Key Points Caveolae, submicroscopic pits of the plasma membrane, consist of caveolin membrane proteins and cytoplasmic cavin proteins. Caveolae can bud from the plasma membrane, fuse with early endosomes and recycle back to the cell surface, or they can be turned over via a ubiquitylation-dependent mechanism and targeted to multivesicular bodies. Mutations in caveolins and cavins have been linked to diverse disease states, including cancer, lipodystrophy, cardiomyopathy and muscular dystrophies. The various diseases linked to caveolae dysfunction suggest a crucial cellular role in lipid regulation, membrane organization and in cell protection against physical stress.
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Advanced Search Caveolae are strikingly abundant in endothelial cells, yet the physiological functions of caveolae in endothelium and other tissues remain incompletely understood. Previous studies suggest a mechanoprotective role, but whether this is relevant under the mechanical forces experienced by endothelial cells in vivo is unclear. In this study we have sought to determine whether endothelial caveolae disassemble under increased hemodynamic forces, and whether caveolae help prevent acute rupture of the plasma membrane under these conditions.
Experiments in cultured cells established biochemical assays for disassembly of caveolar protein complexes, and assays for acute loss of plasma membrane integrity.
In vivo, we demonstrate that caveolae in endothelial cells of the lung and cardiac muscle disassemble in response to acute increases in cardiac output. These data imply that mechanoprotection through disassembly of caveolae is important for endothelial function in vivo. Introduction Caveolae, flask-shaped invaginations of the plasma membrane, are highly abundant in endothelial cells, and are therefore likely to play an important role in endothelial cell biology Tse and Stan, Defining precisely how caveolae function in the endothelium and elsewhere has proved challenging, but phenotypes of mice lacking genes essential for the biogenesis of caveolae provide evidence that caveolae play an important role in the cardiovascular system.
These mice have alterations in the permeability of continuous endothelium Schubert et al. Humans with mutations in the same genes also exhibit cardiac arrhythmias and pulmonary hypertension, and have enlarged blood vessels Rajab et al. Caveolae-deficient mice and humans have metabolic phenotypes consistent with a role for caveolae in adipocytes Pilch and Liu, and muscular dystrophy Galbiati et al.
Adipocytes and muscle cells are also cells where caveolae are abundant; hence, there is a correlation between abundance of caveolae and their importance for cell function. Caveolae are formed by large complexes of caveolin and cavin proteins Hill et al.
Caveolins are multiply acylated, embedded in the inner leaflet of the plasma membrane, and oligomerize to form the most membrane-proximal component of the caveolar coat complex. Cavins are soluble proteins that are recruited to caveolins after biosynthetic delivery of the latter to the plasma membrane. Caveolin 1, caveolin 2, cavin 1, and the additional cavins 2 and 3, can all be purified from cells as a single 80S caveolar coat complex after chemical cross-linking Ludwig et al.
Recent experiments in cultured cells have revived the idea that caveolae may have a simple mechanoprotective role Dulhunty and Franzini-Armstrong, ; Sinha et al. In this model, caveolae act as membrane convolutions that can flatten in response to forces within the membrane, thereby buffering such forces and reducing the chance of critical membrane rupture or loss of cell—cell contact Parton and del Pozo, The model is attractive because it provides a good explanation for the abundance of caveolae in some cell types.
The situation is complicated by data suggesting that endocytosis of caveolae occurs in response to plasma membrane damage Corrotte et al.
Both endocytosis and flattening of caveolae may cause changes in the abundance of these structures at the plasma membrane, thus it is crucial to develop assays for disassembly of caveolae that do not rely solely on morphological criteria. The goal of this study was to address the potential mechanoprotective function of caveolae in endothelial cells in vivo. We designed experiments to test the two key aspects of the hypothesis that disassembly and flattening out of caveolae buffers mechanical forces within the plasma membrane Sinha et al.
First, we asked whether disassembly of caveolae can be detected in intact tissue under physiological conditions. We used tissue culture cell experiments to characterize both biochemical and electron microscopy assays for caveolar disassembly and applied these assays to mice where cardiac output, and hence hemodynamic force on endothelial cells, is increased.
Additional experiments rule out endocytosis as an alternative explanation for changes in caveolar abundance. Results and discussion Biochemical assays for disassembly of caveolae under mechanical force Cells of the endothelium-derived cell line bEnd5 were mechanically stressed by exposure to hypo-osmotic medium, and by cycles of repeated stretching after growth on polydimethylsiloxane, a deformable substrate. We quantified the effect of these treatments on the abundance of morphologically defined caveolae using electron microscopy of complete perimeters of multiple individual cells Fig.
S1, A and B ; Hansen et al. As predicted, both treatments rapidly and significantly reduced the number of morphological caveolae Sinha et al. If the loss of caveolae Fig. S1 were because of disassembly of caveolar coat complexes, then one would predict that this would release soluble caveolar proteins such as cavin 1 from the membrane into the cytoplasm Hayer et al. Western blotting of cytosolic fractions after ultracentrifugation to pellet membranes showed that both hypo-osmotic medium and stretching on a deformable substrate clearly caused a time-dependent increase in the cytosolic pool of cavin 1 Fig.
Caveolin 1 remained in the membrane fraction, and the total amount of cavin 1 present in the cell did not change during the experiment Fig. Interpretation of cytosolic accumulation of cavin 1 as direct evidence for disassembly of caveolae is a central part of our conclusions; therefore, we performed additional experiments to confirm this.
The cavin 1 released into the cytosol under hypo-osmotic conditions was present in two peaks on sucrose velocity gradients. The higher molecular weight peak fraction 8 had the same size as the basal amount of cytosolic cavin 1 detected normally. Crucially, the second peak, comprising smaller complexes fraction 4 , was not observed under isotonic conditions Fig. Therefore, large cavin 1—containing complexes disassemble under mechanical stress induced by hypotonic conditions Gambin et al.
Essentially all of the cavin and caveolin proteins present in caveolae can be purified as a single 80S complex after chemical cross-linking Ludwig et al. If caveolae disassemble, then this complex should be lost. Incubation of cells under hypo-osmotic conditions for 10 min did indeed result in loss of the cross-linked 80S complex because caveolin 1 and cavin 1 no longer cofractionated in high molecular weight fractions on sucrose gradients and instead accumulated in much smaller complexes Fig.
This provides direct evidence for disassembly of the caveolar coat complex, and confirms that the accumulation of cavin 1 in the cytosol reported in Fig. It was possible that changes in the abundance of morphologically defined caveolae observed by electron microscopy could reflect budding from the plasma membrane.
Accordingly, we performed endocytosis assays using total surface protein biotinylation to label all endosomes in bEnd5 cells Bitsikas et al. These experiments were performed using both hypo-osmotic buffer and stretching after growth on a deformable substrate. In hypo-osmotic conditions the extent to which caveolin 1 colocalized with cavin 1 was markedly reduced, consistent with disassembly of caveolae, and under these conditions endocytosis was blocked Fig.
Quantification, using a pixel-mask approach Shvets et al. We conclude that loss of morphologically defined caveolae from the plasma membrane is, therefore, indeed because of disassembly of caveolae. This causes increased heart contractility and output and is used clinically to mimic effects of exercise Marcovitz and Armstrong, Pulse plethysmography showed that dobutamine caused a pronounced elevation of both pulse rate and pulse distension 1 min after i.
S2 A ; Desai et al. After homogenization and cell lysis the soluble and membrane fractions from each tissue were isolated by centrifugation, and the presence of cavin 1 was assayed by Western blotting see Fig. In both the heart and lung dobutamine caused an increase in the amount of soluble cavin 1 Fig.
This was not the case in abdominal muscle. The data suggest that caveolae disassemble in heart and lung in response to dobutamine-induced increases in cardiac output. Electron microscopy was used to confirm that morphologically defined caveolae become less abundant in endothelial cells in response to dobutamine.
The time taken for dissection and fixation of tissue and for heterogeneity in caveolar abundance between different sections of endothelium from the same tissue is a potentially confounding factor in this type of experiment. We focused on the heart and the lung, tissues that can be dissected rapidly, and where the biochemical experiments reported previously suggested that an effect would be found. In both tissues electron microscopy of microvessels revealed that there are considerable stretches of plasma membrane devoid of caveolae in the dobutamine-treated samples, although caveolae are clearly still present Fig.
Quantification of the number of caveolae, defined as distinctive omega shapes open at the luminal face of the endothelial cell, in complete reconstructions of microvessel perimeters from control and dobutamine-treated mice revealed a reduction in response to the drug Fig.
Therefore, endothelial caveolae are indeed likely to disassemble in response to increased cardiac output. Caveolae prevent rupture of endothelial plasma membranes under physiological hemodynamic force If disassembly of caveolae does protect cells from plasma membrane disruption under mechanical stress, then one would predict that, in the absence of caveolae, cells might be more susceptible to acute rupture of the plasma membrane.
In order assay acute membrane rupture we used membrane-impermeant, nucleic-acid-binding dyes, which will confer bright staining of the nucleus only when integrity of the plasma membrane is lost Jones and Singer, In this case, quantification was performed by confocal imaging Fig.
These experiments show that staining with membrane-impermeant nuclear dyes can be used to assay plasma membrane disruption, and that caveolae do indeed protect cells from rupture of the plasma membrane during increased membrane tension. Intravenous injection of FITC-albumin and propidium iodide a membrane-impermeant nuclear dye analogous to SYTOX Green provided a way to image blood vessels in intact tissue, and to label nuclei only where the plasma membrane is damaged.
Loss of plasma membrane integrity in endothelial cells was indicated by nuclear staining with propidium iodide that colocalized with blood vessels highlighted with FITC-albumin. Because caveolin 1 is not expressed in cardiac muscle, one would predict that this could be a result of endothelial cell dysfunction. Given our findings, it seemed likely that ruptured endothelial cells will contribute to the phenotype.
To test this, electron microscopy was used to examine the ultrastructure of endothelial cells from microvessels in cardiac muscle of the right ventricle of mice exposed to hypoxic conditions for 3 wk. Endothelial cells that appeared devoid of cytoplasmic staining, and had a distended membrane outline, were frequently detected Fig. Quantification of the abundance of these abnormal endothelial cells confirmed that caveolae play a significant role in preventing cell damage during hypoxia Fig.
Discussion Two different but nonexclusive mechanisms have been proposed that could explain the mechanoprotective role for caveolae in vivo revealed by our experiments. Caveolae can disassemble, and flattening of the membrane of the caveolar bulb would then increase the effective surface area of the cell, preventing buildup of excessive force within the plane of the membrane Sinha et al.
Additionally, caveolae may bud from the membrane and internalize damaged regions, leading to resealing Corrotte et al. Counting of caveolae in the plasma membrane does not discriminate between the two models because both flattening and budding may decrease the number of surface-connected caveolae.
Our in vivo experiments show that cavin 1 is released from the membrane to cytosolic fractions upon increased blood flow. Given the considerable ex vivo data suggesting that association of cavin 1 with caveolin 1 at the plasma membrane is key for adoption of the characteristic membrane shape of caveolae Hill et al.
There is considerable evidence for endothelial cell dysfunction and cardiovascular phenotypes in mice lacking caveolin 1; however, these phenotypes do not by themselves lend strong support for one specific mechanism by which caveolae act Drab et al. The accumulation of damaged endothelial cells under chronic conditions, such as hypoxia in our experiments, could be attributable to many factors involving cell signaling or other processes.
However, our data show that by using an acute perturbation 1 min of dobutamine treatment and an assay, which specifically detects loss of plasma membrane integrity, that loss of caveolae leads to instantaneous rupture of the membrane under increased hemodynamic stress. We suggest that this mechanism is likely to be a contributing factor to the range of cardiovascular phenotypes consequent to deletion of caveolin 1. Although disassembly of caveolae to release membrane convolutions, and thereby buffer mechanical force within the plasma membrane, is an appealingly simple mechanism by which caveolae may exert mechanoprotective effects, and although this mechanism provides an elegant rationale for the extreme abundance of caveolae within endothelium, it is possible that caveolae may have further functional roles.
Repetitive membrane damage as a result of mechanical force has been well studied in the context of muscle cells and other systems Lammerding and Lee, Similar damage to endothelial cells has been less investigated; however, studies in other systems suggest multiple mechanisms that may allow cells to reseal damaged plasma membrane Lammerding and Lee, ; Han, ; Jimenez et al.
It is probable that these or similar mechanisms act in endothelial cells, thus flattening of caveolae will provide one layer of a complex set of processes involved in mechanoprotection.
However, disassembly of caveolae is conceptually different from mechanisms for resealing damaged plasma membrane because it provides the cell with a method to prevent lesions rather than to repair them. Antibodies and reagents The following antibodies were used: rabbit anti—cavin 1 ab; Abcam and rabbit anti—caveolin 1 ; BD Biosciences.
Cell lysates were spun at 13, rpm for 10 min to remove large cellular debris followed by a final centrifugation step at 55, rpm in a benchtop Optima Max ultracentrifuge Beckman Coulter to isolate membrane and cytosolic fractions.
For analysis of tissues from mice, the animals were killed by cervical dislocation. Immediately, and as rapidly as possible within 30 s of cervical dislocation , the heart, lungs, and abdominal muscle were removed in a defined order and instantly frozen in liquid nitrogen.
Frozen tissues were ground using a pestle and mortar before differential centrifugation as described previously. Sucrose velocity gradients Chemical cross-linking was performed precisely as described Ludwig et al.
Twelve 1-ml fractions were collected from the bottom of the gradient by tube puncture Ludwig et al. Heart and abdominal muscle were dissected for two-photon imaging of nonfixed, fresh whole-mount tissues using a NLO upright microscope Carl Zeiss. Quantification of image overlap was performed as described Bitsikas et al.
Contrast settings were adjusted to facilitate visualization of overlaid pseudo-color images. All such manipulations were applied equally across the whole image.
Caveolae as plasma membrane sensors, protectors and organizers
Caveolins[ edit ] Formation and maintenance of caveolae was initially thought to be primarily due to caveolin ,  a 21 kD protein. There are three homologous genes of caveolin expressed in mammalian cells: Cav1, Cav2 and Cav3. These proteins have a common topology: cytoplasmic N-terminus with scaffolding domain, long hairpin transmembrane domain and cytoplasmic C-terminus. Caveolins are synthesized as monomers and transported to the Golgi apparatus. During their subsequent transport through the secretory pathway, caveolins associate with lipid rafts and form oligomers molecules.
Caveolae protect endothelial cells from membrane rupture during increased cardiac output
No static citation data Abstract Caveolae are submicroscopic, plasma membrane pits that are abundant in many mammalian cell types. The past few years have seen a quantum leap in our understanding of the formation, dynamics and functions of these enigmatic structures. Caveolae have now emerged as vital plasma membrane sensors that can respond to plasma membrane stresses and remodel the extracellular environment. Caveolae at the plasma membrane can be removed by endocytosis to regulate their surface density or can be disassembled and their structural components degraded.