Oligomers of the ATPase EHD2 confine caveolae to the plasma membrane through association with actin

Caveolae are specialized domains present in the plasma membrane (PM) of most mammalian cell types. They function in signalling, membrane regulation, and endocytosis. We found that the Eps-15 homology domain-containing protein 2 (EHD2, an ATPase) associated with the static population of PM caveolae. Recruitment to the PM involved ATP binding, interaction with anionic lipids, and oligomerization into large complexes (60-75S) via interaction of the EH domains with intrinsic NPF/KPF motifs. Hydrolysis of ATP was essential for binding of EHD2 complexes to caveolae. EHD2 was found to undergo dynamic exchange at caveolae, a process that depended on a functional ATPase cycle. Depletion of EHD2 by siRNA or expression of a dominant-negative mutant dramatically increased the fraction of mobile caveolar vesicles coming from the PM. Overexpression of EHD2, in turn, caused confinement of cholera toxin B in caveolae. The confining role of EHD2 relied on its capacity to link caveolae to actin filaments. Thus, EHD2 likely plays a key role in adjusting the balance between PM functions of stationary caveolae and the role of caveolae as vesicular carriers.     

Membrane microdomains,caveolae, and caveolar endocytosis of sphingolipids (Review)

Caveolae are flask-shape membrane invaginations of the plasma membrane that have been implicated in endocytosis, transcytosis, and cell signaling. Recent years have witnessed the resurgence of studies on caveolae because they have been found to be involved in the uptake of some membrane components such as glycosphingolipids and integrins, as well as viruses, bacteria, and bacterial toxins. Accumulating evidence shows that endocytosis mediated by caveolae requires unique structural and signaling machinery (caveolin-1, src kinase), which indicates that caveolar endocytosis occurs through a mechanism which is distinct from other forms of lipid microdomain-associated, clathrin-independent endocytosis. Furthermore, a balance of glycosphingolipids, cholesterol, and caveolin-1 has been shown to be important in regulating caveolae endocytosis.     

Membrane microdomains, caveolae, and caveolar endocytosis of sphingolipids

Caveolae are flask-shape membrane invaginations of the plasma membrane that have been implicated in endocytosis, transcytosis, and cell signaling. Recent years have witnessed the resurgence of studies on caveolae because they have been found to be involved in the uptake of some membrane components such as glycosphingolipids and integrins, as well as viruses, bacteria, and bacterial toxins. Accumulating evidence shows that endocytosis mediated by caveolae requires unique structural and signaling machinery (caveolin-1, src kinase), which indicates that caveolar endocytosis occurs through a mechanism which is distinct from other forms of lipid microdomain-associated, clathrin-independent endocytosis. Furthermore, a balance of glycosphingolipids, cholesterol, and caveolin-1 has been shown to be important in regulating caveolae endocytosis.     

Caveolae,transmembrane signalling and cellular transformation

Caveolae are -50–100 nm membrane micro-invaginations associated with the plasma membrane of a wide variety of cells. Although they were first identified in transmission electron micrographs -40 years ago, their exact function(s) has remained controversial. Two well-established functions include: (1) the transcytosis of both large and small molecules across capillary endothelial cells and (2) the utilization of GPI-linked proteins to concentrate small molecules in caveolae for translocation to the cytoplasm (termed potocytosis). Recently, interest in a ‘third’ proposed caveolar function, namely transmembrane signalling, has been revived by the identification of caveolin — a transformation-dependent v-Src substrate and caveolar marker protein — and the isolation of caveolin-rich membrane domains from cultured cells. Here we will discuss existing evidence that suggests a role for caveolae in signalling events.     

Hormonal regulation of caveolae internalization

Caveolae undergo a cyclic transition from a flat segment of membrane to a vesicle that then returns to the cell surface. Here we present evidence that this cycle depends on a population of protein kinase C- alpha (PKC-alpha) molecules that reside in the caveolae membrane where they phosphorylate a 90-kD protein. This cycle can be interrupted by treatment of the cells with phorbol-12,13-dibutyrate or agents that raise the concentration of diacylglycerol in the cell. Each of these conditions displaces PKC-alpha from caveolae, inhibits the phosphorylation of the 90-kD protein, and prevents internalization. Caveolae also contain a protein phosphatase that dephosphorylates the 90-kD once PKC-alpha is gone. A similar dissociation of PKC-alpha from caveolae and inhibition of invagination was observed when cells were treated with histamine. This effect was blocked by pyrilamine but not cimetidine, indicating the involvement of histamine H1 receptors. These findings suggest that the caveolae internalization cycle is hormonally regulated.     

Increased number of caveolae and caveolin-3 overexpression in Duchenne muscular dystrophy.

Caveolae are small pockets or invaginations localized at the plasma membrane. Caveolins are the principal protein components of caveolae and play an important structural role in the formation of caveolae membranes. Here, we studied by freeze fracture and immunological techniques the spatial organization of caveolae at the muscle cell plasma membrane and the expression of caveolin-3 in Duchenne muscular dystrophy (DMD) muscle fibers. In DMD muscle, we found an increased number of caveolae at the sarcolemma that corresponds to an overexpression of caveolin-3 by immunohistochemistry and by Western blot analysis. These findings suggest a possible role for caveolae and caveolin-3 in the pathogenesis of DMD.     

Role of caveolin and caveolae in insulin signaling and diabetes

Caveolae are specialized membrane microdomains present within the plasma membrane of the vast majority of cell types. They have a unique composition in that they are highly enriched in cholesterol, sphingolipids, and their coat proteins the caveolins (-1, -2, and -3). In recent years it has been recognized that caveolae act as signaling platforms, serving as a concentrating point for numerous signaling molecules, as well as regulating flux through many distinct signaling cascades. Although caveolae are found in a variety of cell types, they are most abundant in adipose tissue. This fact has led to the intense study of the function of these organelles in adipocytes. It has now become apparent that effective insulin signaling in the adipocyte may be strictly dependent on localization of at least two insulin-responsive elements to caveolae (insulin receptor and GLUT4), as well as on a direct functional interaction between caveolin-1 and the insulin receptor. We present a critical discussion of these recent findings.     

Immunoisolation of caveolae with high affinity antibody binding to the oligomeric caveolin cage. Toward understanding the basis of purification.

Defining the molecular composition of caveolae is essential in establishing their molecular architecture and functions. Here, we identify a high affinity monoclonal antibody that is specific for caveolin-1alpha and rapidly binds caveolin oligomerized around intact caveolae. We use this antibody (i) to develop a new simplified method for rapidly isolating caveolae from cell and tissue homogenates without using the silica-coating technology and (ii) to analyze various caveolae isolation techniques to understand how they work and why they yield different compositions. Caveolae are immunoisolated from rat lung plasma membrane fractions subjected to mechanical disruption. Sonication of plasma membranes, isolated with or without silica coating, releases caveolae along with other similarly buoyant microdomains and, therefore, requires immunoisolations to purify caveolae. Shearing of silica-coated plasma membranes provides a homogeneous population of caveolae whose constituents (i) remain unchanged after immunoisolation, (ii) all fractionate bound to the immunobeads, and (iii) appear equivalent to caveolae immunoisolated after sonication. The caveolae immunoisolated from different low density fractions are quite similar in molecular composition. They contain a subset of key signaling molecules (i.e. G protein and endothelial nitric oxide synthase) and are markedly depleted in glycosylphosphatidylinositol-anchored proteins, beta-actin, and angiotensin-converting enzyme. All caveolae isolated from the cell surface of lung microvascular endothelium in vivo appear to be coated with caveolin-1alpha. Caveolin-1beta and -2 can also exist in these same caveolae. The isolation and analytical procedures as well as the time-dependent dissociation of signaling molecules from caveolae contribute to key compositional differences reported in the literature for caveolae. This new, rapid, magnetic immunoisolation procedure provides a consistent preparation for use in the molecular analysis of caveolae.     

Cell surface orifices of caveolae and localization of caveolin to the necks of caveolae in adipocytes

Caveolae are noncoated invaginations of the plasma membrane that form in the presence of the protein caveolin. Caveolae are found in most cells, but are especially abundant in adipocytes. By high-resolution electron microscopy of plasma membrane sheets the detailed structure of individual caveolae of primary rat adipocytes was examined. Caveolin-1 and -2 binding was restricted to the membrane proximal region, such as the ducts or necks attaching the caveolar bulb to the membrane. This was confirmed by transfection with myc-tagged caveolin-1 and -2. Essentially the same results were obtained with human fibroblasts. Hence caveolin does not form the caveolar bulb in these cells, but rather the neck and may thus act to retain the caveolar constituents, indicating how caveolin participates in the formation of caveolae. Caveolae, randomly distributed over the plasma membrane, were very heterogeneous, varying in size between 25 and 150 nm. There was about one million caveolae in an adipocyte, which increased the surface area of the plasma membrane by 50%. Half of the caveolae, those larger than 50 nm, had access to the outside of the cell via ducts and 20-nm orifices at the cell surface. The rest of the caveolae, those smaller than 50 nm, were not open to the cell exterior. Cholesterol depletion destroyed both caveolae and the cell surface orifices.     

The caveolae in rabbit sinus node and atrium

Summary Caveolae or membrane vesicles are commonly observed in smooth and skeletal muscle as well as in working heart muscle. Using sections of fixed tissue and replicas of freeze-cleaved material, we show in this study that caveolae are also very numerous in sinus node cells of the rabbit, and to a lesser degree, in the atrial cells.Caveolae increase the plasma membrane surface area by 115% in the leading sinus node, and by 56% in the atrial cells. In these two cell types, the membrane of the caveolae contains four times fewer intramembranous particles than the rest of the plasma membrane, and this difference applies to both PF and EF faces. The role of the caveolae is still unclear, but it does not seem that they have a pinocytotic function.     

The biology of caveolae: lessons from caveolin knockout mice and implications for human disease

Caveolae, plasma membrane invaginations that serve as membrane organizing centers, are found in most cell types, but are enriched in adipocytes, endothelial cells, and myocytes. Three members of the caveolin family (Cav-1, -2, and -3) are essential for the formation of caveolae. Specialized motifs in the caveolin proteins function to recruit lipids and proteins to caveolae for participation in intracellular trafficking of cellular components and operation in signal transduction. Mutations in the gene encoding CAV-1 are associated with the development and progression of breast cancers, whereas mutations in the CAV-3 gene result in Rippling Muscle Disease and a form of Limb-Girdle Muscular Dystrophy. The generation of caveolin-null mice has confirmed the essential role of these proteins in caveolae biogenesis and in the pathophysiology of diverse tissues. Caveolin-null mice provide new animal models for studying the pathogenesis of a number of human diseases, including cancer, diabetes, atherosclerosis, restrictive lung disease and pulmonary fibrosis, cardiomyopathy, muscular dystrophy, and bladder dysfunction.     

Cytoskeleton Modification and Cholesterol Depletion Affect Membrane Properties and Caveolae Positioning of CHO Cells

The formation of protrusions is necessary for numerous biological processes. It involves extension of the plasma membrane, and the force needed for this is provided by the actin cytoskeleton. Tether pulling with optical tweezers can mimic the formation of a protrusion, so we used this method to investigate the effects of modifying not only actin (with latrunculin A) but also microtubules (with nocodazole) and the plasma membrane itself (with methyl-β-cyclodextrin) on the Chinese hamster ovary cell membrane. After these modifications, the membrane reservoir was supposed to redistribute. Caveolae constitute a small part of the reservoir, so the redistribution of caveolar proteins such as caveolin-1 and cavin-1 that represents caveolae per se was assessed. The main findings concerning protrusion force and membrane reservoir availability were as follows: (1) they correlated inversely, (2) their values underwent the greatest change after microtubule disruption, and (3) membrane composition had a major influence on the parameters studied. F-actin disruption and cholesterol depletion decreased, and microtubule disruption increased the amount of the caveolar proteins (caveolae). Caveolae presented just an example of the membrane reservoir, and from our findings, we suppose that the perturbations caused were too large to be related to caveolae redistribution alone. The integrity of the cytoskeleton and plasma membrane composition are important factors in the formation of protrusions and in determining the availability and distribution of the membrane reservoir.     

Endocytosis via caveolae

Caveolae are flask-shaped invaginations present in the plasma membrane of many cell types. They have long been implicated in endocytosis, transcytosis, and cell signaling. Recent work has confirmed that caveolae are directly involved in the internalization of membrane components (glycosphingolipids and glycosylphosphatidylinositol-anchored proteins), extracellular ligands (folic acid, albumin, autocrine motility factor), bacterial toxins (cholera toxin, tetanus toxin), and several nonenveloped viruses (Simian virus 40, Polyoma virus). Unlike clathrin-mediated endocytosis, internalization through caveolae is a triggered event that involves complex signaling. The mechanism of internalization and the subsequent intracellular pathways that the internalized substances take are starting to emerge.     

A Highlights from MBoC Selection: A phosphoinositide-binding cluster in cavin1 acts as a molecular sensor for cavin1 degradation

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.     

Caveolin-1 and caveolae in atherosclerosis: differential roles in fatty streak formation and neointimal hyperplasia

PURPOSE OF REVIEW: Caveolae are 50-100 nm cell surface plasma membrane invaginations observed in terminally differentiated cells. They are characterized by the presence of the protein marker caveolin-1. Caveolae and caveolin-1 are present in almost every cell type that has been implicated in the development of an atheroma. These include endothelial cells, macrophages, and smooth muscle cells. Caveolae and caveolin-1 are involved in regulating several signal transduction pathways and processes that play an important role in atherosclerosis. RECENT FINDINGS: Several recent studies using genetically engineered mice (Cav-1 (-/-) null animals) have now clearly demonstrated a role for caveolin-1 and caveolae in the development of atherosclerosis. In fact, they suggest a rather complex one, either proatherogenic or antiatherogenic, depending on the cell type examined. For example, in endothelial cells, caveolin-1 and caveolae may play a proatherogenic role by promoting the transcytosis of LDL-cholesterol particles from the blood to the sub-endothelial space. In contrast, in smooth muscle cells, the ability of caveolin-1 to negatively regulate cell proliferation (neointimal hyperplasia) may have an antiatherogenic effect. SUMMARY: Caveolin-1 and caveolae play an important role in several steps involved in the initiation of an atheroma. Development of new drugs that regulate caveolin-1 expression may be important in the prevention or treatment of atherosclerotic vascular disease.     

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.     

Caveolin-1 expression and caveolae biogenesis during cell transdifferentiation in lung alveolar epithelial primary cultures.

Caveolae are omega-shaped invaginations of the plasmalemma possessing a cytoplasmic membrane protein coat of caveolin. Caveolae are present in the in vivo alveolar epithelial type I (ATI) lung cell, but absent in its progenitor, the alveolar epithelial type II (ATII) cell. In primary culture ATII cells grown on a plastic substratum acquire with time an ATI-"like" phenotype. We demonstrate that freshly isolated rat ATII cells lack caveolae and expression of caveolin-1 (a critical caveolae structural protein). As the ATII cells acquire an ATI-like phenotype in primary culture caveolin-1 expression increases, with caveolin-1 signal at 192 h postseeding up to 50-fold greater than at 60 h; caveolae were morphologically evident only after 132 h. When maintaining the differentiated ATII phenotype with time, i.e., culture upon collagen with an apical interface of air, a temporal increase in caveolin-1 expression was not observed, with only very faint signals evident even at 192 h postseeding; at no time did these cultures display caveolae. In late primary ATII cultures caveolin-1 expression and caveolae biogenesis occur as a function of in vitro transformation from the ATII to the ATI-like phenotype. The results have broad implications for the in vitro study of the role of caveolae and caveolin in alveolar epithelial cell biology.     

Osmotic Stress Reduces Ca2+ Signals through Deformation of Caveolae

Caveolae are membrane invaginations that can sequester various signaling proteins. Caveolae have been shown to provide mechanical strength to cells by flattening to accommodate increased volume when cells are subjected to hypo-osmotic stress. We have previously found that caveolin, the main structural component of caveolae, specifically binds Gα_q and stabilizes its activation state resulting in an enhanced Ca²⁺ signal upon activation. Here, we show that osmotic stress caused by decreasing the osmolarity in half reversibly changes the configuration of caveolae without releasing a significant portion of caveolin molecules. This change in configuration due to flattening leads to a loss in Cav1-Gα_q association. This loss in Gα_q/Cav1 association due to osmotic stress results in a significant reduction of Gα_q/phospholipase Cβ-mediated Ca²⁺ signals. This reduced Ca²⁺ response is also seen when caveolae are reduced by treatment with siRNA(Cav1) or by dissolving them by methyl-β-cyclodextran. No change in Ca²⁺ release with osmotic swelling can be seen when growth factor pathways are activated. Taken together, these results connect the mechanical deformation of caveolae to Gα_q-mediated Ca²⁺ signals.     

Caveolar Fatty Acids and Acylation of Caveolin-1

Purpose

Caveolae are cholesterol and sphingolipids rich subcellular domains on plasma membrane. Caveolae contain a variety of signaling proteins which provide platforms for signaling transduction. In addition to enriched with cholesterol and sphingolipids, caveolae also contain a variety of fatty acids. It has been well-established that acylation of protein plays a pivotal role in subcellular location including targeting to caveolae. However, the fatty acid compositions of caveolae and the type of acylation of caveolar proteins remain largely unknown. In this study, we investigated the fatty acids in caveolae and caveolin-1 bound fatty acids.

Methods

Caveolae were isolated from Chinese hamster ovary (CHO) cells. The caveolar fatty acids were extracted with Folch reagent, methyl esterificated with BF3, and analyzed by gas chromatograph-mass spectrometer (GC/MS). The caveolin-1bound fatty acids were immunoprecipitated by anti-caveolin-1 IgG and analyzed with GC/MS.

Results

In contrast to the whole CHO cell lysate which contained a variety of fatty acids, caveolae mainly contained three types of fatty acids, 0.48 µg palmitic acid, 0.61 µg stearic acid and 0.83 µg oleic acid/caveolae preparation/5×10⁷ cells. Unexpectedly, GC/MS analysis indicated that caveolin-1 was not acylated by myristic acid; instead, it was acylated by palmitic acid and stearic acid.

Conclusion

Caveolae contained a special set of fatty acids, highly enriched with saturated fatty acids, and caveolin-1 was acylated by palmitic acid and stearic acid. The unique fatty acid compositions of caveolae and acylation of caveolin-1 may be important for caveolae formation and for maintaining the function of caveolae.