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.     

Functional interaction of caveolin-1 and eNOS in myocardial capillary endothelium revealed by immunoelectron microscopy.

Immunogold labeling on samples of isolated perfused rat hearts embedded by an innovative low-temperature LR White procedure provided detailed insight into the interaction of caveolin-1 and endothelial NOS in myocardial capillary endothelium at the subcellular level. Separately, the localization of caveolin-1 and eNOS at caveolae under steady state conditions was visualized. A double-labeling experiment supported their close co-localization. Short-term bradykinin stimulation caused a detectable dissociation of eNOS from caveolin and its redistribution to different cell compartments, whereas caveolin itself remained stationary at caveolae. Morphometric analysis revealed that more than 80% of detectable eNOS was co-localized with caveolin-1 at caveolae under control conditions. After brief stimulation for 2 min with 10(-7) M bradykinin, only 26% of the eNOS signals were associated with caveolin-1 and randomly distributed over the endothelial cells. After stimulation, eNOS was found at the plasmalemmal and intracellular membranes, freely in the cytoplasm, and at outer mitochondrial membranes.     

Loss of caveolin expression in type I pneumocytes as an indicator of subcellular alterations during lung fibrogenesis

 Caveolin is a major structural protein of caveolae, also known as plasmalemmal vesicles, which are particularly abundant in type I pneumocytes and capillary endothelial cells of lung parenchyma. Here we demonstrate that caveolin expression in the alveolar epithelium of rats and mini pigs is strikingly downregulated after irradiation-induced lung injury. Indirect immunoperoxidase staining with polyclonal anti-caveolin antibodies, confirmed by double fluorescence studies with type I cell-specific monoclonal anti-cytokeratin antibodies or lectins, revealed a dramatic loss of caveolin immunoreactivity in type I pneumocytes. In contrast, caveolin expression increased in endothelial cells. Immunoblotting of lung homogenates from normal and irradiated rats using specific anti-caveolin antibodies confirmed the presence of caveolin in normal tissue and its marked decrease of expression in fibrotic tissue. The loss of caveolin as an important structural protein of caveolae in alveolar epithelial cells may be an early indicator of serious type I cell injury during fibrogenesis. The increase of caveolin immunoreactivity in endothelia of blood vessels may indicate that different types of caveolae and/or different regulatory mechanisms of caveolin expression exist. Accepted: 28 May 1997     

Caveolin and its cellular and subcellular immunolocalisation in lung alveolar epithelium: implications for alveolar epithelial type I cell function

Caveolae are flask-shaped invaginations of the plasmalemma which pinch off to form discrete vesicles within the cell cytoplasm. Biochemically, caveolae may be distinguished by the presence of a protein, caveolin, that is the principal component of filaments constituting their striated cytoplasmic coat. Squamous alveolar epithelial type I (ATI) cells, comprising approximately 95% of the surface area of lung alveolar epithelium, possess numerous plasmalemmal invaginations and cytoplasmic vesicles ultrastructurally indicative of caveolae. However, an ultrastructural appearance does not universally imply the biochemical presence of caveolin. This immunocytochemical study has utilised a novel application of confocal laser scanning and electron microscopy unequivocally to localise caveolin-1 to ATI cells. Further, cytoplasmic vesicles and flask-shaped membrane invaginations in the ATI cell were morphologically identified whose membranes were decorated with anti-caveolin-1 immunogold label. Coexistent with this, however, in both ATI and capillary endothelial cells could be seen membrane invaginations morphologically characteristic of caveolae, but which lacked associated caveolin immunogold label. This could reflect a true biochemical heterogeneity in populations of morphologically similar plasmalemmal invaginations or an antigen threshold requirement for labelling. The cuboidal alveolar epithelial type II cell (ATII) also displayed specific label for caveolin-1 but with no ultrastructural evidence for the formation of caveolae. The biochemical association of caveolin with ATI cell vesicles has broad implications for the assignment and further study of ATI cell function.     

Caveolin‐1 regulates proliferation and osteogenic differentiation of human mesenchymal stem cells

Caveolin‐1 is a scaffolding protein of cholesterol‐rich caveolae lipid rafts in the plasma membrane. In addition to regulating cholesterol transport, caveolin‐1 has the ability to bind a diverse array of cell signaling molecules and regulate cell signal transduction in caveolae. Currently, there is little known about the role of caveolin‐1 in stem cells. It has been reported that the caveolin‐1 null mouse has an expanded population of cells expressing stem cell markers in the gut, mammary gland, and brain, suggestive of a role for caveolin‐1 in stem cell regulation. The caveolin‐1 null mouse also has increased bone mass and an increased bone formation rate, and its bone marrow‐derived mesenchymal stem cells (MSCs) have enhanced osteogenic potential. However, the role of caveolin‐1 in human MSC osteogenic differentiation remains unexplored. In this study, we have characterized the expression of caveolin‐1 in human bone marrow derived MSCs. We show that caveolin‐1 protein is enriched in density gradient‐fractionated MSC plasma membrane, consisting of ～100 nm diameter membrane‐bound vesicles, and is distributed in a punctate pattern by immunofluoresence localization. Expression of caveolin‐1 increases in MSCs induced to undergo osteogenic differentiation, and siRNA‐mediated knockdown of caveolin‐1 expression enhances MSC proliferation and osteogenic differentiation. Taken together, these findings suggest that caveolin‐1 normally acts to regulate the differentiation and renewal of MSCs, and increased caveolin‐1 expression during MSC osteogenesis likely acts as a negative feedback to stabilize the cell phenotype. J. Cell. Biochem. 113: 3773–3787, 2012. © 2012 Wiley Periodicals, Inc.     

The regulation of caveolin expression and localization by serum and heparin in vascular smooth muscle cells

Caveolae have been implicated in growth factor receptor and G-protein coupled receptor signaling in vascular cells. It has been postulated that caveolin, the structural protein of caveolae, may act as a general tyrosine kinase inhibitor by binding and inhibiting signaling molecules involved in the activation of the MAP kinase proliferation cascade. Using an in vitro model of VSMC proliferation, we found that serum stimulation caused a dose dependent decrease in both caveolin-1 and caveolin-2 protein levels in human coronary artery smooth muscle cells. Heparin, an inhibitor of VSMC proliferation, inhibited the serum-induced loss of caveolin-1 and caveolin-2. In addition, heparin caused an increase in both caveolin-1 and caveolin-2 localization to caveolae-enriched sucrose gradient membrane fractions when compared to serum alone. Taken together, caveolin may play an important role in the regulation of VSMC proliferation and heparin and serum have opposing effects on caveolin expression and localization in VSMC.     

Tyrosine-phosphorylated caveolin is a physiological substrate of the low M(r) protein-tyrosine phosphatase

Low M(r) phosphotyrosine-protein phosphatase is involved in the regulation of several tyrosine kinase growth factor receptors. The best characterized action of this enzyme is on the signaling pathways activated by platelet-derived growth factor, where it plays multiple roles. In this study we identify tyrosine-phosphorylated caveolin as a new potential substrate for low M(r) phosphotyrosine-protein phosphatase. Caveolin is tyrosine-phosphorylated in vivo by Src kinases, recruits into caveolae, and hence regulates the activities of several proteins involved in cellular signaling cascades. Our results demonstrate that caveolin and low M(r) phosphotyrosine-protein phosphatase coimmunoprecipitate from cell lysates, and that a fraction of the enzyme localizes in caveolae. Furthermore, in a cell line sensitive to insulin, the overexpression of the C12S dominant negative mutant of low M(r) phosphotyrosine-protein phosphatase (a form lacking activity but able to bind substrates) causes the enhancement of tyrosine-phosphorylated caveolin. Insulin stimulation of these cells induces a strong increase of caveolin phosphorylation. The localization of low M(r) phosphotyrosine-protein phosphatase in caveolae, the in vivo interaction between this enzyme and caveolin, and the capacity of this enzyme to rapidly dephosphorylate phosphocaveolin, all indicate that tyrosine-phosphorylated caveolin is a relevant substrate for this phosphatase.     

The urokinase-type plasminogen activator receptor, a GPI-linked protein, is localized in caveolae

The urokinase plasminogen activator receptor (uPAR), a glycosylphosphatidylinositol-linked glycoprotein, plays a central role in the regulation of pericellular proteolysis and participates in events leading to cell activation. Here, we demonstrate that uPAR, on a human melanoma cell line, is localized in caveolae, flask-shaped microinvaginations of the plasma membrane found in a variety of cell types. Indirect immunofluorescence with anti-uPAR antibodies on the melanoma cells showed a punctated staining pattern that accumulated to stretches along sides of cell-cell contact and membrane ruffles. uPAR colocalized with caveolin, a characteristic protein in the coat of caveolae, as demonstrated by double staining with specific antibodies. Further, uPAR could be directly localized in caveolae by in vivo immunoelectron microscopy. Both uPAR and its ligand, uPA, were present in caveolae enriched low density Triton X-100 insoluble complexes, as shown by immunoblotting. From such complexes, caveolin could be coprecipitated with uPAR-specific antibodies suggesting a close spatial association between uPAR and caveolin that might have implications for the signal transduction mediated by uPAR. Further, functional studies indicated that the localization of uPAR and its ligand in caveolae enhances pericellular plasminogen activation, since treatment of the cells with drugs that interfere with the structural integrity of caveolae, such as nystatin, markedly reduced cell surface plasmin generation. Thus, caveolae promote efficient cell surface plasminogen activation by clustering uPAR, uPA, and possibly other protease receptors in one membrane compartment.     

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.     

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.     

Organization of G Proteins and Adenylyl Cyclase at the Plasma Membrane

There is mounting evidence for the organization and compartmentation of signaling molecules at the plasma membrane. We find that hormone-sensitive adenylyl cyclase activity is enriched in a subset of regulatory G protein-containing fractions of the plasma membrane. These subfractions resemble, in low buoyant density, structures of the plasma membrane termed caveolae. Immunofluorescence experiments revealed a punctate pattern of G protein α and β subunits, consistent with concentration of these proteins at distinct sites on the plasma membrane. Partial coincidence of localization of G protein α subunits with caveolin (a marker for caveolae) was observed by double immunofluorescence. Results of immunogold electron microscopy suggest that some G protein is associated with invaginated caveolae, but most of the protein resides in irregular structures of the plasma membrane that could not be identified morphologically. Because regulated adenylyl cyclase activity is present in low-density subfractions of plasma membrane from a cell type (S49 lymphoma) that does not express caveolin, this protein is not required for organization of the adenylyl cyclase system. The data suggest that hormone-sensitive adenylyl cyclase systems are localized in a specialized subdomain of the plasma membrane that may optimize the efficiency and fidelity of signal transduction.     

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.     

Raft association and lipid droplet targeting of flotillins are independent of caveolin

Lipid rafts are liquid ordered platforms that dynamically compartmentalize membranes. Caveolins and flotillins constitute a group of proteins that are enriched in these domains. Caveolin-1 has been shown to be an essential component of caveolae. Flotillins were also discovered as an integral component of caveolae and have since been suggested to interact with caveolins. However, flotillins are also expressed in non-caveolae-containing cells such as lymphocytes and neuronal cells. Hence, a discrepancy exists in the literature regarding the caveolin dependence of flotillin expression and their subcellular localization. To address this controversy, we used mouse embryonic fibroblasts (MEFs) from caveolin-1 knockout (Cav-1(-/-)) and wild-type mice to study flotillin expression and localization. Here we show that both membrane association and lipid raft partitioning of flotillins are not perturbed in Cav-1(-/-) MEFs, whereas membrane targeting and raft partitioning of caveolin-2, another caveolin family protein, is severely impaired. Moreover, we demonstrate that flotillin-1, but not flotillin-2, associates with lipid droplets upon oleic acid treatment and that this association is completely independent of caveolin. Taken together, our results show that flotillins are localized in lipid rafts independent of caveolin-1 and that translocation of flotillin-1 to lipid droplets is a caveolin-independent process.     

Oxidized low density lipoprotein displaces endothelial nitric-oxide synthase (eNOS) from plasmalemmal caveolae and impairs eNOS activation.

Hypercholesterolemia-induced vascular disease and atherosclerosis are characterized by a decrease in the bioavailability of endothelium-derived nitric oxide. Endothelial nitric-oxide synthase (eNOS) associates with caveolae and is directly regulated by the caveola protein, caveolin. In the present study, we examined the effects of oxidized low density lipoprotein (oxLDL) on the subcellular location of eNOS, on eNOS activation, and on caveola cholesterol in endothelial cells. We found that treatment with 10 microgram/ml oxLDL for 60 min caused greater than 90% of eNOS and caveolin to leave caveolae. Treatment with oxLDL also inhibited acetylcholine-induced activation of eNOS but not prostacyclin production. oxLDL did not affect total cellular eNOS abundance. Oxidized LDL also did not affect the palmitoylation, myristoylation or phosphorylation of eNOS. Oxidized LDL, but not native LDL, or HDL depleted caveolae of cholesterol by serving as an acceptor for cholesterol. Cyclodextrin also depleted caveolae of cholesterol and caused eNOS and caveolin to translocate from caveolae. Furthermore, removal of oxLDL allowed eNOS and caveolin to return to caveolae. We conclude that oxLDL-induced depletion of caveola cholesterol causes eNOS to leave caveolae and inhibits acetylcholine-induced activation of the enzyme. This process may be an important mechanism in the early pathogenesis of atherosclerosis.     

Localization of caveolin-3 in the sinus endothelial cells of the rat spleen

The localization of caveolins in the sinus endothelial cells of the rat spleen has been demonstrated by confocal laser scanning and electron microscopy. Caveolin-3, a muscle-specific caveolin, was detected by Western blot analysis and immunofluorescence microscopy of isolated sinus endothelial cells and tissue cryosections of the spleen. During the immunofluorescence microscopy of isolated endothelial cells, both caveolin-3 and caveolin-1 were found. In tissue cryosections of the spleen, caveolin-3, as well as caveolin-1 and -2, was present in the contours and cytoplasm of the cells. Immunogold electron microscopy of tissue cryosections revealed caveolin-3, -1, and -2 to be present in caveolae in the apical, lateral, and basal plasma membranes and some vesicular profiles in the cytoplasm of sinus endothelial cells. Furthermore, caveolin-3 was colocalized with caveolin-1 in the same caveolae in the apical, lateral, and basal plasma membranes. Stress fibers and tubulovesicular structures were situated in the vicinity of caveolae labeled with anti-caveolin-3, anti-caveolin-1, and anti-caveolin-2 antibodies. It is speculated that caveolae in sinus endothelial cells play an important role in the constriction of stress fibers.     

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.     

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.     

”In vivo cryotechnique” in combination with replica immunoelectron microscopy for caveolin in smooth muscle cells

A novel ”in vivo cryotechnique” with replica immunoelectron microscopy was developed for detecting caveolin localization on replica membranes prepared directly from living smooth muscle cells. After quick-freezing mouse duodenal walls by our ”in vivo cryotechnique”, the specimens were prepared for freeze-fracture and deep-etch replica membranes. Then they were treated with 5% SDS and 0.5% collagenase to keep some antigens on the replica membranes. The immunogold method could be used to clarify the localization of the caveolin antigen in relation to three-dimensional ultrastructures of living smooth muscle cells. Our new cryotechnique can provide native organization of functional molecules in living cells. Accepted: 7 October 1999     

Clustering of GPI-Anchored Folate Receptor Independent of Both Cross-Linking and Association with Caveolin

The distribution of the glycosyl-phosphatidylinositol (GPI)-anchored folate receptor (FR) in a diffuse pattern vs. functional clusters associated with caveolae has been debated. The equivocal nature of direct localization studies is due to possible experimental artifacts such as cross-linking of the protein by the antibody probes prior to fixation and alternatively the use of a disruptive fixation method. Such studies have also been complicated by the use of cells that vastly overexpress FR. In this study a monovalent probe, i.e., a biotinylated folate affinity analogue was used to covalently label FR. Cells expressing moderate levels of FR, i.e., JAR epithelial cells expressing FR-α and recombinant CHO fibroblasts expressing FR-β, were used. The affinity label and either caveolin or antigenic sites on FR were localized by electron microscopy using colloidal gold conjugated antibody probes post-embedding in the relatively permeable LR White resin. The method avoided both receptor cross-linking and early fixation steps and also enabled the use of transport permissive conditions while labeling FR at the cell surface. The results indicate that in steady-state FR is not significantly colocalized with caveolin. However, the receptor molecules occur predominantly in clusters, independent of cross-linking, providing a physical basis for the observed kinetics of receptor internalization and recycling during folate transport. Evidence is also presented to suggest that early mild fixation will disrupt the clustering of FR. Received: 26 September 1996/Revised: 7 May 1997