Caveolar Neck Research Articles

From Flat to Bulb ‐ Novel Insights in Caveolae Membrane Curvature

Caveolae are 60‐100 nm sized bulb‐shaped plasma membrane invaginations found in various cell types including adipocytes, endothelial cells and myocytes. Their cellular functions comprise endocytosis and signaling processes, cellular lipid uptake, as well as sensing and regulation of membrane tension. Thereby, it was shown previously that caveolae can change their membrane shape from invaginated bulbs to flat domains and vice versa. However it is currently not clear which proteins are responsible for the membrane curvature changes. Here, we report the first detailed structural investigation of caveolae membrane shapes in vivo. To do so, Platinum replica EM images of the plasma membrane in mouse embryonic fibroblasts and endothelial cells were correlated to super‐resolution (STED) fluorescence intensities of proteins associated with caveolae domains. This correlative imaging approach (CLEM) allowed us to precisely localize caveolae related proteins to the underlying membrane curvature. The major structural caveolae proteins such as Caveolins and Cavins were found in all caveolae membrane domains independent of their curvature. Surprisingly, EHD2, a caveolar neck protein, was not only detected at bulb‐shaped invaginations but also at flat caveolae. Contrarily, Pacsin/Syndapin2 and EHBP1 were predominantly surrounding flat caveolae domains indicating that both proteins promote the neck formation. Because dynamin was previously associated with caveolae endocytosis and mobility we analyzed its spatial distribution to caveolae. However, our detailed CLEM analysis failed to determine dynamin to caveolae membrane domains in several cell types. Additionally, dynamin lacking cells did not show severe morphological changes of caveolae suggesting that dynamin is not involved in caveolae membrane curvature changes. Taken together, this study gives novel insights in caveolae structure and their formation from flat to invaginated membrane domains.

The Lipids that Get the Invite to a Caveolar Neck Party

Caveolae are plasma membrane (PM) invaginations that appear as bulbs protruding into the cytoplasm. individual bulbs are connected to the bulk membrane by a highly curved neck with an internal pore. Like other highly curved biological structures (e.g., pores created by membrane compartment fusion and clathrin coated endocytic sites before the vesicle pinches off), caveolar necks are created by cellular protein machinery, and the work done by proteins to create and sustain the high curvature depends on the lipid membrane material properties and distributions. Understanding and quantifying lipid equilibrium distributions requires atomistic detail to resolve specific interactions, but also long timescales to allow sufficient lipid diffusion. To meet both of these demands, we used the special-use Anton supercomputer to attain continuous, long timescale trajectories of lipid diffusion on two pores with different internal radii and a planar bilayer as a control. We used a PM mimetic that balances the necessary lipidic complexity with the necessary simplicity to quantify important lipid-lipid interaction motifs and redistributions on a timescale accessible to all-atom simulations. The multi-microsecond simulations allowed us to test two core hypotheses: i) that ganglioside GM3 and palmitoyl sphingomyelin redistribute to areas of positive curvature, and ii) that cholesterol will preferentially flip-flop into the outer leaflet. Because of the diverse curvature accessible on a caveolar pore, the simulations also allow insight into whether lipids redistribute with respect to both net curvature and Gaussian curvature. Preliminary results indicate that cholesterol is depleted in the narrower neck pore and that phosphatidylcholine lipids are enriched in pore neck pores.

Regulation of caveolae through cholesterol-depletion-dependent tubulation mediated by PACSIN2.

The membrane-shaping ability of PACSIN2 (also known as syndapin II), which is mediated by its F-BAR domain, has been shown to be essential for caveolar morphogenesis, presumably through the shaping of the caveolar neck. Caveolar membranes contain abundant cholesterol. However, the role of cholesterol in PACSIN2-mediated membrane deformation remains unclear. Here, we show that the binding of PACSIN2 to the membrane can be negatively regulated by cholesterol. We prepared reconstituted membranes based on the lipid composition of caveolae. The reconstituted membrane with cholesterol had a weaker affinity for the F-BAR domain of PACSIN2 than a membrane without cholesterol. Consistent with this, upon depletion of cholesterol from the plasma membrane, PACSIN2 localized at tubules that had caveolin-1 at their tips, suggesting that cholesterol inhibits membrane tubulation mediated by PACSIN2. The tubules induced by PACSIN2 could be representative of an intermediate of caveolae endocytosis. Consistent with this, the removal of caveolae from the plasma membrane upon cholesterol depletion was diminished in the PACSIN2-deficient cells. These data suggest that PACSIN2-mediated caveolae internalization is dependent on the amount of cholesterol, providing a mechanism for cholesterol-dependent regulation of caveolae.This article has an associated First Person interview with the first author of the paper.

Tagging and Deleting of Endogenous Caveolar Components Using CRISPR/Cas9 Technology.

Here, we describe how to utilize CRISPR/Cas9 technology in the generation of tissue culture cells with fluorescently tagged caveolar components as well as cells deleted of endogenous caveolar components. As one example, we will describe tagging of EHD2, caveolar neck protein, with Green Fluorescent protein (eGFP) from endogenous loci (knock-in, KI). As another example, we will describe deletion (knock-out, KO) of Caveolin1 (Cav1), an essential caveolar component in NIH/3T3 cells. In both instances, the modifications were achieved by using Cas9 delivery on plasmid DNA by electroporation and by utilizing FACS cell sorting for selection or enrichment of edited population of cells. We also provide a list with tested gRNA sequences to successfully produce KI and KO of other caveolar components.

Caveolae

Caveolae are one of the most abundant and striking features of the plasma membrane of many mammalian cell types. These surface pits have fascinated biologists since their discovery by the pioneers of electron microscopy in the middle of the last century, but we are only just starting to understand their multiple functions. Molecular understanding of caveolar formation is advancing rapidly and we now know that sculpting the membrane to generate the characteristic bulb-shaped caveolar pit involves the coordinated action of integral membrane proteins and peripheral membrane coat proteins in a process dependent on their multiple interactions with membrane lipids. The resulting structure is further stabilised by protein complexes at the caveolar neck. Caveolae can bud to generate an endocytic carrier but can also be disassembled in response to specific stimuli to function as a mechanoprotective device. These structures have also been linked to numerous signalling pathways. Here, we will briefly summarise the current molecular and structural understanding of caveolar formation and dynamics, discuss how the crucial structural components of caveolae work together to generate a dynamic sensing domain, and discuss the implications of recent studies on the diverse roles proposed for caveolae in different cells and tissues.

EHD Proteins Cooperate to Generate Caveolar Clusters and to Maintain Caveolae during Repeated Mechanical Stress

SummaryCaveolae introduce flask-shaped convolutions into the plasma membrane and help to protect the plasma membrane from damage under stretch forces. The protein components that form the bulb of caveolae are increasingly well characterized, but less is known about the contribution of proteins that localize to the constricted neck. Here we make extensive use of multiple CRISPR/Cas9-generated gene knockout and knockin cell lines to investigate the role of Eps15 Homology Domain (EHD) proteins at the neck of caveolae. We show that EHD1, EHD2, and EHD4 are recruited to caveolae. Recruitment of the other EHDs increases markedly when EHD2, which has been previously detected at caveolae, is absent. Construction of knockout cell lines lacking EHDs 1, 2, and 4 confirms this apparent functional redundancy. Two striking sets of phenotypes are observed in EHD1,2,4 knockout cells: (1) the characteristic clustering of caveolae into higher-order assemblies is absent; and (2) when the EHD1,2,4 knockout cells are subjected to prolonged cycles of stretch forces, caveolae are destabilized and the plasma membrane is prone to rupture. Our data identify the first molecular components that act to cluster caveolae into a membrane ultrastructure with the potential to extend stretch-buffering capacity and support a revised model for the function of EHDs at the caveolar neck.

Molecular Composition and Ultrastructure of the Caveolar Coat Complex

Author SummaryCaveolae are flask-shaped invaginations in the plasma membrane of many mammalian cell types, and are particularly abundant in fat cells, muscle cells, and the cells that line blood vessels. Although caveolae are likely to be important for cellular responses to mechanical stress, intracellular trafficking, and signaling events, we still lack an understanding of the precise molecular mechanisms for how they form and carry out these functions. Here we address the question of how caveolae are made. Recent years have seen a considerable expansion of the catalogue of known protein components present in caveolae. Our study shows that the main protein components, cavins and caveolins, assemble into one specific complex. We reveal how different amounts of two caveolar proteins, cavin 2 and cavin 3, may be incorporated into this single type of complex, thereby potentially conferring different functional properties on caveolae. Using electron microscopy, we demonstrate that the protein complex is distributed all around the membrane bulb of caveolae, and so can be truly described as the caveolar coat. The caveolar coat excludes the protein EHD2, which regulates the dynamics of caveolae—this protein has a distinct distribution at the caveolar neck. These findings provide the basis for a more complete understanding of the network of protein interactions that produces caveolae.

Regulation of dynamin-2 assembly-disassembly and function through the SH3A domain of intersectin-1s

Intersectin-1s (ITSN-1s), a five Src homology 3 (SH3) domain-containing protein, is critically required for caveolae and clathrin-mediated endocytosis (CME), due to its interactions with dynamin (dyn). Of the five SH3A-E domains, SH3A is unique because of its high affinity for dyn and potent inhibition of CME. However, the molecular mechanism by which SH3A integrates in the overall function of ITSN-1s to regulate the endocytic process is not understood. Using biochemical and functional approaches as well as high-resolution electron microscopy, we show that SH3A exogenously expressed in human lung endothelial cells caused abnormal endocytic structures, distorted caveolae clusters, frequent staining-dense rings around the caveolar necks and 60% inhibition of caveolae internalization. In vitro studies further revealed that SH3A, similar to full-length ITSN-1s stimulates dyn2 oligomerization and guanosine triphosphatase (GTP)ase activity, effects not detected when other SH3 domains of ITSN-1s were used as controls. Strikingly, in the presence of SH3A, dyn2–dyn2 interactions are stabilized and despite continuous GTP hydrolysis, dyn2 oligomers cannot disassemble. SH3A may hold up caveolae release from the plasma membrane and formation of free-transport vesicles, by prolonging the lifetime of assembled dyn2. Altogether, our results indicate that ITSN-1s, via its SH3A has the unique ability to regulate dyn2 assembly–disassembly and function during endocytosis.

High‐Resolution 3D Quantitative Analysis of Caveolar Ultrastructure and Caveola–Cytoskeleton Interactions

Caveolae are characteristic invaginations of the mammalian plasma membrane (PM) implicated in lipid regulation, signal transduction and endocytosis. We have employed electron microscope tomography (ET) to quantify caveolae structure-function relationships in three-dimension (3D) at high resolution both in conventionally fixed and in fast-frozen/freeze-substituted (intact) cells as well as immunolabelled PM lawns. Our findings provide a detailed quantitative comparison of the average caveola dimensions for different cell types including tissue endothelial cells and cultured 3T3-L1 adipocytes. These studies revealed the presence of a spiked caveolar coat and a wide caveolar neck open to the extracellular milieu that is sensitive to conventional fixation; the neck region appeared to form a specialized microdomain with associated cytoplasmic material. In endothelial cells in situ in pancreatic islets of Langerhans, the diaphragm spanning the caveolar opening was clearly resolved by ET, and the involuted 3D topology of the cell surface mapped to measure the contribution of caveolar membranes to local increases in the surface area of the PM. The complexity of connections among caveolae and to the actin cytoskeleton and microtubules suggests that individual caveolae may be interconnected through a complex filamentous network to form a single functional unit.

Regulation of caveolar cardiac sodium current by a single Gsα histidine residue

Cardiac sodium channels (voltage-gated Na(+) channel subunit 1.5) reside in both the plasmalemma and membrane invaginations called caveolae. Opening of the caveolar neck permits resident channels to become functional. In cardiac myocytes, caveolar opening can be stimulated by applying beta-receptor agonists, which initiates an interaction between the stimulatory G protein subunit-alpha (G(s)alpha) and caveolin-3. This study shows that, in adult rat ventricular myocytes, a functional G(s)alpha-caveolin-3 interaction occurs, even in the absence of the caveolin-binding sequence motif of G(s)alpha. Consistent with previous data, whole cell experiments conducted in the presence of intracellular PKA inhibitor stimulation with beta-receptor agonists increased the sodium current (I(Na)) by 35.9 +/- 8.6% (P < 0.05), and this increase was mimicked by application of G(s)alpha protein. Inclusion of anti-caveolin-3 antibody abolished this effect. These findings suggest that G(s)alpha and caveolin-3 are components of a PKA-independent pathway that leads to the enhancement of I(Na). In this study, alanine scanning mutagenesis of G(s)alpha (40THR42), in conjunction with voltage-clamp studies, demonstrated that the histidine residue at position 41 of G(s)alpha (H41) is a critical residue for the functional increase of I(Na). Protein interaction assays suggest that G(s)alphaFL (full length) binds to caveolin-3, but the enhancement of I(Na) is observed only in the presence of G(s)alpha H41. We conclude that G(s)alpha H41 is a critical residue in the regulation of the increase in I(Na) in ventricular myocytes.

Intersectin regulates fission and internalization of caveolae in endothelial cells.

Intersectin, a multiple Eps15 homology and Src homology 3 (SH3) domain-containing protein, is a component of the endocytic machinery in neurons and nonneuronal cells. However, its role in endocytosis via caveolae in endothelial cells (ECs) is unclear. We demonstrate herein by coimmunoprecipitation, velocity sedimentation on glycerol gradients, and cross-linking that intersectin is present in ECs in a membrane-associated protein complex containing dynamin and SNAP-23. Electron microscopy (EM) immunogold labeling studies indicated that intersectin associated preferentially with the caveolar necks, and it remained associated with caveolae after their fission from the plasmalemma. A cell-free system depleted of intersectin failed to support caveolae fission from the plasma membrane. A biotin assay used to quantify caveolae internalization and extensive EM morphological analysis of ECs overexpressing wt-intersectin indicated a wide range of morphological changes (i.e., large caveolae clusters marginated at cell periphery and pleiomorphic caveolar necks) as well as impaired caveolae internalization. Biochemical evaluation of caveolae-mediated uptake by ELISA showed a 68.4% inhibition by reference to control. We also showed that intersectin interaction with dynamin was important in regulating the fission and internalization of caveolae. Taken together, the results indicate the crucial role of intersectin in the mechanism of caveolae fission in endothelial cells.

Rat atrial myocyte plasmalemmal caveolae in situ. Reversible experimental increases in caveolar size and in surface density of caveolar necks.

The structure, size, and surface density of the conspicuous flask-shaped structures called caveolae that are located under the plasma membrane of cardiac myocytes in intact rat atria were studied by electron microscopy after physiological perturbations designed to examine whether caveolae and/or their necks are fixed or mobile and whether the caveolar lumen is always open or can close off from the interstitial space. We showed that, in stretched and unstretched atria, horseradish peroxidase could enter or be washed out of caveolae at 37 degrees, 18 degrees, and 4 degrees C, but this finding does not rule out that caveolae and/or their necks can cycle rapidly between states closed and open to the interstitial space. Electron microscopy of thin sections revealed that exposure of atria at 37 degrees or 18 degrees C to physiological salt solutions made hypertonic by adding 150 mM sucrose or mannitol resulted in a striking enlargement of caveolar profiles within 1 to 5 minutes. Caveolar enlargement was rapidly reversible on return to control saline. After freeze fracture of atria exposed to these hypertonic solutions, quantitative analysis of electron micrographs of the fracture faces revealed statistically significant increases in cross-sectional diameter of cross-fractured caveolar necks and in mean number of caveolar necks penetrating per unit area of plasmalemmal fracture face. These results suggest that atrial myocyte caveolae are dynamic structures whose necks may be reversibly inserted into and withdrawn from the plasmalemma, possibly (but not necessarily) corresponding to states in which caveolae are, respectively, open and closed to the interstitial spaces.

Evaluation of the Intramembrane Particle Count in Biopsied Myocardium from Patients with Idiopathic Cardiomyopathy.

To clarify the abnormalities of myocardial membrane structure in cardiomyopathy, the density of intramembranous particles (IMP) of mitochondria, sarcoplasmic reticulum and sarcolemma, and the number of caveolar necks was evaluated by freeze-fracture morphology. The subjects consisted of 17 patients with hypertrophic cardiomyopathy (HCM), 6 patients with dilated cardiomyopathy (DCM), and 7 patients as controls. The results showed that the numbers of IMPs on the membrane faces of mitochondria, sarcoplasmic reticulum and sarcolemma were significantly decreased in HCM and DCM, and that they were decreased more markedly in DCM than in HCM. The number of caveolar necks was significantly decreased in DCM. Thus, the decreased numbers of IMPs on myocardial membranes and the decrease in caveolar necks may be important characteristics resulting from abnormal myocardial membrane metabolism in cardiomyopathies, HCM and DCM.

Quantitative studies on plasmalemmal folds and caveolae of rabbit ventricular myocardial cells.

Plasmalemmal folds and caveolae were investigated by qualitative and quantitative analysis of electron micrographs obtained by freeze fracture and transmission electron microscopy (TEM) of rabbit right ventricular papillary muscles whose mean sarcomere lengths ranged from 1.64 to 2.28 micron. In passively extended muscles, folds were observed at sarcomere lengths of 2.3 micron and could be shown by extrapolation to become completely extended at a maximum sarcomere length of 2.8 micron. It was concluded that the plasmalemma does not contribute to resting tension in the physiological range of sarcomere lengths. Caveolae are present in both the external plasmalemmal envelope and T-tubular plasmalemma. They show no preferential distribution with respect to underlying myofibrillar striations or membrane folds and are nearly devoid of membrane particles in freeze-fractured material. The surface density of caveolar necks (4.0/micron2 apparent plasmalemmal area) is only 16-20% of that reported for frog skeletal muscle. Caveolae augment plasmalemmal area by 21-32%, assuming two or three caveolae per neck, respectively. Caveolar membrane does not serve as a reservoir of membrane to be recruited into external plasmalemma, at least over the physiological range of sarcomere lengths. In heart muscle they do not account for the T-tubular access resistance, and their function in this tissue remains unknown.

Inpocketings of the cell membrane (caveolae) in the rat myocardium

The surface membrane of cardiac muscle cells of the rat papillary muscle shows, in addition to the openings of the T tubules, many small inpocketings (caveolae). Some caveolae are flask shaped, but many others are composite in shape and nonuniform in size; they all open into the intercellular space through a single, narrow neck. Their packing density is 6.1/ μ m 2 of cell surface and it has been culculated that they increase the cell membrane area by about 27%. A network of sarcoplasmic reticulum tubules lies close to the caveolae. Intramembranous particles, 9 nm in diameter, are fewer in the membrane of the caveolae than in the surface membrane proper and have no special arrangement around the caveolar necks. By comparison, the caveolae of endothelial cells are smaller and uniform in size, and have a greater packing density (67.5/ μ m 2 ). The caveolae of vascular smooth muscle cells are small and uniform in size, are arranged in rows, are associated with sarcoplasmic reticulum, and have a characteristic distribution of intramembranous particles around their necks.