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.     

PC12 cells have caveolae that contain TrkA. Caveolae-disrupting drugs inhibit nerve growth factor-induced, but not epidermal growth factor-induced, MAPK phosphorylation

Nerve growth factor (NGF) induces survival and differentiation of the neural crest-derived PC12 cell line. Caveolae are cholesterol-enriched, caveolin-containing plasma membrane microdomains involved in vesicular transport and signal transduction. Here we demonstrate the presence of caveolae in PC12 cells and their involvement in NGF signaling. Our results showed the expression of caveolin-1 by Western blot and confocal immuno-microscopy. The presence of plasma membrane caveolae was directly shown by rapid-freeze deep-etching electron microscopy. Moreover, combined deep-etching and immunogold techniques revealed the presence of the NGF receptor TrkA in the caveolae of PC12 cells. These data together with the cofractionation of Shc, Ras, caveolin, and TrkA in the caveolae fraction supported a role for these plasma membrane microdomains in NGF signaling. To approach this hypothesis, caveolae were disrupted by treatment of PC12 cells with cholesterol binding drugs. Either filipin or cyclodextrin treatment increased basal levels of MAPK phosphorylation. In contrast, pretreatment of PC12 cells with these drugs inhibited the NGF- but not the epidermal growth factor-induced MAPK phosphorylation without affecting the TrkA autophosphorylation. Taken together, our results demonstrate the presence of caveolae in PC12 cells, which contain the high affinity NGF receptor TrkA, and the specific involvement of these cholesterol-enriched plasma membrane microdomains in the propagation of the NGF-induced signal.     

Caveolin interacts with the angiotensin II type 1 receptor during exocytic transport but not at the plasma membrane

The mechanisms involved in angiotensin II type 1 receptor (AT1-R) trafficking and membrane localization are largely unknown. In this study, we examined the role of caveolin in these processes. Electron microscopy of plasma membrane sheets shows that the AT1-R is not concentrated in caveolae but is clustered in cholesterol-independent microdomains; upon activation, it partially redistributes to lipid rafts. Despite the lack of AT1-R in caveolae, AT1-R.caveolin complexes are readily detectable in cells co-expressing both proteins. This interaction requires an intact caveolin scaffolding domain because mutant caveolins that lack a functional caveolin scaffolding domain do not interact with AT1-R. Expression of an N-terminally truncated caveolin-3, CavDGV, that localizes to lipid bodies, or a point mutant, Cav3-P104L, that accumulates in the Golgi mislocalizes AT1-R to lipid bodies and Golgi, respectively. Mislocalization results in aberrant maturation and surface expression of AT1-R, effects that are not reversed by supplementing cells with cholesterol. Similarly mutation of aromatic residues in the caveolin-binding site abrogates AT1-R cell surface expression. In cells lacking caveolin-1 or caveolin-3, AT1-R does not traffic to the cell surface unless caveolin is ectopically expressed. This observation is recapitulated in caveolin-1 null mice that have a 55% reduction in renal AT1-R levels compared with controls. Taken together our results indicate that a direct interaction with caveolin is required to traffic the AT1-R through the exocytic pathway, but this does not result in AT1-R sequestration in caveolae. Caveolin therefore acts as a molecular chaperone rather than a plasma membrane scaffold for AT1-R.     

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.     

Constitutive formation of caveolae in a bacterium

Caveolin plays an essential role in the formation of characteristic surface pits, caveolae, which cover the surface of many animal cells. The fundamental principles of caveola formation are only slowly emerging. Here we show that caveolin expression in a prokaryotic host lacking any intracellular membrane system drives the formation of cytoplasmic vesicles containing polymeric caveolin. Vesicle formation is induced by expression of wild-type caveolins, but not caveolin mutants defective in caveola formation in mammalian systems. In addition, cryoelectron tomography shows that the induced membrane domains are equivalent in size and caveolin density to native caveolae and reveals a possible polyhedral arrangement of caveolin oligomers. The caveolin-induced vesicles or heterologous caveolae (h-caveolae) form by budding in from the cytoplasmic membrane, generating a membrane domain with distinct lipid composition. Periplasmic solutes are encapsulated in the budding h-caveola, and purified h-caveolae can be tailored to be targeted to specific cells of interest.     

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.     

Caveolin cycles between plasma membrane caveolae and the Golgi complex by microtubule-dependent and microtubule-independent steps

Caveolin is a protein associated with the characteristic coats that decorate the cytoplasmic face of plasma membrane caveolae. Recently it was found that exposure of human fibroblasts to cholesterol oxidase (CO) rapidly induces caveolin to redistribute to the ER and then to the Golgi complex, and that subsequent removal of CO allows caveolin to return to the plasma membrane (Smart, E. J., Y.-S. Ying, P. A. Conrad, R. G. W. Anderson, J. Cell Biol. 1994, 127:1185-1197). We now present evidence that caveolin normally undergoes microtubule-dependent cycling between the plasma membrane and the Golgi. In cells that were treated briefly with nocodazole and then with a mixture of nocodazole plus CO, caveolin relocated from the plasma membrane to the ER and then to the ER/Golgi intermediate compartment (ERGIC), but subsequent movement to the Golgi was not observed. Even in the absence of CO, nocodazole caused caveolin to accumulate in the ERGIC. Nocodazole did not retard the movement of caveolin from the Golgi to the plasma membrane after removal of CO. Incubation of cells at 15 degrees followed by elevation of the temperature to 37 degrees caused caveolin to accumulate first in the ERGIC and then in the Golgi, before finally reestablishing its normal steady state distribution predominantly in plasma membrane caveolae. In cells released from a 15 degrees block, movement of caveolin from the Golgi to the plasma membrane was not inhibited by nocodazole. Taken together, these results imply that caveolin cycles constitutively between the plasma membrane and the Golgi by a multi- step process, one of which, ERGIC-to-Golgi transport, requires microtubules. This novel, bidirectional pathway may indicate roles for microtubules in the maintenance of caveolae, and for caveolin in shuttling fatty acids and cholesterol between the plasma membrane and the ER/Golgi system.     

Regulating c-Ras function. cholesterol depletion affects caveolin association, GTP loading, and signaling.

Cholesterol-rich and caveolin-containing microdomains of the plasma membrane, termed "caveolae," have been implicated in signal transduction. However, the role of caveolae in regulating the Ras-MAP kinase cascade is incompletely understood. The mammalian Ras isoforms (H, N, and K) use different membrane anchors to attach to the plasma membrane and thereby may localize to functionally distinct microdomains, which might explain isoform-specific signaling. Here, we show that, in Cos epithelial cells, endogenous K-Ras colocalizes largely with caveolin, whereas N-Ras localizes to both caveolar and noncaveolar subdomains; H-Ras localization was below detection limits. We find that epidermal growth factor (EGF) activates N-Ras but fails to activate K-Ras in these cells. Extraction of cholesterol with methyl-beta-cyclodextrin disrupts complex formation between caveolin and K- and N-Ras and, strikingly, enables EGF to activate both K-Ras and N-Ras. While cholesterol depletion enhances GTP-loading on total c-Ras, activation of the downstream MEK-MAP kinase cascade by EGF and lysophosphatidic acid but not that by phorbol ester is inhibited. Thus, plasma membrane cholesterol is essential for negative regulation of c-Ras isoforms (complexed to caveolin), as well as for mitogenic signaling downstream of receptor-activated c-Ras.     

Identification of signals and mechanisms of sorting of plasma membrane proteins in intestinal epithelial cells]

In epithelial cells the plasma membrane is divided into domains that are biochemically and functionally different. In intestinal cells for example the apical domain is facing the intestinal lumen and is involved in the uptake of nutriments while the basolateral domain is mediating cell-cell adhesion and signalisation. We are interested in deciphering the mechanisms underlying the creation and maintenance of such specialized domains. As an epithelial model we have used the intestinal cell line Caco-2 and we have studied the transport and sorting of the human neurotrophin receptor (p75 NTR) in these cells. Newly synthesized p75 NTR is first transported to the basolateral membrane and then is accumulated on the apical membrane after transcytosis. This final apical localization is controlled by the presence of a membrane anchor and a cluster of O-glycosylation sites located in the part of the ectodomain close to the membrane. Among the mechanisms likely to be involved in the sorting of apical components we have looked for a role of lipid-protein microdomain formation in the Golgi apparatus. These membrane microdomains are highly enriched in glycosylphosphatidyl inositol (GPI) anchored proteins, glycosphingolipids and apical proteins such as sucrase isomaltase (SI). Such a composition is also found for endocytic structures called caveolae which are made of caveolin 1. We have expressed caveolin 1 in Caco-2 cells which do not express it and also caveolin 2, a related protein of unknown function. Expression of caveolin 1 led to formation of caveolae indicating that this protein is necessary for caveolae formation while caveolin 2 is restricted to the Golgi apparatus and has no effect on caveolae formation. However Caveolin 2 increased the amount of SI incorporated in microdomains suggesting a role in recruitment into the apical pathway. The choice for a site of fusion for transport vesicles is the last step of control during exocytosis. To identify proteins involved in that step we have cloned and characterized two members of the t-SNARE family, namely syntaxin 3 and SNAP23. Syntaxin 3 is present on the apical membrane and forms a complex with SNAP23 which is also localized on the basolateral membrane where it forms a complex with syntaxin 4. Overexpression of syntaxin 3 in Caco-2 led to a decrease of SI exocytosis towards the apical membrane confirming that syntaxin 3 is involved in targeting the fusion of apical transport vesicles to the apical pole of the cells.     

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.     

Colocalization of beta-adrenergic receptors and caveolin within the plasma membrane.

The rapid amplification of beta-adrenergic receptor signaling involves the sequential activation of multiple signaling molecules ranging from the receptor to adenylyl cyclase. The prevailing view of the agonist-induced interaction between signaling molecules is based on random collisions between proteins that diffuse freely in the plasma membrane. The recent identification of G protein alpha- and betagamma-subunits in caveolae and their functional interaction with caveolin suggests that caveolae may participate in G protein-coupled signaling. We have investigated the potential interaction of beta-adrenergic receptors with caveolin under resting conditions. beta1- and beta2-adrenergic receptors were recombinantly overexpressed in COS-7 cells. Caveolae were isolated using the detergent-free sucrose gradient centrifugation method. beta1- and beta2-adrenergic receptors were localized in the same gradient fractions as caveolin, where Gsalpha- and betagamma-subunits were detected as well. Immunofluorescence microscopy demonstrated the colocalization of beta-adrenergic receptors with caveolin, indicating a nonrandom distribution of beta-adrenergic receptors in the plasma membrane. Using polyhistidine-tagged recombinant proteins, beta-adrenergic receptors were copurified with caveolin, suggesting that they were physically bound. Our results suggest that, in addition to clathrin-coated pits, caveolae may act as another plasma membrane microdomain to compartmentalize beta-adrenergic receptors.     

Lipids and glycosphingolipids in caveolae and surrounding plasma membrane of primary rat adipocytes.

We have made a comprehensive and quantitative analysis of the lipid composition of caveolae from primary rat fat cells and compared the composition of plasma membrane inside and outside caveolae. We isolated caveolae from purified plasma membranes using ultrasonication in carbonate buffer to disrupt the membrane, or extraction with nonionic detergent, followed by density gradient ultracentrifugation. The carbonate-isolated caveolae fraction was further immunopurified using caveolin antibodies. Carbonate-isolated caveolae were enriched in cholesterol and sphingomyelin, and the concentration was three- and twofold higher, respectively, in caveolae compared to the surrounding plasma membrane. The concentration of glycerophospholipids was similar suggesting that glycerophospholipids constitute a constant core throughout the plasma membrane. The composition of detergent-insoluble fractions of the plasma membrane was very variable between preparations, but strongly enriched in sphingomyelin and depleted of glycerophospholipids compared to carbonate-isolated caveolae; indicating that detergent extraction is not a suitable technique for caveolae preparation. An average adipocyte caveola contained about 22 x 10(3) molecules of cholesterol, 7.5 x 10(3) of sphingomyelin and 23 x 10(3) of glycerophospholipid. The glycosphingolipid GD3 was highly enriched in caveolae, whereas GM3, GM1 and GD1a were present inside as well as outside the caveolae membrane. GD1b, GT1b, GM2, GQ1b, sulfatide and lactosylceramide sulfate were not detected in caveolae.     

Intracellular retention of caveolin 1 in presenilin-deficient cells

Mutations in genes encoding presenilins (PS1 and PS2) are responsible for the majority of early onset familial Alzheimer's disease. PS, a critical component of gamma-secretase, is responsible for the intramembranous cleavage of amyloid precursor protein and Notch. Other physiological functions have been assigned to PS without any clear identification of the mechanisms underlying these multiple biological roles. The early embryonic lethality of PS1 and PS2 double knock-out (PS1/2 null) mice prevents the evaluation of physiological roles of PS. To investigate new functions for presenilins, we performed a proteomic approach by using cells derived from PS1/2 null blastocysts and wild type controls. We identified a presenilin-dependent cell-surface binding of albumin. Binding of albumin depends on intact caveolae on the cellular surface. Abnormal caveolin 1 localization in PS1/2 null cells was associated with a loss of caveolae and an absence of caveolin 1 expression within lipid rafts. Expressing PS1 or PS2 but not the intracellular form of Notch1 in PS1/2 null cells restored normal caveolin 1 localization, demonstrating that presenilins are required for the subcellular trafficking of caveolin 1 independently from Notch activity. Despite an expression of both caveolin 1 and PS1 within lipid raft-enriched fractions after sucrose density centrifugation in wild type cells, no direct interaction between these two proteins was detected, implying that presenilins affect caveolin 1 trafficking in an indirect manner. We conclude that presenilins are required for caveolae formation by controlling transport of intracellular caveolin 1 to the plasma membrane.     

Caveolin-associated filamentous actin (Cav-actin) defines a novel F-actin structure in adipocytes

Dynamic actin remodeling has been implicated in the translocation of the insulin-responsive glucose transporter 4 (GLUT4) to the plasma membrane in adipocytes. Here we show that fully differentiated 3T3L1 adipocytes have unique cortical filamentous actin structure, designated Cav-actin (caveolae-associated F-actin). During 3T3L1 adipocyte differentiation, rhodamine-phalloidin staining demonstrated the formation of a cortical actin cytoskeleton that is composed of small dot-like F-actin spikes lining the inside of the plasma membrane. Double labeling with a caveolin antibody indicated that these F-actin spikes emanate from organized rosette-like clusters of caveolae/lipid raft microdomains. In contrast, there was no obvious relationship between F-actin and caveolin localization and/or organization in 3T3L1 preadipocytes (fibroblasts). Treatments of differentiated adipocytes with latrunculin B, Clostridium difficile toxin B or a dominant-interfering TC10 mutant (TC10/T31N) disrupted the Cav-actin structure without significantly affecting the organization of clustered caveolae. Similarly, disruption of the clustered caveolae with methyl-beta-cyclodextrin also dispersed the Cav-actin structure. These data demonstrate that this novel Cav-actin structure is organized through clustered caveolae but that the formation of caveolae-rosettes are not dependent upon F-actin.     

Involvement of caveolin-2 in caveolar biogenesis in MDCK cells

Caveolins have been identified as key components of caveolae, specialized cholesterol-enriched raft domains visible as small flask-shaped invaginations of the plasma membrane. In polarized MDCK cells caveolin-1 and -2 are found together on basolateral caveolae whereas the apical membrane, where only caveolin-1 is present, lacks caveolae. Expression of a caveolin mutant prevented the formation of the large caveolin-1/-2 hetero-oligomeric complexes, and led to intracellular retention of caveolin-2 and disappearance of caveolae from the basolateral membrane. Correspondingly, in MDCK cells over-expressing caveolin-2 the basolateral membrane exhibited an increased number of caveolae. These results indicate the involvement of caveolin-2 in caveolar biogenesis.     

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.     

Ultrastructural freeze-fracture immunolabeling identifies plasma membrane-localized syndapin II as a crucial factor in shaping caveolae

Membrane topology control is thought to involve peripheral membrane proteins of the F-BAR domain family including syndapins. These proteins are predestined to shape membranes by partial insertion and by imposing their curved shape onto the lipid bilayer. Direct observation of such functions on cellular membranes, however, was precluded by the difficulty to combine high-resolution imaging with visualization of membrane topology. Here, we report the ultrastructural visualization of endogenous syndapin II at the plasma membrane of NIH 3T3 cells using a combination of freeze-fracturing, immunogold labeling and transmission electron microscopy. Surprisingly, syndapin II was detected at flat and curved membrane areas. Ultrastructural colocalization with caveolin 1 identified syndapin II-positive invaginations as caveolae. Consistent with the syndapin II F-BAR domain interacting with caveolin 1, F-BAR overexpression affected caveolin 1 localization. Syndapin II knockdown did not alter caveolin 1 expression or plasma membrane recruitment. Instead, syndapin II knockdown reduced the density of caveolae and strongly increased the number of caveolin 1 molecules at flat membrane areas. Comparative immunoelectron microscopy and tilt series revealed that syndapin II was asymmetrically localized at the neck of caveolae. Double-immunogold labeling showed that the caveolae-shaping molecule PTRF/cavin 1 behaved similarly and that syndapin II and PTRF/cavin 1 colocalized. Visualization of a transiently membrane-binding F-BAR protein in direct relation to membrane topology of mammalian cells thereby revealed that syndapin II binds to both flat and curved membranes in vivo and that it plays an important role in caveolar shaping, a role that it shares with PTRF/cavin 1.     

Caveolin Transfection Results in Caveolae Formation but Not Apical Sorting of Glycosylphosphatidylinositol (GPI)-anchored Proteins in Epithelial Cells

Most epithelial cells sort glycosylphosphatidylinositol (GPI)-anchored proteins to the apical surface. The “raft” hypothesis, based on data mainly obtained in the prototype cell line MDCK, postulates that apical sorting depends on the incorporation of apical proteins into cholesterol/glycosphingolipid (GSL) rafts, rich in the cholesterol binding protein caveolin/VIP21, in the Golgi apparatus. Fischer rat thyroid (FRT) cells constitute an ideal model to test this hypothesis, since they missort both endogenous and transfected GPI- anchored proteins to the basolateral plasma membrane and fail to incorporate them into cholesterol/glycosphingolipid clusters. Because FRT cells lack caveolin, a major component of the caveolar coat that has been proposed to have a role in apical sorting of GPI- anchored proteins (Zurzolo, C., W. Van't Hoff, G. van Meer, and E. Rodriguez-Boulan. 1994. EMBO [Eur. Mol. Biol. Organ.] J. 13:42–53.), we carried out experiments to determine whether the lack of caveolin accounted for the sorting/clustering defect of GPI- anchored proteins. We report here that FRT cells lack morphological caveolae, but, upon stable transfection of the caveolin1 gene (cav1), form typical flask-shaped caveolae. However, cav1 expression did not redistribute GPI-anchored proteins to the apical surface, nor promote their inclusion into cholesterol/GSL rafts. Our results demonstrate that the absence of caveolin1 and morphologically identifiable caveolae cannot explain the inability of FRT cells to sort GPI-anchored proteins to the apical domain. Thus, FRT cells may lack additional factors required for apical sorting or for the clustering with GSLs of GPI-anchored proteins, or express factors that inhibit these events. Alternatively, cav1 and caveolae may not be directly involved in these processes.     

Changes in membrane cholesterol affect caveolin-1 localization and ICC-pacing in mouse jejunum

Pacing of mouse is dependent on the spontaneous activity of interstitial cells of Cajal in the myenteric plexus (ICC-MP). These ICC, as well as intestinal smooth muscle, contain small membrane invaginations called caveolae. Caveolae are signaling centers formed by insertions of caveolin proteins in the inner aspect of the plasma membrane. Caveolins bind signaling proteins and thereby negatively modulate their signaling. We disrupted caveolae by treating intestinal segments with methyl beta-clodextrin (CD) to remove cholesterol or with water-soluble cholesterol (WSC) to load cholesterol. Both of these treatments reduced pacing frequencies, and these effects were reversed by the other agent. These treatments also inhibited paced contractions, but complete reversal was not observed. To evaluate the specificity of the effects of CD and WSC, additional studies were made of their effects on responses to carbamoyl choline and to stimulation of cholinergic nerves. Neither of these treatments affected these sets of responses compared with their respective time controls. Immunochemical and ultrastructural studies showed that caveolin 1 was present in smooth muscle membranes and ICC-MP. CD depleted both caveolin 1 and caveolae, whereas WSC increased the amount of caveolin 1 immunoreactivity and altered its distribution but failed to increase the number of caveolae. The effects of each agent were reversed in major part by the other. We conclude that signaling through caveolae may play a role in pacing by ICC but does not affect responses to acetylcholine from nerves or when added exogenously.     

Caveolae Are Highly Immobile Plasma Membrane Microdomains, Which Are not Involved in Constitutive Endocytic Trafficking

To investigate whether caveolae are involved in constitutive endocytic trafficking, we expressed N- and C- terminally green fluorescent protein (GFP)-tagged caveolin- 1 fusion proteins in HeLa, A431, and Madin-Darby canine kidney cells. The fusion proteins were shown by immunogold labeling to be sorted correctly to caveolae. By using confocal microscopy and photobleaching techniques, it was found that although intracellular structures labeled with GFP-tagged caveolin were dynamic, GFP-labeled caveolae were very immobile. However, after incubation with methyl- beta-cyclodextrin, distinct caveolae disappeared and the mobility of GFP-tagged caveolin in the plasma membrane increased. Treatment of cells with cytochalasin D caused lateral movement and aggregation of GFP-labeled caveolae. Therefore, both cholesterol and an intact actin cytoskeleton are required for the integrity of GFP-labeled caveolae. Moreover, stimulation with okadaic acid caused increased mobility and internalization of the labeled caveolae. Although the calculated mobile fraction (for t = infinity) of intracellular, GFP-tagged caveolin- associated structures was 70-90%, GFP-labeled caveolae in unstimulated cells had a mobile fraction of <20%, a value comparable to that previously reported for E-cadherin in junctional complexes. We therefore conclude that caveolae are not involved in constitutive endocytosis but represent a highly stable plasma membrane compartment anchored by the actin cytoskeleton.