PTRF-Cavin, a conserved cytoplasmic protein required for caveola formation and function

Caveolae are abundant cell-surface organelles involved in lipid regulation and endocytosis. We used comparative proteomics to identify PTRF (also called Cav-p60, Cavin) as a putative caveolar coat protein. PTRF-Cavin selectively associates with mature caveolae at the plasma membrane but not Golgi-localized caveolin. In prostate cancer PC3 cells, and during development of zebrafish notochord, lack of PTRF-Cavin expression correlates with lack of caveolae, and caveolin resides on flat plasma membrane. Expression of PTRF-Cavin in PC3 cells is sufficient to cause formation of caveolae. Knockdown of PTRF-Cavin reduces caveolae density, both in mammalian cells and in the zebrafish. Caveolin remains on the plasma membrane in PTRF-Cavin knockdown cells but exhibits increased lateral mobility and accelerated lysosomal degradation. We conclude that PTRF-Cavin is required for caveola formation and sequestration of mobile caveolin into immobile caveolae.     

Cavin1; a Regulator of Lung Function and Macrophage Phenotype

Caveolae are cell membrane invaginations that are highly abundant in adipose tissue, endothelial cells and the lung. The formation of caveolae is dependent on the expression of various structural proteins that serve as scaffolding for these membrane invaginations. Cavin1 is a newly identified structural protein whose deficiency in mice leads to loss of caveolae formation and to development of a lipodystrophic phenotype. In this study, we sought to investigate the functional role of Cavin1 in the lung. Cavin1 deficient mice possessed dramatically altered distal lung morphology and exhibited significant physiological alterations, notably, increased lung elastance. The changes in distal lung architecture were associated with hypercellularity and the accumulation of lung macrophages. The increases in lung macrophages occurred without changes to circulating numbers of mononuclear cells and without evidence for increased proliferation. However, the increases in lung macrophages were associated with higher levels of macrophage chemotactic factors CXCL2 and CCL2 in BAL fluid from Cavin1−/− mice suggesting a possible mechanism by which these cells accumulate. In addition, lung macrophages from Cavin1−/− mice were larger and displayed measurable differences in gene expression when compared to macrophages from wild-type mice. Interestingly, macrophages were also increased in adipose tissue but not in liver, kidney or skeletal muscle from Cavin1−/− mice, and similar tissue specificity for macrophage accumulation was observed in lungs and adipose tissue from Caveolin1−/− mice. In conclusion, this study demonstrates an important role for Cavin1 in lung homeostasis and suggests that caveolae structural proteins are necessary for regulating macrophage number and phenotype in the lung.     

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     

Cells respond to mechanical stress by rapid disassembly of caveolae

The functions of caveolae, the characteristic plasma membrane invaginations, remain debated. Their abundance in cells experiencing mechanical stress led us to investigate their role in membrane-mediated mechanical response. Acute mechanical stress induced by osmotic swelling or by uniaxial stretching results in a rapid disappearance of caveolae, in a reduced caveolin/Cavin1 interaction, and in an increase of free caveolins at the plasma membrane. Tether-pulling force measurements in cells and in plasma membrane spheres demonstrate that caveola flattening and disassembly is the primary actin- and ATP-independent cell response that buffers membrane tension surges during mechanical stress. Conversely, stress release leads to complete caveola reassembly in an actin- and ATP-dependent process. The absence of a functional caveola reservoir in myotubes from muscular dystrophic patients enhanced membrane fragility under mechanical stress. Our findings support a new role for caveolae as a physiological membrane reservoir that quickly accommodates sudden and acute mechanical stresses.     

Characterisation of caveolins from cartilage: expression of caveolin-1, -2 and -3 in chondrocytes and in alginate cell culture of the rat tibia

This study was performed to determine if rat articular chondrocytes express caveolin, the structural protein of caveolae, and to determine differences in the distribution of the caveolin subtypes 1, 2 and 3 in knee joints of newborn and adult rats. All three subtypes of caveolin were detected in adult cartilage by immunocytochemical staining. In newborn rats, only caveolin-1 was found in the hyaline cartilage. Caveolin-1, -2 and -3 messenger RNA and protein were also detected in chondrocyte cell cultures. Ultrastructural investigations of cell culture and cartilage tissue revealed the presence of caveolae at the plasma membrane of chondrocytes. These findings represent the first report on the different expression of caveolin isoforms, in particular the expression of the muscle cell-specific caveolin-3 in chondrocytes. There is evidence that caveolin-2 and -3 are upregulated during growth and development of articular cartilage, suggesting a role for caveolins in chondrocyte differentiation. Accepted: 4 May 1999     

Caveolins, caveolae, and lipid rafts in cellular transport, signaling, and disease.

Caveolae were initially described some 50 years ago. For many decades, they remained predominantly of interest to structural biologists. The identification of a molecular marker for these domains, caveolin, combined with the possibility to isolate such cholesterol- and sphingolipid-rich regions as detergent-insoluble membrane complexes paved the way to more rigorous characterization of composition, regulation, and function. Experiments with knock-out mice for the caveolin genes clearly demonstrate the importance of caveolin-1 and -3 in formation of caveolae. Nonetheless, detergent-insoluble domains are also found in cells lacking caveolin expression and are referred to here as lipid rafts. Caveolae and lipid rafts were shown to represent membrane compartments enriched in a large number of signaling molecules whose structural integrity is essential for many signaling processes. Caveolin-1 is an essential structural component of cell surface caveolae, important for regulating trafficking and mobility of these vesicles. In addition, caveolin-1 is found at many other intracellular locations. Variations in subcellular localization are paralleled by a plethora of ascribed functions for this protein. Here, more recent data addressing the role of caveolin-1 in cellular signaling and the development of diseases like cancer will be preferentially discussed.     

Cavin proteins: New players in the caveolae field

Caveolae are specialized lipid microdomains, forming small invaginations in the plasma membrane, known to be implicated in multiple functions including lipid storage, cell signaling and endocytosis. Formation of these wide flask-shaped invaginations is dependent on the expression of a caveolar coat protein, namely caveolin. Until now, the accepted paradigm was that caveolin was the sole and only structural protein of caveolae since its expression was necessary and sufficient to drive caveolae biogenesis. The recent characterizations of PTRF/cavin-1 and subsequently other cavin family members in caveolae formation have highlighted additional levels of complexity in the biogenesis of these plasma membrane invaginations. In this review, recent advances on the role of the different cavin family members in the regulation of caveolae structures as well as potential new functions will be discussed.     

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.     

Cavin4 interacts with Bin1 to promote T-tubule formation and stability in developing skeletal muscle

The cavin proteins are essential for caveola biogenesis and function. Here, we identify a role for the muscle-specific component, Cavin4, in skeletal muscle T-tubule development by analyzing two vertebrate systems, mouse and zebrafish. In both models, Cavin4 localized to T-tubules, and loss of Cavin4 resulted in aberrant T-tubule maturation. In zebrafish, which possess duplicated cavin4 paralogs, Cavin4b was shown to directly interact with the T-tubule–associated BAR domain protein Bin1. Loss of both Cavin4a and Cavin4b caused aberrant accumulation of interconnected caveolae within the T-tubules, a fragmented T-tubule network enriched in Caveolin-3, and an impaired Ca²⁺ response upon mechanical stimulation. We propose a role for Cavin4 in remodeling the T-tubule membrane early in development by recycling caveolar components from the T-tubule to the sarcolemma. This generates a stable T-tubule domain lacking caveolae that is essential for T-tubule function.     

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.     

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.     

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.     

Is caveolin involved in normal proximal tubule function? Presence in model PT systems but absence in situ

Kidney proximal tubule (PT) cells are specialized for the uptake and transport of ions, solutes, peptides, and proteins. These functions are often regulated by hormones that signal at the cell surface and are internalized by clathrin-mediated endocytosis. However, the caveolin/caveolae pathway has also been implicated in normal PT function, often based on data from isolated PTs or PT cells in culture. Although we reported previously that caveolae and caveolin 1 are not detectable in PTs in vivo, reports of caveolin expression and function in PT cells appear periodically in the literature. Therefore, we reexamined caveolin expression in PTs in vivo, in isolated "purified" PTs following collagenase digestion, and in cultured PT cells. Caveolin 1 and 2 protein, mRNA, or immunofluorescence was undetectable in PTs in vivo, but PT cell cultures expressed caveolin 1 and/or 2. Furthermore, caveolin 1 and 2 mRNAs were detected in isolated PTs along with the endothelial markers CD31 and ICAM1. In contrast, no caveolin or endothelial marker mRNAs were detectable in samples isolated from snap-frozen kidneys by laser cut microdissection, which eliminates contamination by other cell types. We conclude 1) caveolin 1 and 2 are not normally expressed by PT cells in situ, 2) caveolin expression is "activated" in cultured PT cells, 3) contamination with non-PT, caveolin-expressing cells is a potential source of caveolin 1 and 2 that must be taken into account when isolated PTs are used in studies to correlate expression of these proteins with PT function.     

Getting rid of caveolins: phenotypes of caveolin-deficient animals

The elucidation of the role of caveolae has been the topic of many investigations which were greatly enhanced after the discovery of caveolin, the protein marker of these flask-shaped plasma membrane invaginations. The generation of mice deficient in the various caveolin genes (cav-1, cav-2 and cav-3) has provided physiological models to unravel the role of caveolins or caveolae at the whole organism level. Remarkably, despite the essential role of caveolins in caveolae biogenesis, all knockout mice are viable and fertile. However, lack of caveolae or caveolins leads to a wide range of phenotypes including muscle, pulmonary or lipid disorders, suggesting their implication in many cellular processes. The aim of this review is to give a broad overview of the phenotypes described for the caveolin-deficient mice and to link them to the numerous functions so far assigned to caveolins/caveolae.     

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.