Final Report Summary - FUNCTION OF CAVEOLAE (MOLECULAR MECHANISM OF CAVEOLAR STRUCTURE AND FUNCTION)
The research goal of this study was to investigate the structure and function of caveolae in-vivo, with specific attention to the contribution of cavin proteins to this process.
Two main objectives of this study included:
1. To characterize the formation of cavin complexes.
2. Study the component regulating caveolar biogenesis and dynamics.
1. To characterize the formation of cavin complexes in-vivo and in-vitro.
Accumulating evidence support the idea that different functions of caveolae in various tissues require diverse set of other protein complexes. Since cavins show tissue-specific expression profiles, we hypothesized that characteristics of cavin complexes in caveolae may vary between different tissues, consequentially affecting caveolae morphology and function. To address this question we analyzed caveolins and cavins complexes, purified from different murine tissues. To test interrelationship as well as role of different cavins in complex formation we analyzed complexes purified from animals with different knockout backgrounds (such as caveolin1, cavin1, cavin2 and cavin3).
We find that cavins form high molecular weight complexes in tissues and that apparent size of these complexes varies between tissues, with lung and fat containing smaller size complexes, as compared to heart and kidney tissues. We next analyzed the formation of caveolin or cavins complexes in caveolin 1, cavin 1, cavin 2 and cavin 3 gene knockout mice. Caveolin 1 was not responsible for cavins complex formation, while cavin 1, in contrast, was essential for normal oligomerisation of caveolin 1. Deletion of cavin 3 was found to slightly reduce the size of cavin1 complex. Deletion of cavin 2, however, resulted in a marked increase in the size or stability of cavin 1-containing complexes. Importantly, deletion of cavin 2, (but not cavin 3), caused loss of endothelial caveolae in lung and adipose tissue, as judged by electron microscopy. Interestingly, cavin 2-knockout animals had no defect in the abundance of endothelial caveolae in heart and other tissues. This phenotype correlated well with tissue-specific changes of endothelial function in cavin 2 null mice. To gain further insight into the role of cavin 2 specifically in lung endothelial cells, we established cultures of endothelial cells and further confirmed that cavin 2 controls the size of cavin complexes, and acts to shape caveolae. Our data reveal that endothelial caveolae are unexpectedly heterogeneous, and identify cavin 2 as a key molecular determinant of this heterogeneity.
2. Study the components regulating caveolar biogenesis and dynamics.
One outstanding and central issue pertaining to the cell biological function of caveolae is the extent to which they are involved in intracellular trafficking, potentially by acting as autonomous vesicles. Recent developments questioned this idea. It was shown that overexpression of caveolin-1-GFP leads to its ubiquitination and, as a consequence, aberrant accumulation in endosomal compartments that may be mistaken for caveolae-positive endosomes. Overexpressed caveolin-1-GFP can readily be observed in motile intracellular structures, but whether these are induced by overexpression, whether they contain endocytosed cargo of some kind, and whether they also contain cavin are therefore have an assembled caveolar coat have all been unclear. It must be mentioned also that the levels of caveolar proteins are tightly co-regulated, thus suggesting that overexpression of any caveolar component might lead to artificial localization/dynamics thus resulting in misinterpretation. Therefore, we decided that any studies done on caveolar dynamics and endocytosis (which by itself became a question-mark) or caveolar assembly has to be done in system where no overexpression of any caveolar proteins takes place. To overcome this issue, we used genome editing technology (both TALENs endonucleases and CRISPR/Cas9 system) to insert fluorescent tagging into genomic DNA. Using these techniques we successfully tagged both caveolin-1 and cavin1 in mouse cell-line. We then used this system to address the three principal questions in caveolar field: the extent to which caveolae are involved directly in endocytosis, the composition of the caveolar membrane and hence likely function of this endocytosis, and the extent to which patterns of cellular membrane traffic are altered without caveolae.
Using these genome-edited cell lines expressing both caveolin-1-GFP and cavin-1-mCherry from endogenous loci we studied the sub-cellular distribution, dynamics, composition and function of caveolae. We used several imaging techniques including Super-resolution microscopy (STED), electron microscopy and live-cell imaging to approach these questions. We found that both caveolar proteins are present in caveolae on dynamic structures. Their motility was regulated by expression of EHD2 protein, previously shown to affect caveolar dynamics. These structures also contained internalized Cholera toxin Beta subunit, previously shown to be marker for caveolar endocytosis, thus revisiting the existence of caveolar endocytosis. However, quantitative analysis of endocytic flux through caveolae shows that this pathway account for only a small fraction of total protein endocytosis.
Prompted by our analysis of a specific protein coat all around the caveolar bulb, we hypothesised that this coat could modify the local protein complement of the plasma membrane, for example via steric exclusion of proteins with cytosolic domains. To address this question we used a construct composed of a signal peptide, mCherry, an artificial trans-membrane domain composed of 7 repeats of ILA, and a cytosolic FLAG epitope (mCherry-TMD). Ratiometric imaging revealed that this protein is clearly less abundant within caveolae than SNAP-GPI (servinf as positive control for this experiment), implying that even a single trans-membrane helix and cytosolic domain of just 8 amino acid residues is depleted within the caveolar bulb. Moreover, ratiometric imaging confirmed that three different trans-membrane proteins (type I (mCh-LGT46), type II (syntaxin-2-mCh), and polytopic (CD9-mCh)) were also all depleted within caveolae. We used electron microscopy to test the conclusion that trans-membrane proteins are excluded from caveolae more directly. CD9 was fused to APEX, a modified form of ascorbate peroxidase that can be used as a genetically encoded tag for electron microscopy. Consistent with our previous finding CD9 was excluded from caveolae, suggesting that bulk plasma membrane proteins are excluded from caveolae.
The observation that caveolae are relatively depleted of trans-membrane proteins, together with previous reports of glycosphingolipid transport within caveolae, is consistent with a role in lipid trafficking. To determine the cellular function of caveolae, caveolin 1-/- MEFs were loaded with an exogenous fluorescent analogs (BODIPY conjugates) of GM1 and lactosyl ceramide by addition of the lipids bound to albumin. The amount of both lipids incorporated into cells, as well as the sub-cellular distribution of both lipids after short periods of uptake was not notably different in the absence of caveolin 1. Given the above, we reasoned that the effects of loss of caveolae on glycosphingolipid homeostasis may have much slower kinetics than total endocytosis, and may only be apparent when cells are loaded with excess lipid. Indeed when cells were loaded with lipids for longer time periods, the intracellular distribution of both lipid analogs was then strikingly different in the caveolin 1-/- cells. In control cells they accumulated in a peri-nuclear structure that previous studies imply is likely to represent the Golgi apparatus and potentially recycling endosomes, while in knockout cells this peri-nuclear pool was hard to detect, and instead BODIPY was observed accumulated in late endosomes or lysosomes. Similarly, electron microscopy revealed that loading cells with non-flourescent GM1 and lactosyl ceramide resulted in abundant multi-lamellar inclusions in lysosomes of the caveolin 1-/- cells. As no detectable defect on lipid metabolism was detected caveolin 1-/- cells, we concluded that loss of caveolae causes mis-sorting of internalised glycosphingolipid to lysosomes and accumulation of lipids in this location. Thus we propose that one cellular function of caveolar endocytosis is to maintain plasma membrane lipid homeostasis.
Two main objectives of this study included:
1. To characterize the formation of cavin complexes.
2. Study the component regulating caveolar biogenesis and dynamics.
1. To characterize the formation of cavin complexes in-vivo and in-vitro.
Accumulating evidence support the idea that different functions of caveolae in various tissues require diverse set of other protein complexes. Since cavins show tissue-specific expression profiles, we hypothesized that characteristics of cavin complexes in caveolae may vary between different tissues, consequentially affecting caveolae morphology and function. To address this question we analyzed caveolins and cavins complexes, purified from different murine tissues. To test interrelationship as well as role of different cavins in complex formation we analyzed complexes purified from animals with different knockout backgrounds (such as caveolin1, cavin1, cavin2 and cavin3).
We find that cavins form high molecular weight complexes in tissues and that apparent size of these complexes varies between tissues, with lung and fat containing smaller size complexes, as compared to heart and kidney tissues. We next analyzed the formation of caveolin or cavins complexes in caveolin 1, cavin 1, cavin 2 and cavin 3 gene knockout mice. Caveolin 1 was not responsible for cavins complex formation, while cavin 1, in contrast, was essential for normal oligomerisation of caveolin 1. Deletion of cavin 3 was found to slightly reduce the size of cavin1 complex. Deletion of cavin 2, however, resulted in a marked increase in the size or stability of cavin 1-containing complexes. Importantly, deletion of cavin 2, (but not cavin 3), caused loss of endothelial caveolae in lung and adipose tissue, as judged by electron microscopy. Interestingly, cavin 2-knockout animals had no defect in the abundance of endothelial caveolae in heart and other tissues. This phenotype correlated well with tissue-specific changes of endothelial function in cavin 2 null mice. To gain further insight into the role of cavin 2 specifically in lung endothelial cells, we established cultures of endothelial cells and further confirmed that cavin 2 controls the size of cavin complexes, and acts to shape caveolae. Our data reveal that endothelial caveolae are unexpectedly heterogeneous, and identify cavin 2 as a key molecular determinant of this heterogeneity.
2. Study the components regulating caveolar biogenesis and dynamics.
One outstanding and central issue pertaining to the cell biological function of caveolae is the extent to which they are involved in intracellular trafficking, potentially by acting as autonomous vesicles. Recent developments questioned this idea. It was shown that overexpression of caveolin-1-GFP leads to its ubiquitination and, as a consequence, aberrant accumulation in endosomal compartments that may be mistaken for caveolae-positive endosomes. Overexpressed caveolin-1-GFP can readily be observed in motile intracellular structures, but whether these are induced by overexpression, whether they contain endocytosed cargo of some kind, and whether they also contain cavin are therefore have an assembled caveolar coat have all been unclear. It must be mentioned also that the levels of caveolar proteins are tightly co-regulated, thus suggesting that overexpression of any caveolar component might lead to artificial localization/dynamics thus resulting in misinterpretation. Therefore, we decided that any studies done on caveolar dynamics and endocytosis (which by itself became a question-mark) or caveolar assembly has to be done in system where no overexpression of any caveolar proteins takes place. To overcome this issue, we used genome editing technology (both TALENs endonucleases and CRISPR/Cas9 system) to insert fluorescent tagging into genomic DNA. Using these techniques we successfully tagged both caveolin-1 and cavin1 in mouse cell-line. We then used this system to address the three principal questions in caveolar field: the extent to which caveolae are involved directly in endocytosis, the composition of the caveolar membrane and hence likely function of this endocytosis, and the extent to which patterns of cellular membrane traffic are altered without caveolae.
Using these genome-edited cell lines expressing both caveolin-1-GFP and cavin-1-mCherry from endogenous loci we studied the sub-cellular distribution, dynamics, composition and function of caveolae. We used several imaging techniques including Super-resolution microscopy (STED), electron microscopy and live-cell imaging to approach these questions. We found that both caveolar proteins are present in caveolae on dynamic structures. Their motility was regulated by expression of EHD2 protein, previously shown to affect caveolar dynamics. These structures also contained internalized Cholera toxin Beta subunit, previously shown to be marker for caveolar endocytosis, thus revisiting the existence of caveolar endocytosis. However, quantitative analysis of endocytic flux through caveolae shows that this pathway account for only a small fraction of total protein endocytosis.
Prompted by our analysis of a specific protein coat all around the caveolar bulb, we hypothesised that this coat could modify the local protein complement of the plasma membrane, for example via steric exclusion of proteins with cytosolic domains. To address this question we used a construct composed of a signal peptide, mCherry, an artificial trans-membrane domain composed of 7 repeats of ILA, and a cytosolic FLAG epitope (mCherry-TMD). Ratiometric imaging revealed that this protein is clearly less abundant within caveolae than SNAP-GPI (servinf as positive control for this experiment), implying that even a single trans-membrane helix and cytosolic domain of just 8 amino acid residues is depleted within the caveolar bulb. Moreover, ratiometric imaging confirmed that three different trans-membrane proteins (type I (mCh-LGT46), type II (syntaxin-2-mCh), and polytopic (CD9-mCh)) were also all depleted within caveolae. We used electron microscopy to test the conclusion that trans-membrane proteins are excluded from caveolae more directly. CD9 was fused to APEX, a modified form of ascorbate peroxidase that can be used as a genetically encoded tag for electron microscopy. Consistent with our previous finding CD9 was excluded from caveolae, suggesting that bulk plasma membrane proteins are excluded from caveolae.
The observation that caveolae are relatively depleted of trans-membrane proteins, together with previous reports of glycosphingolipid transport within caveolae, is consistent with a role in lipid trafficking. To determine the cellular function of caveolae, caveolin 1-/- MEFs were loaded with an exogenous fluorescent analogs (BODIPY conjugates) of GM1 and lactosyl ceramide by addition of the lipids bound to albumin. The amount of both lipids incorporated into cells, as well as the sub-cellular distribution of both lipids after short periods of uptake was not notably different in the absence of caveolin 1. Given the above, we reasoned that the effects of loss of caveolae on glycosphingolipid homeostasis may have much slower kinetics than total endocytosis, and may only be apparent when cells are loaded with excess lipid. Indeed when cells were loaded with lipids for longer time periods, the intracellular distribution of both lipid analogs was then strikingly different in the caveolin 1-/- cells. In control cells they accumulated in a peri-nuclear structure that previous studies imply is likely to represent the Golgi apparatus and potentially recycling endosomes, while in knockout cells this peri-nuclear pool was hard to detect, and instead BODIPY was observed accumulated in late endosomes or lysosomes. Similarly, electron microscopy revealed that loading cells with non-flourescent GM1 and lactosyl ceramide resulted in abundant multi-lamellar inclusions in lysosomes of the caveolin 1-/- cells. As no detectable defect on lipid metabolism was detected caveolin 1-/- cells, we concluded that loss of caveolae causes mis-sorting of internalised glycosphingolipid to lysosomes and accumulation of lipids in this location. Thus we propose that one cellular function of caveolar endocytosis is to maintain plasma membrane lipid homeostasis.