defined scenario. But, for instance, we note that chronic cigarette smoke exposure markedly increases Cav-1 in airway epithelial cells\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. This upregulation correlates with p53 induction and airway mucus hypersecretion\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Given our previous findings on p53/Mdm2 regulation of BSC differentiation, a potential link between MCCs and Cav-1 via p53 protein levels warrants exploration\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBeyond MCC determination, we uncover a phenotype related to MCC maturation and length. Previous studies highlight Cav-1\u0026rsquo;s roles in regulating ciliary membrane composition, function, and primary cilium length\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Intriguingly, depletion of Cav-1 expression promotes primary cilia formation\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. While our experimental model system focuses on airway MCCs, it\u0026rsquo;s essential to consider their broader roles. MCCs extend beyond the airway tract, contributing to ovule movement in the fallopian tubes and cerebrospinal fluid (CSF) flow within brain ventricles. Investigating whether Cav-1 affects reproduction and CSF dynamics in these contexts holds promise for understanding broader physiological implications.\u003c/p\u003e \u003cp\u003eIn summary, our study underscores the multifaceted impact of Cav-1 in airway epithelium, from MCC differentiation to ciliary function, and prompts further exploration of its intricate connections.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eIsolation of mouse tracheal epithelial cells (MTECs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe isolation of MTECs in wild-type and Cav1 constitutive knockout (provided by Miguel \u0026Aacute;ngel del Pozo) C57BL/6J adult mice was carried out using a modified version of the procedure described by You et al (2002). All animal studies have been performed in accordance with the National and European legislation (Spanish Royal Decree RD53/2013 and EU Directive 86/609/CEE as modified by 2003/65/ CE, respectively) and in accordance with the Institute of Laboratory Animal Resources (ILAR) for the protection of animals used for research. Experimental protocols were approved by the Bioethics Committee for Animal Experimentation of the University of Extremadura (Registry July 7, 2017). Firstly, the mice were euthanized, and the tracheas were dissected from the bronchial main to the larynx and placed in cold Ham\u0026rsquo;s F-12 medium supplemented with 1% of penicillin and streptomycin. After resection, vascular, fatty tissues, and muscle were removed in cold media, the clean tracheas were longitudinally excised and incubated with 1.5 mg/ml of pronase (Roche Molecular Biochemicals) in Ham\u0026rsquo;s F-12 medium with penicillin\u0026ndash;streptomycin for 16 hours at 4\u0026deg;C. Afterward, fetal bovine serum (FBS, Gibco) was added to a final concentration of 10%. The processed tracheas were discarded, and the isolated cells were collected by centrifugation at 500\u003cem\u003eg\u003c/em\u003e for 5 min at 4\u0026deg;C. Then, the cells were then incubated in F-12 medium containing 0.5 mg/ml pancreatic DNase I (Sigma-Aldrich) for 10 min and collected again by centrifugation. After this step, cells were seeded within PneumaCult-Ex Plus complete medium (StemCell) in primary tissue culture plates (Corning) for 4 h in 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C to remove fibroblasts. Finally, the supernatant was collected, and cells were seeded in 60-mm plates previously treated with type I rat tail collagen (Gibco) in 0.02 N acetic acid. We used a 60-mm plate for every three tracheas.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Culture and Differentiation of MTECs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMTECs were expanded in PneumaCult-Ex Plus medium (StemCell) at 37\u0026deg;C in 5% CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003euntil 70\u0026ndash;80% confluence (3-5 days after plating). Then, cells were detached from plates by two consecutive incubations, with 0.02% EDTA in PBS for 20 minutes at 37\u0026deg;C (to provide separation of cell-cell junction), and Accutase (Gibco) for 10 min at room temperature (to separate cells from plate). Following that, suspension cells were centrifugued at 1000\u003cem\u003eg\u003c/em\u003e for 5 min and pellet containing cells was resuspended in 1 mL of Pneumacult-Ex plus and counted. For MTECs differentiation, 9 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e were seeded in supported polyester porous membranes (Transwell 0.4 \u0026mu;m pores, Corning). The upper and lower chambers were filled with PneumaCult-Ex Plus medium, which was changed every 2 days. Cells proliferated during 4-6 days with media in the upper chamber and lower chamber with expansion media. Once the epithelial monolayer reached the medium was removed from the upper chamber and changed in the lower chamber to ALI medium (Air-Liquid Interface Medium, StemCell) to simulate the organization of respiratory tract tissue. ALI was replaced every 2 days and maintained until the end of the differentiation, 14 days (ALI 14).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLentivirus Production and Infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShort hairpin RNAs (shRNAs) against Cav1 were cloned in the pLKO.1 vector which drives the expression of shRNAs from the U6 human promoter and contains a puromycin-IRESmCherry selection cassette. The shRNA against luciferase was provided by Miguel Fidalgo\u0026rsquo;s laboratory. The shRNAs (Supplementary Table I) were cloned into the pLKO.1 using EcoRI and AgeI sites as described by Woo et al., (2019)\u003csup\u003e43\u003c/sup\u003e. Purified DNAs were transfected with the packaging and envelope vectors psPAX2 and PMD2.G into HEK-293T cells using polyethylenimine (PEI) (Sigma). Viruses were collected 48 and 72 h post-transfection and concentrated with Amicon Ultra-15 (Merck) by centrifugation at 3500\u003cem\u003eg\u003c/em\u003e for 30 min at 4\u0026deg;C. The approximate concentration of infectious virus particles was adjusted to 2 x 10\u003csup\u003e5\u003c/sup\u003e infectious virus particles/ml. We added a volume of 75 \u0026mu;l of concentrated virus in 3 ml of medium in the presence of 8 \u0026mu;g/ml polybrene (Sigma) in a 60-mm plate. Infected MTECs were selected with puromycin 3 \u0026mu;g/ml during 48 h. Finally, cells were expanded and plated for experiments. For caveolin-1 overexpression, we used pRRL-IRES-eGPF-hCav1 lentiviral vector (provided by Miguel A. Del Pozo\u0026apos;s laboratory) and followed the same protocol for lentivirus generation and MTEC infection as was used for knockdown generation. However, this vector does not allow for cell selection using puromycin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCulture of mouse airway organoids (AOs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMTECs were expanded and prepared as described previously to culture mouse airway organoids \u003csup\u003e17\u0026ndash;19\u003c/sup\u003e. When cells reached 60%-70% confluence, they were detached and prepared for organoid formation. For mouse organoids, 96-well plates were coated with 100 \u0026mu;L of 30% matrigel in PneumaCult-Ex Plus medium and incubated at 37\u0026deg;C for 20 minutes until gelation occurred. Then, 500 MTECs control or Cav1-KD were suspended in 100 \u0026mu;L of 2% matrigel in PneumaCult-Ex Plus and were seeded onto the matrigel layer. After 5 days, images of the airway organoids were captured using an EVOS Floid Cell Imaging Station (Invitrogen).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence (IF)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMTECs cultures were fixed in 4% paraformaldehyde (PFA) (PolyScience) for 10 min at room temperature, permeabilized in PBS-Triton X-100 0.1% for 10 min and blocked with PBS-Triton X-100 with 2% bovine serum albumin (BSA, Roche) for 1h. Primary antibodies were incubated overnight at 4\u0026deg;C in the blocking buffer. The primary antibodies used for immunofluorescence (IF) were anti-p63 (Abcam #ab124762, 1:200), anti-Foxj1 (Invitrogen, #14-9965-82, 1:200), anti-Scgb1a1 (Abcam, ab213203, 1:500), anti-Acetylated tubulin (Sigma, #T6793,1:100), anti-Caveolin1 (Cell Signaling, #3238, 1:400), anti-Centriolin (Santa Cruz, SE-365521, 1:100), anti-Notch1 (CellSignalling, #3608, 1:200) and anti-E-Cadherin (BD Biosciences, #610182, 1:1000). After the incubation with primary antibodies, the samples were washed five times in 0.1% Triton and then incubated for 1 hour with fluorescent secondary antibodies diluted in the same buffer. The secondary antibodies used for IF were, Alexa Fluor 488 anti-Rabbit (Invitrogen, #A21206, 1:500), and Alexa Fluor 594 anti-Mouse (Invitrogen, #A11005, 1:500). Nuclei were stained with DAPI at a concentration of 0.5 \u0026mu;g/mL (Thermo Fisher Scientific, #62248, 1:1000) and actin were stained with Phalloidin (Invitrogen, A12381, 1:500). Five additional washes were performed in PBS-Triton 0.1%. Slides were mounted in Vectashield (Vector Labs), and images were captured using an Olympus FV 1000 confocal microscope. After acquisition, the images were processed using ImageJ (Fiji) and Adobe Photoshop CC 2023.\u003c/p\u003e\n\u003cp\u003eFor the quantitative assessment of cells expressing a specific cell-type marker, we counted the number of positive cells in a minimum of four fields of view captured using a confocal microscope and then projected them into a single image. To determine the percentage of positive cells, DAPI staining was employed to calculate the total number of cells in a given field of view.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene expression analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated using the illustra RNAspin Mini kit (GE Healthcare). Following elution, for quantitative PCR (qPCR) 200-400 ng of RNA was subjected to reverse transcription using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer\u0026apos;s instructions. qPCR was performed to analyze gene expression using the oligonucleotides listed in Supplementary Table II and the Power SYBR Green PCR Master Mix (Applied Biosystems) as per the provided instructions. Melt curve analyses were performed to confirm the specificity of the PCR reactions. For RNA-seq analysis, libraries from RNA samples were built following Illumina recommendations at Novogene UK and then sequenced. Raw reads were pseudoaligned to \u003cem\u003eMus musculus\u003c/em\u003e (mm10) genome using kallisto\u003csup\u003e44\u003c/sup\u003e. An \u003cem\u003ead hoc\u003c/em\u003e script transformed these outputs to the specific format required to be analyzed by Gene Set Enrichment Analysis (GSEA)\u003csup\u003e45\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot (WB)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were lysed using ice-cold lysis buffer composed of 50 mM Tris-HCl (pH 7.5), 1 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 5 mM sodium pyrophosphate, 10 mM sodium fluoride, 0.27 M sucrose, 0.1 mM phenylmethylsulphonyl fluoride, 0.1% (v/v) 2-mercaptoethanol, 1% (v/v) Triton X-100, and complete protease inhibitor cocktail (Roche). The protein concentration was determined using the Bio-Rad protein assay, and 20 \u0026mu;g of protein was subjected to SDS-PAGE electrophoresis and transferred onto nitrocellulose membranes (Bio-Rad Laboratories). The membranes were then blocked with 5% dry milk in TBS-Tween (Tris-buffered saline containing 0.05% Tween-20) and incubated with primary antibodies overnight at 4\u0026deg;C. The primary antibodies for WB were diluted in blocking solution. The antibodies used anti-Caveolin1 (Cell Signaling, #3238, 1:1000), anti-GAPDH (Cell Singnaling, #5174, 1:1000), anti-Flotillin (Cell Signaling, #18634 1:1000), anti-Vinculin (Sigma, #V4505, 1:1000), anti-Notch1 (CellSignalling, #3608, 1:1000), anti-E-Cadherin (BD Biosciences, #610182, 1:1000) and anti-Actin (Sigma, #A2066, 1:1000) in blocking solution. The membranes were washed five times with TBS-Tween and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit-HRP, Cell Signaling, #7074; anti-mouse-HRP, Cell Signaling, #7076) in blocking solution (1:1000) for 1 h at room temperature. After additional washes with TBS-Tween, the proteins were visualized using a chemiluminescence detection system (SuperSignal West Dura, Thermo Fisher Scientific) and detected with iBright CL1000.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSubcellular Protein Fractionation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe subcellular fractionation kit (#78840, Thermo Fisher Scientific) was used for separation and preparation of cytoplasmic, membrane and chromatin-bound protein extracts. The procedure was performed according to the manufacturer\u0026rsquo;s instructions. Protein concentrations were determined, and equal amounts of protein (10 \u0026micro;g per fraction) were subjected to western blot analysis. For SDS-PAGE we have used 4-20% Mini-PROTEAN\u0026reg; TGX gels (Bio-Rad, #4561094) which allows for the separation of a wider range of protein size. The primary antibodies used were anti-Notch1 (CellSignalling, #3608, 1:1000), anti-GAPDH (CellSignalling, #2118, 1:2500) and Histone 3 (CellSignalling, #4499, 1:10000) incubated 1h at room temperature. The HRP-conjugated secondary antibodies that were used are anti-Rabbit-HRP (ThermoFisher, #31460, 1:10000) and anti-Mouse-HRP (ThermoFisher, #62-6520, 1:5000) incubated 1h at room temperature. Ponceau S staining was used to confirm homogeneous loading.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell cycle assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor quantitative assessment of the cell cycle, MTECs were detached as described above and fixed with cold 70% ethanol. Then, the cells were treated with RNAsa and PI overnight. 1 x 10\u003csup\u003e5\u0026nbsp;\u003c/sup\u003ecells were analyzed on the MACSQuant X flow cytometer (Miltenyi Biotec) and the data were processed in Flowlogic software (Miltenyi Biotec).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransepithelial resistance measurements (TEER)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe permeability of tight junction along the differentiation process of MTECs was tested by TEER in ALI 2, ALI 4, ALI 6 and ALI 14. First, the upper chamber of Transwells was washed once in PBS. Then, DMEM (Dulbecco\u0026apos;s Modified Eagle \u003cem\u003eMedium\u003c/em\u003e) at 37\u0026deg;C was added in the upper and lower chambers of Transwells and TEER was measured with an epithelial Ohm-voltmeter (EVOM3, WPI), positioning the electrode in the upper chamber. Resistance was recorded in kilohms (kW). For each time point, a fold change relative to control cells was calculated. Finally, graphs were obtained using Microsoft Excel and GraphPad Prism 8.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScanning electron microscopy (SEM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMTECs were differentiated up to ALI 14 and prepared for SEM by fixation with 2.5% glutaraldehyde for 1h and 30 min at 4\u0026deg;C, washed in 0.2 M cacodylate buffer and stained with 1% osmium tetroxide (Sigma) in 0.2 M cacodylate for 2 hours at 4\u0026deg;C. Cells were further dehydrated by incubation in increasing concentrations of ethanol (10%, 30%, 50%, 70%, 90% and 100%) for 20 minutes each. Finally, samples were dried using the technique of liquid carbon dioxide critical point, gold sputter coated, and visualized in a Quanta 3D FEG (ESEM-FIB; FEI Company) electron microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCilia beating frequency measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCilia beating frequency was measured in ALI 14 MTECs wild-type and Cav1 knockdown. The upper chamber of 12-mm transwells was incubated with 5 \u0026mu;L of Dynabeads Streptavidin C1 (Invitrogen) in 300 \u0026mu;L of Ca\u003csup\u003e2+\u003c/sup\u003e/Mg\u003csup\u003e2+\u003c/sup\u003e-PBS for 10 min at 37\u0026deg;C. After, the medium from the upper chamber was removed and transwells were incubated for 1h and 30 min at 37\u0026deg;C. Before movie recording, 100 \u0026mu;L of Ca\u003csup\u003e2+\u003c/sup\u003e/Mg\u003csup\u003e2+\u003c/sup\u003e-PBS at 37\u0026deg;C was added on top of the transwell. Cilia beating movie was obtained using Motic AE20 microscope and an iPhone 14 at 240 frames per second (fps), and a custom MATLAB function was developed to assess the movement of the beads as well as to quantify the cilia beating frequencies using FFT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCilia length measurement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCilia lengths were measured using Fiji software. Images were acquired at 8,000x magnification using SEM, providing sufficient resolution for detailed morphological analysis. To convert pixel measurements to micrometers (\u0026micro;m), a known scale embedded in the images was used. The Set Scale function in the Analyze menu of Fiji was employed, where the known scale length was entered, and the unit of measurement was set to micrometers. Measurements of the lengths of the structures were conducted using the Segmented Lines tool, which allowed tracing over morphological features in the images to record their lengths in micrometers. For each phenotype (wild type and Cav1-KO) analyzed, at least 50 independent measurements were performed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromatin immunoprecipitation sequencing (ChIP-seq)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo analyze the binding of NICD to different promoters, ChIP was performed as described above\u003csup\u003e46,47\u003c/sup\u003e. After, libraries for ChIP-seq were built following Illumina recommendations at STAB VIDA. 20M reads were obtained per input and IP sample, respectively. They were aligned to the \u003cem\u003eM. musculus\u003c/em\u003e (mm10) genome using BWA, and peaks were retrieved with MACS2\u003csup\u003e48\u003c/sup\u003e. ChIPseeker\u003csup\u003e49\u003c/sup\u003e and HOMER\u003csup\u003e50\u003c/sup\u003e were used to annotate the peaks, and custom MATLAB functions were developed for other analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSingle cell RNA sequencing (scRNA-seq)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe used the GSE102580 dataset\u003csup\u003e14\u003c/sup\u003e from NCBI GEO to analyze the cell-type expression of specific genes in mouse airway epithelia. A custom MATLAB function was developed in order to generate a dot plot showing average expression and cell percentage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferent experimental groups (GraphPad Prism) were compared against control conditions by means of two-tailed \u003cem\u003et\u003c/em\u003e-tests. The n value in figure legends for WB and qPCR represents independent samples which are single transwells coming from at least four differentiation sets. In qPCR experiments, each independent sample is the result of 2-3 technical replicates. Relative expression was obtained using EIF1a as a housekeeping gene.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData from NGS experiments are publicly available at figshare server (10.6084/m9.figshare.26417137).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConfocal microscopy and scanning electron microscopy were performed at the UEX microscopy core facilities. This work was supported by BFU2017-85547-P, TED2021-130560B-I00 and PID2021-126905NB-I00 grants from the Ministry of Economy, IB18014 from Junta de Extremadura to J.M. C-G and GR21140 from Junta de Extremadura to S.M-N. This study was also supported by grants to M.A.d.P. from the Spanish Ministry of Science and Innovation (MICIIN)/Agencia Estatal de Investigaci\u0026oacute;n (AEI)/European Regional Development Fund (ARDF/FEDER) \u0026ldquo;A way to make Europe\u0026rdquo; PID2020-118658RB-I00 and PDC2021-121572-I00, and Fundaci\u0026oacute;n Obra Social La Caixa (AtheroConvergence, HR20-00075. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovaci\u0026oacute;n (MCIN) and the Pro CNIC Foundation) and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). S.G-J. was a recipient of a Fellowship from the Universidad de Extremadura. S. D-Ch and C.M.N-Q were recipients of a Fellowship from Junta de Extremadura. All Spanish funding is co-sponsored by the European Union FEDER program. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.O.-G., G. C.-L., J.F. B.-L., N. V.-P., A. D.-P., M. T.-D., and M. C.-M. performed all of the experiments. G. C.-L. and D.A.B. designed and performed the data analysis. S.M.-N., A.C. R., M. A.P. and J.M.C.-G. designed the experiments, analyzed data and wrote the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure and competing interest statement\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003c/strong\u003eThe authors declare no competing financial interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHewitt, R. 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Recent studies have illuminated the intricate interplay between basal cell self-renewal, differentiation, and epithelial homeostasis following injury. Notably, expression of caveolin-1 (Cav-1) has been linked to specific cell types within the airway epithelium, primarily basal stem cells (BSCs) and multiciliated cells (MCCs). Despite its specific expression, the precise function of Cav-1 in BSC differentiation remains largely enigmatic. In this study, we investigate Cav-1\u0026rsquo;s function within the airway epithelium, exploring the molecular mechanisms underlying BSC differentiation into MCCs and secretory cells (SCs). Our results reveal limited Cav-1 expression in mouse airway epithelial BSCs, with additional enrichment observed in MCCs. Notably, deficiency of Cav-1 accelerates MCC differentiation and maturation. Additionally, we found that Cav-1 downregulation dramatically affects Notch intracellular domain (NICD) transcriptional activity. That leads us to propose that Cav-1 participates indirectly in a transcriptional program orchestrated by NICD, thereby modulating both BSC differentiation and MCC maturation.\u003c/p\u003e","manuscriptTitle":"Caveolin-1 Modulates Notch Transcriptional Activity During Respiratory Multiciliated Cell Maturation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-07 08:10:49","doi":"10.21203/rs.3.rs-6189425/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-24T06:46:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-17T15:21:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"35266639442480007452546421522077900509","date":"2025-07-05T09:26:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-17T16:36:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"58373604984550277860364981165191605690","date":"2025-04-14T03:28:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"266942297477313063076468810266674522911","date":"2025-03-26T10:31:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-25T19:32:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-25T19:13:12+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-03-19T15:58:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-19T02:10:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-09T15:13:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d9d74827-0139-47eb-826c-b27b8de38028","owner":[],"postedDate":"April 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46466541,"name":"Biological sciences/Stem cells"},{"id":46466542,"name":"Biological sciences/Stem cells/Adult stem cells"},{"id":46466543,"name":"Biological sciences/Stem cells/Stem cell differentiation"}],"tags":[],"updatedAt":"2026-02-16T16:13:39+00:00","versionOfRecord":{"articleIdentity":"rs-6189425","link":"https://doi.org/10.1038/s41598-026-40201-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-02-15 15:58:48","publishedOnDateReadable":"February 15th, 2026"},"versionCreatedAt":"2025-04-07 08:10:49","video":"","vorDoi":"10.1038/s41598-026-40201-6","vorDoiUrl":"https://doi.org/10.1038/s41598-026-40201-6","workflowStages":[]},"version":"v1","identity":"rs-6189425","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6189425","identity":"rs-6189425","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}