Dynamin at the Neck of Caveolae Mediates Their Budding to Form Transport Vesicles by GTP-driven Fission from the Plasma Membrane of Endothelium

The molecular mechanisms mediating cell surface trafficking of caveolae are unknown. Caveolae bud from plasma membranes to form free carrier vesicles through a “pinching off” or fission process requiring cytosol and driven by GTP hydrolysis (Schnitzer, J.E., P. Oh, and D.P. McIntosh. 1996. Science. 274:239–242). Here, we use several independent techniques and functional assays ranging from cell-free to intact cell systems to establish a function for dynamin in the formation of transport vesicles from the endothelial cell plasma membrane by mediating fission at the neck of caveolae. This caveolar fission requires interaction with cytosolic dynamin as well as its hydrolysis of GTP. Expression of dynamin in cytosol as well as purified recombinant dynamin alone supports GTP-induced caveolar fission in a cell-free assay whereas its removal from cytosol or the addition to the cytosol of specific antibodies for dynamin inhibits this fission. Overexpression of mutant dynamin lacking normal GTPase activity not only inhibits GTP-induced fission and budding of caveolae but also prevents caveolae-mediated internalization of cholera toxin B chain in intact and permeabilized endothelial cells. Analysis of endothelium in vivo by subcellular fractionation and immunomicroscopy shows that dynamin is concentrated on caveolae, primarily at the expected site of action, their necks. Thus, through its ability to oligomerize, dynamin appears to form a structural collar around the neck of caveolae that hydrolyzes GTP to mediate internalization via the fission of caveolae from the plasma membrane to form free transport vesicles.     

Assembly and trafficking of caveolar domains in the cell: caveolae as stable, cargo-triggered, vesicular transporters

Using total internal reflection fluorescence microscopy (TIR-FM), fluorescence recovery after photobleaching (FRAP), and other light microscopy techniques, we analyzed the dynamics, the activation, and the assembly of caveolae labeled with fluorescently tagged caveolin-1 (Cav1). We found that when activated by simian virus 40 (SV40), a non-enveloped DNA virus that uses caveolae for cell entry, the fraction of mobile caveolae was dramatically enhanced both in the plasma membrane (PM) and in the caveosome, an intracellular organelle that functions as an intermediate station in caveolar endocytosis. Activation also resulted in increased microtubule (MT)-dependent, long-range movement of caveolar vesicles. We generated heterokaryons that contained GFP- and RFP-tagged caveolae by fusing cells expressing Cav1-GFP and -RFP, respectively, and showed that even when activated, individual caveolar domains underwent little exchange of Cav1. Only when the cells were subjected to transient cholesterol depletion, did the caveolae domain exchange Cav1. Thus, in contrast to clathrin-, or other types of coated transport vesicles, caveolae constitute stable, cholesterol-dependent membrane domains that can serve as fixed containers through vesicle traffic. Finally, we identified the Golgi complex as the site where newly assembled caveolar domains appeared first.     

Caveolin moves from caveolae to the Golgi apparatus in response to cholesterol oxidation

Caveolae are a membrane specialization used to internalize molecules by potocytosis. Caveolin, an integral membrane protein, is associated with the striated coat present on the cytoplasmic surface of the caveolae membrane. We now report that oxidation of caveolar cholesterol with cholesterol oxidase rapidly displaces the caveolin from the plasma membrane to intracellular vesicles that colocalize with Golgi apparatus markers. After the enzyme is removed from the medium, caveolin returns to caveolae. When untreated cells are gently homogenized, caveolin on the plasma membrane is accessible to both anti-caveolin IgG and trypsin. After cholesterol oxidase treatment, however, Golgi-associated caveolin is inaccessible to both of these molecules. Brefeldin A, which inhibits ER to Golgi trafficking, blocks the appearance of caveolin in the Golgi apparatus but does not prevent caveolin from leaving the plasma membrane. Indirect immunogold localization experiments show that in the presence of cholesterol oxidase caveolin leaves the plasma membrane and becomes associated with endoplasmic reticulum and Golgi compartments. Surprisingly, the loss of caveolin from the plasma membrane does not affect the number or morphology of the caveolae.     

Selective stimulation of caveolar endocytosis by glycosphingolipids and cholesterol

Internalization of some plasma membrane constituents, bacterial toxins, and viruses occurs via caveolae; however, the factors that regulate caveolar internalization are still unclear. Here, we demonstrate that a brief treatment of cultured cells with natural or synthetic glycosphingolipids (GSLs) or elevation of cholesterol (either by acute treatment with mbeta-cyclodextrin/cholesterol or by alteration of growth conditions) dramatically stimulates caveolar endocytosis with little or no effect on other endocytic mechanisms. These treatments also stimulated the movement of GFP-labeled vesicles in cells transfected with caveolin-1-GFP and reduced the number of surface-connected caveolae seen by electron microscopy. In contrast, overexpression of caveolin-1 decreased caveolar uptake, but treatment with GSLs reversed this effect and stimulated caveolar endocytosis. Stimulation of caveolar endocytosis did not occur using ceramide or phosphatidylcholine and was not due to GSL degradation because similar results were obtained using a nonhydrolyzable GSL analog. Stimulated caveolar endocytosis required src kinase and PKC-alpha activity as shown by i) use of pharmacological inhibitors, ii) expression of kinase inactive src or dominant negative PKCalpha, and iii) stimulation of src kinase activity upon addition of GSLs or cholesterol. These results suggest that caveolar endocytosis is regulated by a balance of caveolin-1, cholesterol, and GSLs at the plasma membrane.     

Membrane structure of caveolae and isolated caveolin-rich vesicles

 Caveolae are specialized invaginated domains of the plasma membrane. Using freeze-fracture electron microscopy, the shape of caveolae and the distribution of intramembrane particles (integral membrane proteins) were analyzed. The caveolar membrane is highly curved and forms flask-like invaginations with a diameter of 80–120 nm with an open porus of 30–50 nm in diameter. The fracture faces of caveolar membranes are nearly free of intramembrane particles. Protein particles in a circular arrangement surrounding the caveolar opening were found on plasma membrane fracture faces. For isolation of caveolin-enriched membrane vesicles, the method of Triton X-100 solubilization, as well as a detergent-free isolation method, was used. The caveolin-rich vesicles had an average size of between 100 and 200 nm. No striated coat could be detected on the surface of isolated caveolin-rich vesicles. Areas of clustered intramembrane particles were found frequently on membrane fracture faces of caveolin-rich vesicles. The shape of these membrane protein clusters is often ring-like with a diameter of 30–50 nm. Membrane openings were found to be present in the caveolin-rich membrane vesicles, mostly localized in the areas of the clustered membrane proteins. Immunogold labeling of caveolin showed that the protein is a component within the membrane protein clusters and is not randomly distributed on the membrane of caveolin-rich vesicles. Accepted: 16 September 1998     

Caveolin-1 is a negative regulator of caveolae-mediated endocytosis to the endoplasmic reticulum.

Caveolae are flask-shaped invaginations at the plasma membrane that constitute a subclass of detergent-resistant membrane domains enriched in cholesterol and sphingolipids and that express caveolin, a caveolar coat protein. Autocrine motility factor receptor (AMF-R) is stably localized to caveolae, and the cholesterol extracting reagent, methyl-beta-cyclodextrin, inhibits its internalization to the endoplasmic reticulum implicating caveolae in this distinct receptor-mediated endocytic pathway. Curiously, the rate of methyl-beta-cyclodextrin-sensitive endocytosis of AMF-R to the endoplasmic reticulum is increased in ras- and abl-transformed NIH-3T3 cells that express significantly reduced levels of caveolin and few caveolae. Overexpression of the dynamin K44A dominant negative mutant via an adenovirus expression system induces caveolar invaginations sensitive to methyl-beta-cyclodextrin extraction in the transformed cells without increasing caveolin expression. Dynamin K44A expression further inhibits AMF-R-mediated endocytosis to the endoplasmic reticulum in untransformed and transformed NIH-3T3 cells. Adenoviral expression of caveolin-1 also induces caveolae in the transformed NIH-3T3 cells and reduces AMF-R-mediated endocytosis to the endoplasmic reticulum to levels observed in untransformed NIH-3T3 cells. Cholesterol-rich detergent-resistant membrane domains or glycolipid rafts therefore invaginate independently of caveolin-1 expression to form endocytosis-competent caveolar vesicles via rapid dynamin-dependent detachment from the plasma membrane. Caveolin-1 stabilizes the plasma membrane association of caveolae and thereby acts as a negative regulator of the caveolae-mediated endocytosis of AMF-R to the endoplasmic reticulum.     

The shape of caveolae is omega-like after glutaraldehyde fixation and cup-like after cryofixation

Caveolae were defined as flask- or omega-shaped plasma membrane invaginations, abundant in adipocytes, fibroblasts, endothelial and smooth muscle cells. The major protein component of caveolar membranes is an integral membrane protein named caveolin. We compared the freeze-fracture behavior of caveolae in glutaraldehyde-fixed and cryofixed mouse fibroblast cells and found distinct differences. In glutaraldehyde-fixed cells almost all caveolae were cross-fractured through their pore and only very few caveolar membranes were membrane-fractured. We found the reverse situation in rapid frozen cells without any chemical fixation where most of the caveolae were membrane-fractured, showing different degrees of invagination from nearly flat to deeply invaginated. In ultrathin sections of glutaraldehyde-fixed heart endothelial cells, caveolae exhibit the well known omega-like shape. In high-pressure frozen, freeze-substituted and low temperature embedded heart endothelial cells, the caveolae frequently exhibit a cup-like shape without any constriction or pore. The cup-like caveolar shape could also be shown by tilt series analysis of freeze-fracture replicas obtained from cryofixed cells. Freeze-fracture immunolabeling of caveolin-1 revealed a lateral belt-like caveolin alignment. These findings point out that the constricted “neck” region of caveolae in most cases is an effect that is caused and intensified by the glutaraldehyde fixation. Our data indicate that caveolae in vivo show all degrees of invagination from nearly flat via cup-like depressed to in a few cases omega-like.     

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.     

Spiral Coating of the Endothelial Caveolar Membranes as Revealed by Electron Tomography and Template Matching

Caveolae are invaginations of the plasma membrane involved in multiple cellular processes, including transcytosis. In this paper we present an extensive 3-D electron tomographic study of the endothelial caveolar system in situ . Analysis of large cellular volumes of (high-pressure frozen, freeze-substituted and epon-embedded) human umbilical vein endothelial cells (HUVECs) provided a notable view on the architecture of the caveolar system that comprises – as confirmed by 3-D immunolabeling for caveolin of 'intact' cells – bona fide caveolae, free plasmalemmal vesicles, racemose invaginations and free multi-caveolar bodies. Application of template matching to tomograms allowed the 3-D localization of caveolar membrane coatings in a robust manner. In this way we observed that bona fide endothelial caveolae, cryofixed and embedded in their cellular context, show a spiral organization of the coating as shown in the past for chemically fixed and freeze-etched caveolae from fibroblasts. Meticulous 3-D analysis further revealed that the coatings are distributed in triads of spirals over the caveolar bulb and neck. Remarkably, this coating distribution is consistently present over the membranes of the other members of the caveolar system in HUVECs. The novel observations that we present clarify the ultrastructural complexity of the 'intact' caveolar system, setting a detailed morphological basis for its functional diversity.     

Gangliosides and β1‐Integrin Are Required for Caveolae and Membrane Domains

Caveolae are plasma membrane domains involved in the uptake of certain pathogens and toxins. Internalization of some cell surface integrins occurs via caveolae suggesting caveolae may play a crucial role in modulating integrin‐mediated adhesion and cell migration. Here we demonstrate a critical role for gangliosides (sialo‐glycosphingolipids) in regulating caveolar endocytosis in human skin fibroblasts. Pretreatment of cells with endoglycoceramidase (cleaves glycosphingolipids) or sialidase (modifies cell surface gangliosides and glycoproteins) selectively inhibited caveolar endocytosis by >70%, inhibited the formation of plasma membrane domains enriched in sphingolipids and cholesterol (‘lipid rafts'), reduced caveolae and caveolin‐1 at the plasma membrane by approximately 80%, and blunted activation of β1‐integrin, a protein required for caveolar endocytosis in these cells. These effects could be reversed by a brief incubation with gangliosides (but not with asialo‐gangliosides or other sphingolipids) at 10°C, suggesting that sialo‐lipids are critical in supporting caveolar endocytosis. Endoglycoceramidase treatment also caused a redistribution of focal adhesion kinase, paxillin, talin, and PIP Kinase Iγ away from focal adhesions. The effects of sialidase or endoglycoceramidase on membrane domains and the distribution of caveolin‐1 could be recapitulated by β1‐integrin knockdown. These results suggest that both gangliosides and β1‐integrin are required for maintenance of caveolae and plasma membrane domains.     

Caveolae and sorting in the trans-Golgi network of epithelial cells.

VIP21 is a 21 kDa membrane protein present in TGN-derived transport vesicles isolated from the epithelial MDCK cell line. The membrane topology and subcellular localization of VIP21 were studied using antibodies against the N- and C-terminal domains. The protein was found to have a structure with little or no exposure to the exoplasmic side of the membrane. VIP21 was localized to the TGN, consistent with its presence in TGN-derived transport vesicles. Unexpectedly, it was also very abundant in the non-clathrin-coated plasma membrane invaginations called caveolae. We have previously proposed that VIP21 is associated with glycosphingolipid-enriched membrane domains in the TGN which may be involved in the sorting of proteins into vesicles directed to the apical plasma membrane. Caveolae are specialized lipid structures with similarities to the glycolipid microdomains in the TGN. The presence of VIP21 in both locations suggests that the mechanisms governing inclusion of proteins into caveolar plasma membrane domains are related to the processes of protein and lipid sorting at the TGN. This connection is confirmed by the recent finding that the amino acid sequence of VIP21 is almost identical to that of caveolin, a protein previously localized to caveolae.     

Belt-like localisation of caveolin in deep caveolae and its re-distribution after cholesterol depletion

Caveolae are specialised vesicular microdomains of the plasma membrane. Using freeze-fracture immunogold labelling and stereoscopic imaging, the distribution of labelled caveolin 1 in caveolae of 3T3-L1 mouse fibroblast cells was shown. Immunogold-labelled caveolin structures surrounded the basolateral region of deeply invaginated caveolae like a belt whereas in the apical region distal to the plasma membrane, the caveolin labelling was nearly absent. Shallow caveolar membranes showed a dispersed caveolin labelling. After membrane cholesterol reduction by methyl-ß-cyclodextrin treatment, a dynamic re-distribution of labelled caveolin 1 and a flattening of caveolar structures was found. The highly curved caveolar membrane got totally flat, and the initial belt-like caveolin labelling disintegrated to a ring-like structure and later to a dispersed order. Intramembrane particle-free domains were still observable after cholesterol depletion and caveolin re-distribution. These results indicate that cholesterol interacting with caveolin structures at the basolateral part of caveolae is necessary for the maintenance of the deeply invaginated caveolar membranes.     

Caveolar endocytosis and virus entry

The endocytic function of caveolae has been controversial for a long time. However, a real-time-imaging analysis of Simian virus 40 (SV40) 's entry in cells has indicated the existence of caveolar endocytosis during virus entry. The caveolae engulfed SV40 virions begin budding from plasma membrane depending on dynamin. SV40 enclosed in caveolae vesicles move to the caveosome, then to the endoplasmic reticulum. In addition, it was demonstrated that human coronavirus-229E enters the cell through caveolae. This review examines the involvement of caveolae in endocytosis used by the viral entry system.     

Integrin-linked kinase controls microtubule dynamics required for plasma membrane targeting of caveolae

Caveolae are specialized compartments of the plasma membrane that are involved in signaling, endocytosis, and cholesterol transport. Their formation requires the transport of caveolin-1 to the plasma membrane, but the molecular mechanisms regulating the transport are largely unknown. Here, we?identify a critical role for adhesion-mediated signaling through β1 integrins and integrin-linked kinase (ILK) in caveolae formation. Mice lacking β1 integrins or ILK in keratinocytes have dramatically reduced numbers of plasma membrane caveolae in?vivo, which is due to impaired transport of caveolin-1-containing vesicles along microtubules (MT) to the plasma membrane. Mechanistically, ILK promotes the recruitment of the F-actin binding protein IQGAP1 to the cell cortex, which, in turn, cooperates with its?effector mDia1 to locally stabilize MTs and to allow?stable insertion of caveolae into the plasma membrane. Our results assign an important role to the integrin/ILK complex for caveolar trafficking to the cell surface.     

Spatiotemporal analysis of endocytosis and membrane distribution of fluorescent sterols in living cells

Distribution and dynamics of cholesterol in the plasma membrane as well as internalization pathways for sterol from the cell surface are of great cell biological interest. Here, UV-sensitive wide field microscopy of the intrinsically fluorescent sterols, dehydroergosterol (DHE) and cholestatrienol (CTL) combined with advanced image analysis were used to study spatiotemporal sterol distribution in living macrophages, adipocytes and fibroblasts. Sterol endocytosis was directly visualized by time-lapse imaging and noise-robust tracking revealing confined motion of DHE containing vesicles in close proximity to the cell membrane. Spatial surface intensity patterns of DHE as well as that of the lipid marker DiIC12 being assessed by statistical image analysis persisted over several minutes in cells having a constant overall curvature. Sites of sterol endocytosis appeared indistinguishable from other regions of the cell surface, and endocytosis contributed by 62% to total sterol uptake in J774 cells. DHE co-localized with fluorescent transferrin (Tf) in vesicles right after onset of endocytosis and in deepened surface patches of energy depleted cells. Surface caveolae labeled with GFP-tagged caveolin were not particularly enriched in DHE or CTL. Some sterol co-localized with internalized caveolin suggesting that caveolar endocytosis contributes to vesicular sterol uptake. These findings demonstrate that plasma membrane sterol is internalized by several endocytic pathways. Sterol endocytosis does not require formation of microscopically resolvable sterol clusters or enrichment of sterol in surface caveolae. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Caveolin regulation of endothelial function

Caveolae are the sites in the cell membrane responsible for concentrating an array of signaling molecules critical for cell function. Recent studies have begun to identify the functions of caveolin-1, the 22-kDa caveolar protein that oligomerizes and inserts into the cytoplasmic face of the plasma membrane. Caveolin-1 appears to regulate caveolar internalization by stabilizing caveolae at the plasma membrane rather than controlling the shape of the membrane invagination. Because caveolin-1 is a scaffolding protein, it has also been hypothesized to function as a "master regulator" of signaling molecules in caveolae. Deletion of the caveolin-1 gene in mice resulted in cardiac hypertrophy and lung fibrosis, indicating its importance in cardiac and lung development. In the endothelium, caveolin-1 regulates nitric oxide signaling by binding to and inhibiting endothelial nitric oxide synthase (eNOS). Increased cytosolic Ca2+ or activation of the kinase Akt leads to eNOS activation and its dissociation from caveolin-1. Caveolae have also been proposed as the vesicle carriers responsible for transcellular transport (transcytosis) in endothelial cells. Transcytosis, the primary means of albumin transport across continuous endothelia, occurs by fission of caveolae from the membrane. This event is regulated by tyrosine phosphorylation of caveolin-1 and dynamin. As Ca2+ influx channels and pumps are localized in caveolae, caveolin-1 is also an important determinant of Ca2+ signaling in endothelial cells. Many of these findings were presented in San Diego, CA, at the 2003 Experimental Biology symposium "Caveolin Regulation of Endothelial Function" and are reviewed in this summary.     

Distinct mechanisms of clathrin-independent endocytosis have unique sphingolipid requirements

Sphingolipids (SLs) play important roles in membrane structure and cell function. Here, we examine the SL requirements of various endocytic mechanisms using a mutant cell line and pharmacological inhibitors to disrupt SL biosynthesis. First, we demonstrated that in Chinese hamster ovary cells we could distinguish three distinct mechanisms of clathrin-independent endocytosis (caveolar, RhoA, and Cdc42 dependent) which differed in cargo, sensitivity to pharmacological agents, and dominant negative proteins. General depletion of SLs inhibited endocytosis by each clathrin-independent mechanism, whereas clathrin-dependent uptake was unaffected. Depletion of glycosphingolipids (GSLs; a subgroup of SLs) selectively blocked caveolar endocytosis and decreased caveolin-1 and caveolae at the plasma membrane. Caveolar endocytosis and PM caveolae could be restored in GSL-depleted cells by acute addition of exogenous GSLs. Disruption of RhoA- and Cdc42-regulated endocytosis by SL depletion was shown to be related to decreased targeting of these Rho proteins to the plasma membrane and could be partially restored by exogenous sphingomyelin but not GSLs. Both the in vivo membrane targeting and in vitro binding to artificial lipid vesicles of RhoA and Cdc42 were shown to be dependent upon sphingomyelin. These results provide the first evidence that SLs are differentially required for distinct mechanisms of clathrin-independent endocytosis.     

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.     

Tyrosine phosphorylation of caveolin-1 in the endothelium

Caveolin-1, a scaffolding protein of caveolae, is known to be tyrosine-phosphorylated by Src kinases. Recently we generated a specific antibody to caveolin-1 phosphorylated at tyrosine-14 (PY14) (R. Nomura and T. Fujimoto, 1999, Mol. Biol. Cell 10, 975-986). In the present study, by applying PY14 to sections of normal rat tissues, we found that tyrosine phosphorylation of caveolin-1 occurred in limited locations, including the endothelium of the continuous capillaries and small venules. Cultured endothelial cells were not labeled by PY14 under a standard culture condition, but became positively labeled when exposed to oxidative stresses and/or tyrosine phosphatase inhibitors. The reaction was prohibited by pretreating the cells with herbimycin A or genistein. Vasoactive reagents or physical stimuli did not cause the phosphorylation. Concomitant with the tyrosine phosphorylation, the number of invaginated caveolae decreased drastically, and vesicles labeled intensely for caveolin-1 appeared in the cytoplasm; the average diameter of the vesicles was larger than that of caveolae. The result implies that tyrosine phosphorylation of caveolin-1 occurs at tyrosine-14 in the normal rat endothelium in vivo and may induce caveolar vesiculation and/or fusion.