Model for the architecture of caveolae based on a flexible, net-like assembly of Cavin1 and Caveolin discs.
ABSTRACT: Caveolae are invaginated plasma membrane domains involved in mechanosensing, signaling, endocytosis, and membrane homeostasis. Oligomers of membrane-embedded caveolins and peripherally attached cavins form the caveolar coat whose structure has remained elusive. Here, purified Cavin1 60S complexes were analyzed structurally in solution and after liposome reconstitution by electron cryotomography. Cavin1 adopted a flexible, net-like protein mesh able to form polyhedral lattices on phosphatidylserine-containing vesicles. Mutating the two coiled-coil domains in Cavin1 revealed that they mediate distinct assembly steps during 60S complex formation. The organization of the cavin coat corresponded to a polyhedral nano-net held together by coiled-coil segments. Positive residues around the C-terminal coiled-coil domain were required for membrane binding. Purified caveolin 8S oligomers assumed disc-shaped arrangements of sizes that are consistent with the discs occupying the faces in the caveolar polyhedra. Polygonal caveolar membrane profiles were revealed in tomograms of native caveolae inside cells. We propose a model with a regular dodecahedron as structural basis for the caveolae architecture.
Project description:Several human diseases are associated with a lack of caveolae. Yet, the functions of caveolae and the molecular mechanisms critical for shaping them still are debated. We show that muscle cells of syndapin III KO mice show severe reductions of caveolae reminiscent of human caveolinopathies. Yet, different from other mouse models, the levels of the plasma membrane-associated caveolar coat proteins caveolin3 and cavin1 were both not reduced upon syndapin III KO. This allowed for dissecting bona fide caveolar functions from those supported by mere caveolin presence and also demonstrated that neither caveolin3 nor caveolin3 and cavin1 are sufficient to form caveolae. The membrane-shaping protein syndapin III is crucial for caveolar invagination and KO rendered the cells sensitive to membrane tensions. Consistent with this physiological role of caveolae in counterpoising membrane tensions, syndapin III KO skeletal muscles showed pathological parameters upon physical exercise that are also found in CAVEOLIN3 mutation-associated muscle diseases.
Project description:Quantitative approaches to analyze the large data sets generated by single molecule localization super-resolution microscopy (SMLM) are limited. We developed a computational pipeline and applied it to analyzing 3D point clouds of SMLM localizations (event lists) of the caveolar coat protein, caveolin-1 (Cav1), in prostate cancer cells differentially expressing CAVIN1 (also known as PTRF), that is also required for caveolae formation. High degree (strongly-interacting) points were removed by an iterative blink merging algorithm and Cav1 network properties were compared with randomly generated networks to retain a sub-network of geometric structures (or blobs). Machine-learning based classification extracted 28 quantitative features describing the size, shape, topology and network characteristics of ?80,000 blobs. Unsupervised clustering identified small S1A scaffolds corresponding to SDS-resistant Cav1 oligomers, as yet undescribed larger hemi-spherical S2 scaffolds and, only in CAVIN1-expressing cells, spherical, hollow caveolae. Multi-threshold modularity analysis suggests that S1A scaffolds interact to form larger scaffolds and that S1A dimers group together, in the presence of CAVIN1, to form the caveolae coat.
Project description:Caveolae are abundant surface organelles implicated in a range of cellular processes. Two classes of proteins work together to generate caveolae: integral membrane proteins termed caveolins and cytoplasmic coat proteins called cavins. Caveolae respond to membrane stress by releasing cavins into the cytosol. A crucial aspect of this model is tight regulation of cytosolic pools of cavin under resting conditions. We now show that a recently identified region of cavin1 that can bind phosphoinositide (PI) lipids is also a major site of ubiquitylation. Ubiquitylation of lysines within this site leads to rapid proteasomal degradation. In cells that lack caveolins and caveolae, cavin1 is cytosolic and rapidly degraded as compared with cells in which cavin1 is associated with caveolae. Membrane stretching causes caveolar disassembly, release of cavin complexes into the cytosol, and increased proteasomal degradation of wild-type cavin1 but not mutant cavin1 lacking the major ubiquitylation site. Release of cavin1 from caveolae thus leads to exposure of key lysine residues in the PI-binding region, acting as a trigger for cavin1 ubiquitylation and down-regulation. This mutually exclusive PI-binding/ubiquitylation mechanism may help maintain low levels of cytosolic cavin1 in resting cells, a prerequisite for cavins acting as signaling modules following release from caveolae.
Project description:Caveolae are plasma membrane invaginations involved in transport, signalling and mechanical membrane sensing in metazoans. Their formation depends upon multiple interactions between membrane-embedded caveolins, lipids and cytosolic cavin proteins. Of the four cavin family members, only cavin1 is strictly required for caveola formation. Here, we demonstrate that an eleven residue (undecad) repeat sequence (UC1) exclusive to cavin1 is essential for caveolar localization and promotes membrane remodelling through binding to phosphatidylserine. In the notochord of mechanically stimulated zebrafish embryos, the UC1 domain is required for caveolar stability and resistance to membrane stress. The number of undecad repeats in the cavin1 UC1 domain varies throughout evolution, and we find that an increased number also correlates with increased caveolar stability. Lastly, we show that the cavin1 UC1 domain induces dramatic remodelling of the plasma membrane when grafted into cavin2 suggesting an important role in membrane sculpting. Overall, our work defines a novel conserved cavin1 modular domain that controls caveolar assembly and stability.
Project description:Eps15 homology domain-containing 2 (EHD2) belongs to the EHD-containing protein family of dynamin-related ATPases involved in membrane remodeling in the endosomal system. EHD2 dimers oligomerize into rings on highly curved membranes, resulting in stimulation of the intrinsic ATPase activity. In this paper, we report that EHD2 is specifically and stably associated with caveolae at the plasma membrane and not involved in clathrin-mediated endocytosis or endosomal recycling, as previously suggested. EHD2 interacts with pacsin2 and cavin1, and ordered membrane assembly of EHD2 is dependent on cavin1 and caveolar integrity. While the EHD of EHD2 is dispensable for targeting, we identified a loop in the nucleotide-binding domain that, together with ATP binding, is required for caveolar localization. EHD2 was not essential for the formation or shaping of caveolae, but high levels of EHD2 caused distortion and loss of endogenous caveolae. Assembly of EHD2 stabilized and constrained caveolae to the plasma membrane to control turnover, and depletion of EHD2, resulting in endocytic and more dynamic and short-lived caveolae. Thus, following the identification of caveolin and cavins, EHD2 constitutes a third structural component of caveolae involved in controlling the stability and turnover of this organelle.
Project description:The mechanisms controlling the abundance and sub-cellular distribution of caveolae are not well described. A first step towards determining such mechanisms would be identification of relevant proteins that interact with known components of caveolae. Here, we applied proximity biotinylation (BioID) to identify a list of proteins that may interact with the caveolar protein cavin1. Screening of these candidates using siRNA to reduce their expression revealed that one of them, CSDE1, regulates the levels of mRNAs and protein expression for multiple components of caveolae. A second candidate, CD2AP, co-precipitated with cavin1. Caveolar proteins were observed in characteristic and previously un-described linear arrays adjacent to cell-cell junctions in both MDCK cells, and in HeLa cells overexpressing an active form of the small GTPase Rac1. CD2AP was required for the recruitment of caveolar proteins to these linear arrays. We conclude that BioID will be useful in identification of new proteins involved in the cell biology of caveolae, and that interaction between CD2AP and cavin1 may have an important role in regulating the sub-cellular distribution of caveolae.
Project description:MOTIVATION:Network analysis and unsupervised machine learning processing of single-molecule localization microscopy of caveolin-1 (Cav1) antibody labeling of prostate cancer cells identified biosignatures and structures for caveolae and three distinct non-caveolar scaffolds (S1A, S1B and S2). To obtain further insight into low-level molecular interactions within these different structural domains, we now introduce graphlet decomposition over a range of proximity thresholds and show that frequency of different subgraph (k = 4 nodes) patterns for machine learning approaches (classification, identification, automatic labeling, etc.) effectively distinguishes caveolae and scaffold blobs. RESULTS:Caveolae formation requires both Cav1 and the adaptor protein CAVIN1 (also called PTRF). As a supervised learning approach, we applied a wide-field CAVIN1/PTRF mask to CAVIN1/PTRF-transfected PC3 prostate cancer cells and used the random forest classifier to classify blobs based on graphlet frequency distribution (GFD). GFD of CAVIN1/PTRF-positive (PTRF+) and -negative Cav1 clusters showed poor classification accuracy that was significantly improved by stratifying the PTRF+ clusters by either number of localizations or volume. Low classification accuracy (<50%) of large PTRF+ clusters and caveolae blobs identified by unsupervised learning suggests that their GFD is specific to caveolae. High classification accuracy for small PTRF+ clusters and caveolae blobs argues that CAVIN1/PTRF associates not only with caveolae but also non-caveolar scaffolds. At low proximity thresholds (50-100 nm), the caveolae groups showed reduced frequency of highly connected graphlets and increased frequency of completely disconnected graphlets. GFD analysis of single-molecule localization microscopy Cav1 clusters defines changes in structural organization in caveolae and scaffolds independent of association with CAVIN1/PTRF. SUPPLEMENTARY INFORMATION:Supplementary data are available at Bioinformatics online.
Project description:Caveolin-1 (Cav1), the coat protein for caveolae, also forms non-caveolar Cav1 scaffolds. Single molecule Cav1 super-resolution microscopy analysis previously identified caveolae and three distinct scaffold domains: smaller S1A and S2B scaffolds and larger hemispherical S2 scaffolds. Application here of network modularity analysis of SMLM data for endogenous Cav1 labeling in HeLa cells shows that small scaffolds combine to form larger scaffolds and caveolae. We find modules within Cav1 blobs by maximizing the intra-connectivity between Cav1 molecules within a module and minimizing the inter-connectivity between Cav1 molecules across modules, which is achieved via spectral decomposition of the localizations adjacency matrix. Features of modules are then matched with intact blobs to find the similarity between the module-blob pairs of group centers. Our results show that smaller S1A and S1B scaffolds are made up of small polygons, that S1B scaffolds correspond to S1A scaffold dimers and that caveolae and hemispherical S2 scaffolds are complex, modular structures formed from S1B and S1A scaffolds, respectively. Polyhedral interactions of Cav1 oligomers, therefore, leads progressively to the formation of larger and more complex scaffold domains and the biogenesis of caveolae.
Project description:Caveolae are bulb-shaped nanodomains of the plasma membrane that are enriched in cholesterol and sphingolipids. They have many physiological functions, including endocytic transport, mechanosensing, and regulation of membrane and lipid transport. Caveola formation relies on integral membrane proteins termed caveolins (Cavs) and the cavin family of peripheral proteins. Both protein families bind anionic phospholipids, but the precise roles of these lipids are unknown. Here, we studied the effects of phosphatidylserine (PtdSer), phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) on caveolar formation and dynamics. Using live-cell, single-particle tracking of GFP-labeled Cav1 and ultrastructural analyses, we compared the effect of PtdSer disruption or phosphoinositide depletion with caveola disassembly caused by cavin1 loss. We found that PtdSer plays a crucial role in both caveola formation and stability. Sequestration or depletion of PtdSer decreased the number of detectable Cav1-GFP puncta and the number of caveolae visualized by electron microscopy. Under PtdSer-limiting conditions, the co-localization of Cav1 and cavin1 was diminished, and cavin1 degradation was increased. Using rapamycin-recruitable phosphatases, we also found that the acute depletion of PtdIns4P and PtdIns(4,5)P2 has minimal impact on caveola assembly but results in decreased lateral confinement. Finally, we show in a model of phospholipid scrambling, a feature of apoptotic cells, that caveola stability is acutely affected by the scrambling. We conclude that the predominant plasmalemmal anionic lipid PtdSer is essential for proper Cav clustering, caveola formation, and caveola dynamics and that membrane scrambling can perturb caveolar stability.
Project description:The Hippo pathway plays major roles in development, regeneration, and cancer. Its activity is tightly regulated by both diffusible chemical ligands and mechanical stimuli. The pathway consists of a series of kinases that can control the sub-cellular localization and stability of YAP or TAZ, homologous transcriptional co-factors. Caveolae, small (60-100 nm) bulb-like invaginations of the plasma membrane, are comprised predominantly of caveolin and cavin proteins and can respond to mechanical stimuli. Here, we show that YAP/TAZ, the major transcriptional mediators of the Hippo pathway, are critical for expression of caveolae components and therefore caveolae formation in both mammalian cells and zebrafish. In essence, without YAP/TAZ, the cell loses an entire organelle. CAVEOLIN1 and CAVIN1, the two essential caveolar genes, are direct target genes of YAP/TAZ, regulated via TEA domain (TEAD) transcription factors. Notably, YAP/TAZ become nuclear enriched and facilitate target gene transcription in cells with diminished levels of caveolae. Furthermore, caveolar-mediated shear stress response activates YAP/TAZ. These data link caveolae to Hippo signaling in the context of cellular responses to mechanical stimuli and suggest activity-based feedback regulation between components of caveolae and the outputs of the Hippo pathway.