Upregulation of caveolae-associated structural proteins in the hair follicle bulge of lichen planopilaris and frontal fibrosing alopecia.

Lichen planopilaris (LPP) and frontal fibrosing alopecia (FFA) are primary cicatricial alopecia that cause a major impact on quality of life due to irreversible hair loss and symptoms as itching, burning and pain. They are characterized by permanent loss of hair follicle stem cells (HFSCs) by pathomechanisms still poorly understood, resulting in poor efficacy of currently available treatments. Caveolae are flask-shaped lipid rafts invaginated within the plasma membrane of multiple cell types. Although their role in the HF physiology and pathophysiology is relatively unknown, we have previously demonstrated that the primary structural component of caveolae (caveolin-1 or Cav1) is upregulated in FFA. Thus, we propose to investigate the expression and localization of caveolae-associated structural proteins (Cav1, Cav2, and Cavin-1) and HFSCs (identified by K15) in both LPP and FFA. We analyzed 4 patients with LPP biopsied in affected and non-affected (NA) scalp, 4 patients with FFA biopsied in affected scalp and 4 healthy controls. Affected scalp of LPP and FFA demonstrated increased levels of Cav1 and Cavin-1 compared with HC and LPP-NA. Moreover, Cav1, Cav2 and Cavin1 all exhibit high colocalization with K15 and their expression appears to be negatively correlated, supporting the hypothesis that these proteins are important players in LPP/FFA and may serve as therapeutic targets in future treatments.

Scaffolds and the scaffolding domain: an alternative paradigm for caveolin-1 signaling.

Caveolin-1 (Cav1) is a 22 kDa intracellular protein that is the main protein constituent of bulb-shaped membrane invaginations known as caveolae. Cav1 can be also found in functional non-caveolar structures at the plasma membrane called scaffolds. Scaffolds were originally described as SDS-resistant oligomers composed of 10-15 Cav1 monomers observable as 8S complexes by sucrose velocity gradient centrifugation. Recently, cryoelectron microscopy (cryoEM) and super-resolution microscopy have shown that 8S complexes are interlocking structures composed of 11 Cav1 monomers each, which further assemble modularly to form higher-order scaffolds and caveolae. In addition, Cav1 can act as a critical signaling regulator capable of direct interactions with multiple client proteins, in particular, the endothelial nitric oxide (NO) synthase (eNOS), a role believed by many to be attributable to the highly conserved and versatile scaffolding domain (CSD). However, as the CSD is a hydrophobic domain located by cryoEM to the periphery of the 8S complex, it is predicted to be enmeshed in membrane lipids. This has led some to challenge its ability to interact directly with client proteins and argue that it impacts signaling only indirectly via local alteration of membrane lipids. Here, based on recent advances in our understanding of higher-order Cav1 structure formation, we discuss how the Cav1 CSD may function through both lipid and protein interaction and propose an alternate view in which structural modifications to Cav1 oligomers may impact exposure of the CSD to cytoplasmic client proteins, such as eNOS.

Caveolin-1 protects endothelial cells from extensive expansion of transcellular tunnel by stiffening the plasma membrane.

Large transcellular pores elicited by bacterial mono-ADP-ribosyltransferase (mART) exotoxins inhibiting the small RhoA GTPase compromise the endothelial barrier. Recent advances in biophysical modeling point toward membrane tension and bending rigidity as the minimal set of mechanical parameters determining the nucleation and maximal size of transendothelial cell macroaperture (TEM) tunnels induced by bacterial RhoA-targeting mART exotoxins. We report that cellular depletion of caveolin-1, the membrane-embedded building block of caveolae, and depletion of cavin-1, the master regulator of caveolae invaginations, increase the number of TEMs per cell. The enhanced occurrence of TEM nucleation events correlates with a reduction in cell height due to the increase in cell spreading and decrease in cell volume, which, together with the disruption of RhoA-driven F-actin meshwork, favor membrane apposition for TEM nucleation. Strikingly, caveolin-1 specifically controls the opening speed of TEMs, leading to their dramatic 5.4-fold larger widening. Consistent with the increase in TEM density and width in siCAV1 cells, we record a higher lethality in CAV1 KO mice subjected to a catalytically active mART exotoxin targeting RhoA during staphylococcal bloodstream infection. Combined theoretical modeling with independent biophysical measurements of plasma membrane bending rigidity points toward a specific contribution of caveolin-1 to membrane stiffening in addition to the role of cavin-1/caveolin-1-dependent caveolae in the control of membrane tension homeostasis.

Nutrient scavenging-fueled growth in pancreatic cancer depends on caveolae-mediated endocytosis under nutrient-deprived conditions.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by its nutrient-scavenging ability, crucial for tumor progression. Here, we investigated the roles of caveolae-mediated endocytosis (CME) in PDAC progression. Analysis of patient data across diverse datasets revealed a strong association of high caveolin-1 (Cav-1) expression with higher histologic grade, the most aggressive PDAC molecular subtypes, and worse clinical outcomes. Cav-1 loss markedly promoted longer overall and tumor-free survival in a genetically engineered mouse model. Cav-1-deficient tumor cell lines exhibited significantly reduced proliferation, particularly under low nutrient conditions. Supplementing cells with albumin rescued the growth of Cav-1-proficient PDAC cells, but not in Cav-1-deficient PDAC cells under low glutamine conditions. In addition, Cav-1 depletion led to significant metabolic defects, including decreased glycolytic and mitochondrial metabolism, and downstream protein translation signaling pathways. These findings highlight the crucial role of Cav-1 and CME in fueling pancreatic tumorigenesis, sustaining tumor growth, and promoting survival through nutrient scavenging.

Listeria adhesion protein orchestrates caveolae-mediated apical junctional remodeling of epithelial barrier for Listeria monocytogenes translocation.

The cellular junctional architecture remodeling by Listeria adhesion protein-heat shock protein 60 (LAP-Hsp60) interaction for Listeria monocytogenes (Lm) passage through the epithelial barrier is incompletely understood. Here, using the gerbil model, permissive to internalin (Inl) A/B-mediated pathways like in humans, we demonstrate that Lm crosses the intestinal villi at 48 h post-infection. In contrast, the single isogenic (lap- or ΔinlA) or double (lap-ΔinlA) mutant strains show significant defects. LAP promotes Lm translocation via endocytosis of cell-cell junctional complex in enterocytes that do not display luminal E-cadherin. In comparison, InlA facilitates Lm translocation at cells displaying apical E-cadherin during cell extrusion and mucus expulsion from goblet cells. LAP hijacks caveolar endocytosis to traffic integral junctional proteins to the early and recycling endosomes. Pharmacological inhibition in a cell line and genetic knockout of caveolin-1 in mice prevents LAP-induced intestinal permeability, junctional endocytosis, and Lm translocation. Furthermore, LAP-Hsp60-dependent tight junction remodeling is also necessary for InlA access to E-cadherin for Lm intestinal barrier crossing in InlA-permissive hosts. IMPORTANCE: Listeria monocytogenes (Lm) is a foodborne pathogen with high mortality (20%-30%) and hospitalization rates (94%), particularly affecting vulnerable groups such as pregnant women, fetuses, newborns, seniors, and immunocompromised individuals. Invasive listeriosis involves Lm's internalin (InlA) protein binding to E-cadherin to breach the intestinal barrier. However, non-functional InlA variants have been identified in Lm isolates, suggesting InlA-independent pathways for translocation. Our study reveals that Listeria adhesion protein (LAP) and InlA cooperatively assist Lm entry into the gut lamina propria in a gerbil model, mimicking human listeriosis in early infection stages. LAP triggers caveolin-1-mediated endocytosis of critical junctional proteins, transporting them to early and recycling endosomes, facilitating Lm passage through enterocytes. Furthermore, LAP-Hsp60-mediated junctional protein endocytosis precedes InlA's interaction with basolateral E-cadherin, emphasizing LAP and InlA's cooperation in enhancing Lm intestinal translocation. This understanding is vital in combating the severe consequences of Lm infection, including sepsis, meningitis, encephalitis, and brain abscess.

Allosteric inhibitor of SHP2 enhances macrophage endocytosis and bacteria elimination by increasing caveolae activation and protects against bacterial sepsis.

The uncontrolled bacterial infection-induced cytokine storm and sequential immunosuppression are commonly observed in septic patients, which indicates that the activation of phagocytic cells and the efficient and timely elimination of bacteria are crucial for combating bacterial infections. However, the role of dysregulated immune cells and their disrupted function in sepsis remains unclear. Here, we found that macrophages exhibited the impaired endocytosis capabilities in sepsis by Single-cell RNA sequencing and bulk RNA sequencing. Caveolae protein Caveolin-1 (Cav-1) of macrophages was inactivated by SHP2 rapidly during Escherichia coli (E.coli) infection. Allosteric inhibitor of SHP2 effectively maintains Cav-1 phosphorylation to enhance macrophage to endocytose and eliminate bacteria. Additionally, TLR4 endocytosis of macrophage was also enhanced upon E.coli infection by SHP099, inducing an increased and rapidly resolved inflammatory response. In vivo, pretreatment or posttreatment with inhibitor of SHP2 significantly reduced the bacterial burden in organs and mortality of mice subjected E.coli infection or CLP-induced sepsis. The cotreatment of inhibitor of SHP2 with an antibiotic conferred complete protection against mortality in mice. Our findings suggest that Cav-1-mediated endocytosis and bacterial elimination may play a critical role in the pathogenesis of sepsis, highlighting inhibitor of SHP2 as a potential therapeutic agent for sepsis.

C-kit controls blood-brain barrier permeability by regulating caveolae-mediated transcytosis after chronic cerebral hypoperfusion.

Blood-brain barrier (BBB) dysfunction plays a pivotal role in the pathology of chronic cerebral hypoperfusion (CCH)-related neurodegenerative diseases. Continuous endothelial cells (EC) that line the blood vessels of the brain are important components of the BBB to strictly control the flow of substances and maintain the homeostatic environment of the brain. However, the molecular mechanisms from the perspective of EC-induced BBB dysfunction after CCH are largely unknown. In this study, the BBB function was assessed using immunostaining and transmission electron microscopy. The EC dysfunction profile was screened by using EC enrichment followed by RNA sequencing. After identified the key EC dysfunction factor, C-kit, we used the C-kit inhibition drug (imatinib) and C-kit down-regulation method (AAV-BR1-C-kit shRNA) to verify the role of C-kit on BBB integrity and EC transcytosis after CCH. Furthermore, we also activated C-kit with stem cell factor (SCF) to observe the effects of C-kit on BBB following CCH. We explored that macromolecular proteins entered the brain mainly through EC transcytosis after CCH and caused neuronal loss. Additionally, we identified receptor tyrosine kinase C-kit as a key EC dysfunction molecule. Furthermore, the pharmacological inhibition of C-kit with imatinib counteracted BBB leakage by reducing caveolae-mediated transcytosis. Moreover, treatment with AAV-BR1-C-kit shRNA, which targets brain EC to inhibit C-kit expression, also ameliorated BBB leakage by reducing caveolae-mediated transcytosis. Furthermore, the SCF increased the permeability of the BBB by actively increasing caveolae-mediated transcytosis. This study provides evidence that C-kit is a key BBB permeability regulator through caveolae-mediated transcytosis in EC after CCH.

Differential roles of eNOS in late effects of VEGF-A on hyperpermeability in different types of endothelial cells.

Vascular endothelial growth factor (VEGF)-A induces endothelial hyperpermeability, but the molecular pathways remain incompletely understood. Endothelial nitric oxide synthase (eNOS) regulates acute effects of VEGF-A on permeability of endothelial cells (ECs), but it remains unknown whether and how eNOS regulates late effects of VEGF-A-induced hyperpermeability. Here we show that VEGF-A induces hyperpermeability via eNOS-dependent and eNOS-independent mechanisms at 2 days after VEGF-A stimulation. Silencing of expression of the eNOS gene (NOS3) reduced VEGF-A-induced permeability for dextran (70 kDa) and 766 Da-tracer in human dermal microvascular ECs (HDMVECs), but not in human retinal microvascular ECs (HRECs) and human umbilical vein ECs (HUVECs). However, silencing of NOS3 expression in HRECs increased permeability to dextran, BSA and 766 Da-tracer in the absence of VEGF-A stimulation, suggesting a barrier-protective function of eNOS. We also investigated how silencing of NOS3 expression regulates the expression of permeability-related transcripts, and found that NOS3 silencing downregulates the expression of PLVAP, a molecule associated with trans-endothelial transport via caveolae, in HDMVECs and HUVECs, but not in HRECs. Our findings underscore the complexity of VEGF-A-induced permeability pathways in ECs and the role of eNOS therein, and demonstrate that different pathways are activated depending on the EC phenotype.

Unraveling the Cave: A Seventy-Year Journey into the Caveolar Network, Cellular Signaling, and Human Disease.

In the mid-1950s, a groundbreaking discovery revealed the fascinating presence of caveolae, referred to as flask-shaped invaginations of the plasma membrane, sparking renewed excitement in the field of cell biology. Caveolae are small, flask-shaped invaginations in the cell membrane that play crucial roles in diverse cellular processes, including endocytosis, lipid homeostasis, and signal transduction. The structural stability and functionality of these specialized membrane microdomains are attributed to the coordinated activity of scaffolding proteins, including caveolins and cavins. While caveolae and caveolins have been long appreciated for their integral roles in cellular physiology, the accumulating scientific evidence throughout the years reaffirms their association with a broad spectrum of human disorders. This review article aims to offer a thorough account of the historical advancements in caveolae research, spanning from their initial discovery to the recognition of caveolin family proteins and their intricate contributions to cellular functions. Furthermore, it will examine the consequences of a dysfunctional caveolar network in the development of human diseases.

Adiposomes from Obese-Diabetic Individuals Promote Endothelial Dysfunction and Loss of Surface Caveolae.

Glycosphingolipids (GSLs) are products of lipid glycosylation that have been implicated in the development of cardiovascular diseases. In diabetes, the adipocyte microenvironment is characterized by hyperglycemia and inflammation, resulting in high levels of GSLs. Therefore, we sought to assess the GSL content in extracellular vesicles derived from the adipose tissues (adiposomes) of obese-diabetic (OB-T2D) subjects and their impact on endothelial cell function. To this end, endothelial cells were exposed to adiposomes isolated from OB-T2D versus healthy subjects. Cells were assessed for caveolar integrity and related signaling, such as Src-kinase and caveolin-1 (cav-1) phosphorylation, and functional pathways, such as endothelial nitric oxide synthase (eNOS) activity. Compared with adiposomes from healthy subjects, OB-T2D adiposomes had higher levels of GSLs, especially LacCer and GM3; they promoted cav-1 phosphorylation coupled to an obvious loss of endothelial surface caveolae and induced eNOS-uncoupling, peroxynitrite generation, and cav-1 nitrosylation. These effects were abolished by Src kinase inhibition and were not observed in GSL-depleted adiposomes. At the functional levels, OB-T2D adiposomes reduced nitric oxide production, shear response, and albumin intake in endothelial cells and impaired flow-induced dilation in healthy arterioles. In conclusion, OB-T2D adiposomes carried a detrimental GSL cargo that disturbed endothelial caveolae and the associated signaling.

Role of rafts in neurological disorders.

INTRODUCTION: Rafts are protein-lipid structural nanodomains involved in efficient signal transduction and the modulation of physiological processes of the cell plasma membrane. Raft disruption in the nervous system has been associated with a wide range of disorders. DEVELOPMENT: We review the concept of rafts, the nervous system processes in which they are involved, and their role in diseases such as Parkinson's disease, Alzheimer disease, and Huntington disease. CONCLUSIONS: Based on the available evidence, preservation and/or reconstitution of rafts is a promising treatment strategy for a wide range of neurological disorders.

A mechanosensitive caveolae-invadosome interplay drives matrix remodelling for cancer cell invasion.

Invadosomes and caveolae are mechanosensitive structures that are implicated in metastasis. Here, we describe a unique juxtaposition of caveola clusters and matrix degradative invadosomes at contact sites between the plasma membrane of cancer cells and constricting fibrils both in 2D and 3D type I collagen matrix environments. Preferential association between caveolae and straight segments of the fibrils, and between invadosomes and bent segments of the fibrils, was observed along with matrix remodelling. Caveola recruitment precedes and is required for invadosome formation and activity. Reciprocally, invadosome disruption results in the accumulation of fibril-associated caveolae. Moreover, caveolae and the collagen receptor ß1 integrin co-localize at contact sites with the fibrils, and integrins control caveola recruitment to fibrils. In turn, caveolae mediate the clearance of ß1 integrin and collagen uptake in an invadosome-dependent and collagen-cleavage-dependent mechanism. Our data reveal a reciprocal interplay between caveolae and invadosomes that coordinates adhesion to and proteolytic remodelling of confining fibrils to support tumour cell dissemination.

Polymer-Initiating Caveolae-Mediated Endocytosis and GSH-Responsive MiR-34a Gene Delivery System for Enhanced Orthotopic Triple Negative Breast Cancer Therapy.

Gene therapy based on miRNAs has broad application prospects in the treatment of tumors. However, due to degradation and ineffective release during intracellular transport, current gene delivery vectors used for miRNAs limited their actual transfection efficiency. This study develops a novel nonviral vector PEI-SPDP-Man (PSM) that can simultaneously target cellular uptake pathways and intracellular responsive release for miR-34a. PSM is synthesized by connected mannitol (Man) to branched polyethylenimine (PEI) using a disulfide bond. The prepared PSM/miR-34a gene delivery system can induce and enter to tumor cells through caveolae-mediated endocytosis to reduce the degradation of miR-34a in lysosomes. The disulfide bond is sensed at high concentration of glutathione (GSH) in the tumor cells and miR-34a is released, thereby reducing the expression of Bcl-2 and CD44 to suppress the proliferation and invasion of tumor cells. In vitro and in vivo experiments show that through the targeted cellular uptake and the efficient release of miR-34a, an effective antitumor and antimetastasis profiles for the treatment of orthotopic triple negative breast cancer (TNBC) are achieved. This strategy of controlling intracellular transport pathways by targeting cellular uptake pathways in the gene therapy is an approach that could be developed for highly effective cancer therapy.

Senolytics prevent caveolar CaV 3.2-RyR axis malfunction in old vascular smooth muscle.

Aging is a major risk factor for cardiovascular diseases. Our previous studies demonstrate that aging impairs the caveolar T-type CaV 3.2-RyR axis for extracellular Ca2+ influx to trigger Ca2+ sparks in vascular smooth muscle cells (VSMCs). We hypothesize that the administration of senolytics, which can selectively clear senescent cells, could preserve the caveolar CaV 3.2-RyR axis in aging VSMCs. In this study, 10-month-old mice were administered the senolytics cocktail consisting of dasatinib (5 mg/kg) and quercetin (50 mg/kg) or vehicle bi-weekly for 4 months. Using VSMCs from mouse mesenteric arteries, we found that Ca2+ sparks were diminished after caveolae disruption by methyl-ß-cyclodextrin (10 mM) in cells from D + Q treated but not vehicle-treated 14-month-old mice. D + Q treatment promoted the expression of CaV 3.2 in 14-month-old mesenteric arteries. Structural analysis using electron tomography and immunofluorescence staining revealed the remodeling of caveolae and co-localization of CaV 3.2-Cav-1 in D + Q treatment aged mesenteric arteries. In keeping with theoretical observations, Cav 3.2 channel inhibition by Ni2+ (50 µM) suppressed Ca2+ in VSMCs from the D + Q group, with no effect observed in vehicle-treated arteries. Our study provides evidence that age-related caveolar CaV 3.2-RyR axis malfunction can be alleviated by pharmaceutical intervention targeting cellular senescence. Our findings support the potential of senolytics for ameliorating age-associated cardiovascular disease.

Caveolin-1 and lipids: Association and their dualism in oncogenic regulation.

Caveolin-1 (Cav-1) is a structural protein of caveolae that functions as a molecular organizer for different cellular functions including endocytosis and cellular signaling. Cancer cells take advantage of the physical position of Cav-1, as it can communicate with extracellular matrix, help to organize growth factor receptors, redistribute cholesterol and glycosphingolipids, and finally transduce signals within the cells for oncogenesis. Recent studies emphasize the exceeding involvement of Cav-1 with different lipid bodies and in altering the metabolism, especially lipid metabolism. However, the association of Cav-1 with different lipid bodies like lipid rafts, lipid droplets, cholesterols, sphingolipids, and fatty acids is remarkably dynamic. The lipid-Cav-1 alliance plays a dual role in carcinogenesis. Both cancer progression and regression are modified and affected by the type of lipid molecule's association with Cav-1. Accordingly, this Cav-1-lipid cooperation exemplifies a cancer-type-specific treatment strategy for a better prognosis of the disease. In this review, we first present Cav-1 as an oncogenic molecule and its communication via lipid raft. We discussed the involvement of Cav-1 with lipid droplets, Cholesterol, sphingolipids, gangliosides, and ceramides. Further, we describe the Cav-1-mediated altered Fatty acid metabolism in cancer and the strategic therapeutic approaches toward Cav-1 targeting.

Understanding the Role of Caveolae in Oxygen Buffering: The Effect of Membrane Curvature.

The "oxygen paradox" can be explained as two opposing biological processes with oxygen (O2) as a reactant. On the one hand, oxygen is essential to aerobic metabolism, powering oxidative phosphorylation in mitochondria. On the other hand, an excess supply of oxygen will generate reactive species which are harmful for the cell. In healthy tissues, the first process must be maximized relative to the second one. We have hypothesized that curved and cholesterol-enriched membrane invaginations called caveolae help maintain the proper oxygen level by taking up oxygen and attenuating its release to the mitochondria. The mechanism by which caveolae may help to buffer the oxygen level in cells is still unclear. Here, we aim to assess how structural aspects of caveolae, the curvature of the membrane, influence the local oxygen abundance and the membrane partitioning. We have modelled a flat bilayer and a liposome composed of dipalmitoylphosphatidylcholine (DPPC), using molecular dynamics simulation. Associated changes in the membrane-level oxygen partition coefficient and free energy profiles will be presented.

Caveolae-associated cAMP/Ca2+-mediated mechano-chemical signal transduction in mouse atrial myocytes.

Caveolae are tiny invaginations in the sarcolemma that buffer extra membrane and contribute to mechanical regulation of cellular function. While the role of caveolae in membrane mechanosensation has been studied predominantly in non-cardiomyocyte cells, caveolae contribution to cardiac mechanotransduction remains elusive. Here, we studied the role of caveolae in the regulation of Ca2+ signaling in atrial cardiomyocytes. In Langendorff-perfused mouse hearts, atrial pressure/volume overload stretched atrial myocytes and decreased caveolae density. In isolated cells, caveolae were disrupted through hypotonic challenge that induced a temporal (<10 min) augmentation of Ca2+ transients and caused a rise in Ca2+ spark activity. Similar changes in Ca2+ signaling were observed after chemical (methyl-ß-cyclodextrin) and genetic ablation of caveolae in cardiac-specific conditional caveolin-3 knock-out mice. Acute disruption of caveolae, both mechanical and chemical, led to the elevation of cAMP level in the cell interior, and cAMP-mediated augmentation of protein kinase A (PKA)-phosphorylated ryanodine receptors (at Ser2030 and Ser2808). Caveolae-mediated stimulatory effects on Ca2+ signaling were abolished via inhibition of cAMP production by adenyl cyclase antagonists MDL12330 and SQ22536, or reduction of PKA activity by H-89. A compartmentalized mathematical model of mouse atrial myocytes linked the observed changes to a microdomain-specific decrease in phosphodiesterase activity, which disrupted cAMP signaling and augmented PKA activity. Our findings add a new dimension to cardiac mechanobiology and highlight caveolae-associated cAMP/PKA-mediated phosphorylation of Ca2+ handling proteins as a novel component of mechano-chemical feedback in atrial myocytes.

Caveola mechanotransduction reinforces the cortical cytoskeleton to promote epithelial resilience.

As physical barriers, epithelia must preserve their integrity when challenged by mechanical stresses. Cell-cell junctions linked to the cortical cytoskeleton play key roles in this process, often with mechanotransduction mechanisms that reinforce tissues. Caveolae are mechanosensitive organelles that buffer tension via disassembly. Loss of caveolae, through caveolin-1 or cavin1 depletion, causes activation of PtdIns(4, 5)P2 signaling, recruitment of FMNL2 formin, and enhanced-cortical actin assembly. How this equates to physiological responses in epithelial cells containing endogenous caveolae is unknown. Here we examined the effect of mechanically inducing acute disassembly of caveolae in epithelia. We show that perturbation of caveolae, through direct mechanical stress, reinforces the actin cortex at adherens junctions. Increasing interactions with membrane lipids by introducing multiple phosphatidylserine-binding undecad cavin1 (UC1) repeat domains into cavin1 rendered caveolae more stable to mechanical stimuli. This molecular stabilization blocked cortical reinforcement in response to mechanical stress. Cortical reinforcement elicited by the mechanically induced disassembly of caveolae increased epithelial resilience against tensile stresses. These findings identify the actin cortex as a target of caveola mechanotransduction that contributes to epithelial integrity.

Cell surface plasticity in response to shape change in the whole organism.

Plasma membrane rupture can result in catastrophic cell death. The skeletal muscle fiber plasma membrane, the sarcolemma, provides an extreme example of a membrane subject to mechanical stress since these cells specifically evolved to generate contraction and movement. A quantitative model correlating ultrastructural remodeling of surface architecture with tissue changes in vivo is required to understand how membrane domains contribute to the shape changes associated with tissue deformation in whole animals. We and others have shown that loss of caveolae, small invaginations of the plasma membrane particularly prevalent in the muscle sarcolemma, renders the plasma membrane more susceptible to rupture during stretch.1,2,3 While it is thought that caveolae are able to flatten and be absorbed into the bulk membrane to buffer local membrane expansion, a direct demonstration of this model in vivo has been unachievable since it would require measurement of caveolae at the nanoscale combined with detailed whole-animal morphometrics under conditions of perturbation. Here, we describe the development and application of the "active trapping model" where embryonic zebrafish are immobilized in a curved state that mimics natural body axis curvature during an escape response. The model is amenable to multiscale, multimodal imaging including high-resolution whole-animal three-dimensional quantitative electron microscopy. Using the active trapping model, we demonstrate the essential role of caveolae in maintaining sarcolemmal integrity and quantify the specific contribution of caveolar-derived membrane to surface expansion. We show that caveolae directly contribute to an increase in plasma membrane surface area under physiologically relevant membrane deformation conditions.

Early proteostasis of caveolins synchronizes trafficking, degradation, and oligomerization to prevent toxic aggregation.

Caveolin-1 (CAV1) and CAV3 are membrane-sculpting proteins driving the formation of the plasma membrane (PM) caveolae. Within the PM mosaic environment, caveola assembly is unique as it requires progressive oligomerization of newly synthesized caveolins while trafficking through the biosynthetic-secretory pathway. Here, we have investigated these early events by combining structural, biochemical, and microscopy studies. We uncover striking trafficking differences between caveolins, with CAV1 rapidly exported to the Golgi and PM while CAV3 is initially retained in the endoplasmic reticulum and laterally moves into lipid droplets. The levels of caveolins in the endoplasmic reticulum are controlled by proteasomal degradation, and only monomeric/low oligomeric caveolins are exported into the cis-Golgi with higher-order oligomers assembling beyond this compartment. When any of those early proteostatic mechanisms are compromised, chemically or genetically, caveolins tend to accumulate along the secretory pathway forming non-functional aggregates, causing organelle damage and triggering cellular stress. Accordingly, we propose a model in which disrupted proteostasis of newly synthesized caveolins contributes to pathogenesis.