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.

The adaptable caveola coat generates a plasma membrane sensory system.

Caveolae are atypical plasma membrane invaginations that take part in lipid sorting and regulation of oxidative and mechanical plasma membrane stress. Caveola formation requires caveolin, cavin, and specific lipid types. The recent advances in understanding the structure and assembly of caveolin and cavin complexes within the membrane context have clarified the fundamental processes underlying caveola biogenesis. In addition, the curvature of the caveola membrane is controlled by the regulatory proteins EHD2, pacsin2, and dynamin2, which also function to restrain the scission of caveolae from the plasma membrane (PM). Here, this is integrated with novel insights on caveolae as lipid and mechanosensing complexes that can dynamically flatten or disassemble to counteract mechanical, and oxidative stress.

LINC08148 promotes the caveola-mediated endocytosis of Zika virus through upregulating transcription of Src.

Long non-coding RNAs (lncRNAs) represent a new group of host factors involved in viral infection. Current study identified an intergenic lncRNA, LINC08148, as a proviral factor of Zika virus (ZIKV) and Dengue virus 2 (DENV2). Knockout (KO) or silencing of LINC08148 decreases the replication of ZIKV and DENV2. LINC08148 mainly acts at the endocytosis step of ZIKV but at a later stage of DENV2. RNA-seq analysis reveals that LINC08148 knockout downregulates the transcription levels of five endocytosis-related genes including AP2B1, CHMP4C, DNM1, FCHO1, and Src. Among them, loss of Src significantly decreases the uptake of ZIKV. Trans-complementation of Src in the LINC08148KO cells largely restores the caveola-mediated endocytosis of ZIKV, indicating that the proviral effect of LINC08148 is exerted through Src. Finally, LINC08148 upregulates the Src transcription through associating with its transcription factor SP1. This work establishes an essential role of LINC08148 in the ZIKV entry, underscoring a significance of lncRNAs in the viral infection. IMPORTANCE: Long non-coding RNAs (lncRNAs), like proteins, participate in viral infection. However, functions of most lncRNAs remain unknown. In this study, we performed a functional screen based on microarray data and identified a new proviral lncRNA, LINC08148. Then, we uncovered that LINC08148 is involved in the caveola-mediated endocytosis of ZIKV, rather than the classical clathrin-mediated endocytosis. Mechanistically, LINC08148 upregulates the transcription of Src, an initiator of caveola-mediated endocytosis, through binding to its transcription factor SP1. This study identifies a new lncRNA involved in the ZIKV infection, suggesting lncRNAs and cellular proteins are closely linked and cooperate to regulate viral infection.

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.

Cancer cell invasion: Caveolae and invadosomes are partners in crime.

During cancer progression, tumor cells need to disseminate by remodeling the extracellular tumor matrix. A recent study sheds light on the intricate cooperation between caveolae and invadosomes that facilitates the spread of cancer cells.

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.

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.

Sugar alcohol-modified polyester nanoparticles for gene delivery via selective caveolae-mediated endocytosis.

Nucleic acid-based drugs are changing the scope of emerging medicine in preventing and treating diseases. Nanoparticle systems based on lipids and polymers developed to navigate tissue-level and cellular-level barriers are now emerging as vector systems that can be translated to clinical settings. A class of polymers, poly(ß-amino esters) (PBAEs) known for their chemical flexibility and biodegradability, has been explored for gene delivery. These polymers are sensitive to changes in the monomer composition affecting transfection efficiency. Hence to add functionality to these polymers, we partially substituted ligands to an identified effective polymer chemistry. We report here a new series of statistical copolymers based on PBAEs where the backbone is modified with sugar alcohols to selectively facilitate the caveolae-mediated endocytosis pathway of cellular transport. These ligands are grafted at the polymer's backbone, thereby establishing a new strategy of modification in PBAEs. We demonstrate that these polymers form nanoparticles with DNA, show effective complexation and cargo release, enter the cell via selective caveolae-mediated endocytosis, exhibit low cytotoxicity, and increase transfection in neuronal cells.

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.

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.

Caveolae-mediated endocytosis of extracellular QSOX1b modulates the migration of fibroblasts.

Quiescin/sulfhydryl oxidase (QSOX1) is a secreted flavoprotein that modulates cellular proliferation, migration and adhesion, roles attributed to its ability to organize the extracellular matrix. We previously showed that exogenously added QSOX1b induces smooth muscle cells migration in a process that depends on its enzymatic activity and that is mediated by hydrogen peroxide derived from Nox1, a catalytic subunit of NAD(P)H oxidases. Here, we report that exogenous QSOX1b also stimulates the migration of L929 fibroblasts and that this effect is regulated by its endocytosis. The use of endocytosis inhibitors and caveolin 1-knockdown demonstrated that this endocytic pathway is caveola-mediated. QSOX1b colocalized with Nox1 in intracellular vesicles, as detected by confocal fluorescence, suggesting that extracellular QSOX1b is endocytosed with the transmembrane Nox1. These results reveal that endosomal QSOX1b is a novel intracellular redox regulator of cell migration.

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.

Single-Cell Diagnosis of Cancer Drug Resistance through the Differential Endocytosis of Nanoparticles between Drug-Resistant and Drug-Sensitive Cancer Cells.

Single-cell diagnosis of cancer drug resistance is highly relevant for cancer treatment, as it can be used to identify the subpopulations of drug-resistant cancer cells, reveal the sensitivity of cancer cells to treatment, and monitor the progress of cancer drug resistance. However, simple and effective methods for cancer drug resistance detection at the single-cell level are still lacking in laboratory and clinical studies. Inspired by the fact that nanoparticles with diverse physicochemical properties would generate distinct and specific interactions with drug-resistant and drug-sensitive cancer cells, which have distinctive molecular signatures, here, we have synthesized a library of fluorescent nanoparticles with various sizes, surface charges, and compositions (SiO2 nanoparticles (SNPs), organic PS-co-PAA nanoparticles (ONPs), and ZIF-8 nanoparticles (ZNPs)), thus demonstrating that the composition has a critical influence on the interaction of nanoparticles with drug-resistant cancer cells. Furthermore, the clathrin/caveolae-independent endocytosis of ZNPs together with the P-glycoprotein-related decreased cell membrane fluidity resulted in a lower cellular accumulation of ZNPs in drug-resistant cancer cells, consequently causing the distinct cellular accumulation of ZNPs between the drug-resistant and drug-sensitive cancer cells. This difference was further quantified by detecting the fluorescence signals generated by the accumulation of nanoparticles at the single-cell level via flow cytometry. Our findings provide another insight into the nanoparticle-cell interactions and offer a promising platform for the diagnosis of cancer drug resistance of various cancer cells and clinical cancer samples at the single-cell level.

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.