Fenestral and stomatal diaphragms are endothelial subcellular structures of unknown function that... more Fenestral and stomatal diaphragms are endothelial subcellular structures of unknown function that form on organelles implicated in vascular permeability: fenestrae, transendothelial channels, and caveolae. PV1 protein is required for diaphragm formation in vitro. Here, we report that deletion of the PV1-encoding Plvap gene in mice results in the absence of diaphragms and decreased survival. Loss of diaphragms did not affect the fenestrae and transendothelial channels formation but disrupted the barrier function of fenestrated capillaries, causing a major leak of plasma proteins. This disruption results in early death of animals due to severe noninflammatory protein-losing enteropathy. Deletion of PV1 in endothelium, but not in the hematopoietic compartment, recapitulates the phenotype of global PV1 deletion, whereas endothelial reconstitution of PV1 rescues the phenotype. Taken together, these data provide genetic evidence for the critical role of the diaphragms in fenestrated capillaries in the maintenance of blood composition.
Fenestral and stomatal diaphragms are endothelial subcellular structures of unknown function that... more Fenestral and stomatal diaphragms are endothelial subcellular structures of unknown function that form on organelles implicated in vascular permeability: fenestrae, transendothelial channels, and caveolae. PV1 protein is required for diaphragm formation in vitro. Here, we report that deletion of the PV1-encoding Plvap gene in mice results in the absence of diaphragms and decreased survival. Loss of diaphragms did not affect the fenestrae and transendothelial channels formation but disrupted the barrier function of fenestrated capillaries, causing a major leak of plasma proteins. This disruption results in early death of animals due to severe noninflammatory protein-losing enteropathy. Deletion of PV1 in endothelium, but not in the hematopoietic compartment, recapitulates the phenotype of global PV1 deletion, whereas endothelial reconstitution of PV1 rescues the phenotype. Taken together, these data provide genetic evidence for the critical role of the diaphragms in fenestrated capillaries in the maintenance of blood composition.
PV1 protein is an essential component of stomatal and fenestral diaphragms, which are formed at t... more PV1 protein is an essential component of stomatal and fenestral diaphragms, which are formed at the plasma membrane of endothelial cells (ECs), on structures such as caveolae, fenestrae and transendothelial channels. Knockout of PV1 in mice results in in utero and perinatal mortality. To be able to interpret the complex PV1 knockout phenotype, it is critical to determine whether the formation of diaphragms is the only cellular role of PV1. We addressed this question by measuring the effect of complete and partial removal of structures capable of forming diaphragms on PV1 protein level. Removal of caveolae in mice by knocking out caveolin-1 or cavin-1 resulted in a dramatic reduction of PV1 protein level in lungs but not kidneys. The magnitude of PV1 reduction correlated with the abundance of structures capable of forming diaphragms in the microvasculature of these organs. The absence of caveolae in the lung ECs did not affect the transcription or translation of PV1, but it caused a sharp increase in PV1 protein internalization rate via a clathrin-and dynamin-independent pathway followed by degradation in lysosomes. Thus, PV1 is retained on the cell surface of ECs by structures capable of forming diaphragms, but undergoes rapid internalization and degradation in the absence of these structures, suggesting that formation of diaphragms is the only role of PV1.
The functions of caveolae, the characteristic plasma membrane invaginations, remain debated. Thei... more 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.
PV-1 is a novel protein component of the endothelial fenestral and stomatal diaphragms. PV-1 cDNA... more PV-1 is a novel protein component of the endothelial fenestral and stomatal diaphragms. PV-1 cDNA and protein sequences are highly conserved across species. The conserved extracellular domain features found in rat, mouse, and human PV-1 protein are four N-glycosylation sites, two coiled-coil domains, a proline-rich region, and even cysteine spacing. No consensus site in the intracellular domain was found. Northern blotting of mouse and human tissues is in agreement with and expands those performed in rat and correlated well with the postulated presence of PV-1 in the endothelial diaphragms. The genomic organization of the human and mouse genes (HGMWapproved symbol PLVAP) has been determined, and the analysis of their 5 flanking regions has found a highly conserved classical TATA-driven promoter that shows several transcription factor consensus binding sites. Radiation hybrid panel mapping has localized the human and mouse PLVAP genes to chromosomes 19p13.2 and 8B3-C1, respectively.
The specific role of VEGFA-induced permeability and vascular leakage in physiology and pathology ... more The specific role of VEGFA-induced permeability and vascular leakage in physiology and pathology has remained unclear. Here we show that VEGFA-induced vascular leakage depends on signalling initiated via the VEGFR2 phosphosite Y949, regulating dynamic c-Src and VE-cadherin phosphorylation. Abolished Y949 signalling in the mouse mutant Vegfr2(Y949F/Y949F) leads to VEGFA-resistant endothelial adherens junctions and a block in molecular extravasation. Vessels in Vegfr2(Y949F/Y949F) mice remain sensitive to inflammatory cytokines, and vascular morphology, blood pressure and flow parameters are normal. Tumour-bearing Vegfr2(Y949F/Y949F) mice display reduced vascular leakage and oedema, improved response to chemotherapy and, importantly, reduced metastatic spread. The inflammatory infiltration in the tumour micro-environment is unaffected. Blocking VEGFA-induced disassembly of endothelial junctions, thereby suppressing tumour oedema and metastatic spread, may be preferable to full vascula...
Caveolin-1 negatively regulates eNOS derived NO production and this has been mapped to several re... more Caveolin-1 negatively regulates eNOS derived NO production and this has been mapped to several residues on Cav-1 including F92. Herein, we reasoned that endothelial expression of an F92ACav-1 transgene would let us decipher the mechanisms and relationships between caveolae structure and intracellular signaling. This study was designed to separate caveolae formation from its downstream signaling effects. An endothelial specific doxycycline-regulated mouse model for the expression of Cav 1 F92A was developed. Blood pressure by telemetry and nitric oxide bioavailability by electron paramagnetic resonance and phosphorylation of VASP were determined. Caveolae integrity in the presence of Cav-1-F92A was measured by stabilization of Cav 2, sucrose gradient and electron microscopy. Histological analysis of heart and lung, echocardiography and signaling were performed. This study shows that mutant Cav-1-F92A forms caveolae structures similar to WT but leads to increases in NO bioavailability...
CMGH Cellular and Molecular Gastroenterology and Hepatology, 2015
Background & Aims, Methods: Severe intestinal diseases observed in very young children are often ... more Background & Aims, Methods: Severe intestinal diseases observed in very young children are often the result of monogenic defects. We used whole exome sequencing (WES) to examine the genetic cause in a patient with a distinct severe form of protein losing enteropathy (PLE) characterized by hypoproteinemia, hypoalbuminemia, and hypertriglyceridemia.
Page 48. 2 Anatomy of the Pulmonary Endothelium Radu V. Stan Departments of Pathology and Microbi... more Page 48. 2 Anatomy of the Pulmonary Endothelium Radu V. Stan Departments of Pathology and Microbiology & Immunology, Dartmouth Medical School, Lebanon, NH, USA INTRODUCTION Vascular endothelium is a highly ...
ABSTRACT Tissues with high light absorption, such as melanomas, present a significant challenge t... more ABSTRACT Tissues with high light absorption, such as melanomas, present a significant challenge to fluorescence imaging approaches that seek to estimate molecular expression in vivo, since any fluorescence originating in the tissue will suffer substantial attenuation prior to detection. This can lead to sizable underestimations in estimated fluorescent tracer concentration in these tissues using conventional fluorescence imaging. In this study, a dual-tracer fluorescence imaging approach was employed to correct for severe tissue absorption by 1) using simultaneous injection and imaging of an untargeted tracer to normalize tissue absorption effects on the targeted tracer, and 2) using kinetic modeling that capitalizes on subtle differences in the dynamics of targeted and untargeted tracer uptake to quantify targeted molecule concentrations in the high absorbing tissue. Monte Carlo simulation and kinetic models demonstrated that the effect of optical properties on the approach could be eliminated by a pixel-by-pixel normalization of the targeted and untargeted tracer uptakes prior to 5 min post-tracer injection for fluorescence planar dynamic imaging.
American journal of physiology. Lung cellular and molecular physiology, 2003
Caveolae are the sites in the cell membrane responsible for concentrating an array of signaling m... more Caveolae are the sites in the cell membrane responsible for concentrating an array of signaling molecules critical for cell function. Recent studies have begun to identify the functions of caveolin-1, the 22-kDa caveolar protein that oligomerizes and inserts into the cytoplasmic face of the plasma membrane. Caveolin-1 appears to regulate caveolar internalization by stabilizing caveolae at the plasma membrane rather than controlling the shape of the membrane invagination. Because caveolin-1 is a scaffolding protein, it has also been hypothesized to function as a "master regulator" of signaling molecules in caveolae. Deletion of the caveolin-1 gene in mice resulted in cardiac hypertrophy and lung fibrosis, indicating its importance in cardiac and lung development. In the endothelium, caveolin-1 regulates nitric oxide signaling by binding to and inhibiting endothelial nitric oxide synthase (eNOS). Increased cytosolic Ca2+ or activation of the kinase Akt leads to eNOS activati...
Proceedings of the National Academy of Sciences, 2002
Caveolins are important components of caveolae, which have been implicated in vesicular trafficki... more Caveolins are important components of caveolae, which have been implicated in vesicular trafficking and signal transduction. To investigate the in vivo significance of Caveolins in mammals, we generated mice deficient in the caveolin-1 (cav-1) gene and have shown that, in the absence of Cav-1, no caveolae structures were observed in several nonmuscle cell types. Although cav-1 ؊/؊ mice are viable, histological examination and echocardiography identified a spectrum of characteristics of dilated cardiomyopathy in the left ventricular chamber of the cav-1-deficient hearts, including an enlarged ventricular chamber diameter, thin posterior wall, and decreased contractility. These animals also have marked right ventricular hypertrophy, suggesting a chronic increase in pulmonary artery pressure. Direct measurement of pulmonary artery pressure and histological analysis revealed that the cav-1 ؊/؊ mice exhibit pulmonary hypertension, which may contribute to the right ventricle hypertrophy. In addition, the loss of Cav-1 leads to a dramatic increase in systemic NO levels. Our studies provided in vivo evidence that cav-1 is essential for the control of systemic NO levels and normal cardiopulmonary function.
Caveolae are spherical invaginations of the plasma membrane and associated vesicles that are foun... more Caveolae are spherical invaginations of the plasma membrane and associated vesicles that are found at high surface densities in most cells, endothelia included. Their structural framework has been shown to consist of oligomerized caveolin molecules interacting with cholesterol and sphingolipids. Caveolae have been involved in many cellular functions such as endocytosis, signal transduction, mechano-transduction, potocytosis, and cholesterol trafficking. Some confusion still persists in the field with respect to the relationship between caveolae and the lipid rafts, which have been involved in many of the above functions. In addition to all these, endothelial caveolae have been involved in capillary permeability by their participation in the process of transcytosis. This short review will focus on their structure and components, methods used to determine these components, and the role of caveolae in the transendothelial exchanges between blood plasma and the interstitial fluid.
Caveolin is the principal component of caveolae in vivo. In addition to a structural role, it is ... more Caveolin is the principal component of caveolae in vivo. In addition to a structural role, it is believed to play a scaffolding function to organize and inactivate signaling molecules that are concentrated on the cytoplasmic surface of caveolar membranes. The large GTPase dynamin has been shown to mediate the scission of caveolae from the plasma membrane, although it is unclear if dynamin interacts directly with caveolin or via accessory proteins. Therefore, the goal of this study was to test whether dynamin associates with caveolae via a direct binding to the caveolin 1 (Cav1) protein. Immunoelectron microscopy of lung endothelium or a cultured hepatocyte cell line stained with antibodies for Dyn2 and Cav1 shows that these proteins co-localize to caveolae. To further define this interaction biochemically, in vitro experiments were performed using glutathione-S-transferase (GST)-Dyn2 and GST-Cav1 fusion proteins, which demonstrated a direct interaction between these proteins. This interaction appears to be mediated by the proline-arginine-rich domain (PRD) of Dyn2, as a GST-PRD fragment binds Cav1 while GST-Dyn2DPRD does not. Further, in vitro binding studies using two Dyn2 spliced forms and Cav1 peptides immobilized on paper identify specific domains of Cav1 that bind Dyn2. Interestingly, these Cav1-binding domains differ markedly between two spliced variant forms of Dyn2. In support of these distinctive physical interactions, we find that the different Dyn2 forms, when expressed as GTPase-defective mutants, exert markedly different inhibitory effects on caveolae internalization, as assayed by cholera toxin uptake. These studies provide the first evidence for a direct interaction between dynamin and the caveolin coat, and demonstrate a selectivity of one Dyn2 form toward the caveolae-mediated endocytosis.
Fenestral and stomatal diaphragms are endothelial subcellular structures of unknown function that... more Fenestral and stomatal diaphragms are endothelial subcellular structures of unknown function that form on organelles implicated in vascular permeability: fenestrae, transendothelial channels, and caveolae. PV1 protein is required for diaphragm formation in vitro. Here, we report that deletion of the PV1-encoding Plvap gene in mice results in the absence of diaphragms and decreased survival. Loss of diaphragms did not affect the fenestrae and transendothelial channels formation but disrupted the barrier function of fenestrated capillaries, causing a major leak of plasma proteins. This disruption results in early death of animals due to severe noninflammatory protein-losing enteropathy. Deletion of PV1 in endothelium, but not in the hematopoietic compartment, recapitulates the phenotype of global PV1 deletion, whereas endothelial reconstitution of PV1 rescues the phenotype. Taken together, these data provide genetic evidence for the critical role of the diaphragms in fenestrated capillaries in the maintenance of blood composition.
Fenestral and stomatal diaphragms are endothelial subcellular structures of unknown function that... more Fenestral and stomatal diaphragms are endothelial subcellular structures of unknown function that form on organelles implicated in vascular permeability: fenestrae, transendothelial channels, and caveolae. PV1 protein is required for diaphragm formation in vitro. Here, we report that deletion of the PV1-encoding Plvap gene in mice results in the absence of diaphragms and decreased survival. Loss of diaphragms did not affect the fenestrae and transendothelial channels formation but disrupted the barrier function of fenestrated capillaries, causing a major leak of plasma proteins. This disruption results in early death of animals due to severe noninflammatory protein-losing enteropathy. Deletion of PV1 in endothelium, but not in the hematopoietic compartment, recapitulates the phenotype of global PV1 deletion, whereas endothelial reconstitution of PV1 rescues the phenotype. Taken together, these data provide genetic evidence for the critical role of the diaphragms in fenestrated capillaries in the maintenance of blood composition.
PV1 protein is an essential component of stomatal and fenestral diaphragms, which are formed at t... more PV1 protein is an essential component of stomatal and fenestral diaphragms, which are formed at the plasma membrane of endothelial cells (ECs), on structures such as caveolae, fenestrae and transendothelial channels. Knockout of PV1 in mice results in in utero and perinatal mortality. To be able to interpret the complex PV1 knockout phenotype, it is critical to determine whether the formation of diaphragms is the only cellular role of PV1. We addressed this question by measuring the effect of complete and partial removal of structures capable of forming diaphragms on PV1 protein level. Removal of caveolae in mice by knocking out caveolin-1 or cavin-1 resulted in a dramatic reduction of PV1 protein level in lungs but not kidneys. The magnitude of PV1 reduction correlated with the abundance of structures capable of forming diaphragms in the microvasculature of these organs. The absence of caveolae in the lung ECs did not affect the transcription or translation of PV1, but it caused a sharp increase in PV1 protein internalization rate via a clathrin-and dynamin-independent pathway followed by degradation in lysosomes. Thus, PV1 is retained on the cell surface of ECs by structures capable of forming diaphragms, but undergoes rapid internalization and degradation in the absence of these structures, suggesting that formation of diaphragms is the only role of PV1.
The functions of caveolae, the characteristic plasma membrane invaginations, remain debated. Thei... more 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.
PV-1 is a novel protein component of the endothelial fenestral and stomatal diaphragms. PV-1 cDNA... more PV-1 is a novel protein component of the endothelial fenestral and stomatal diaphragms. PV-1 cDNA and protein sequences are highly conserved across species. The conserved extracellular domain features found in rat, mouse, and human PV-1 protein are four N-glycosylation sites, two coiled-coil domains, a proline-rich region, and even cysteine spacing. No consensus site in the intracellular domain was found. Northern blotting of mouse and human tissues is in agreement with and expands those performed in rat and correlated well with the postulated presence of PV-1 in the endothelial diaphragms. The genomic organization of the human and mouse genes (HGMWapproved symbol PLVAP) has been determined, and the analysis of their 5 flanking regions has found a highly conserved classical TATA-driven promoter that shows several transcription factor consensus binding sites. Radiation hybrid panel mapping has localized the human and mouse PLVAP genes to chromosomes 19p13.2 and 8B3-C1, respectively.
The specific role of VEGFA-induced permeability and vascular leakage in physiology and pathology ... more The specific role of VEGFA-induced permeability and vascular leakage in physiology and pathology has remained unclear. Here we show that VEGFA-induced vascular leakage depends on signalling initiated via the VEGFR2 phosphosite Y949, regulating dynamic c-Src and VE-cadherin phosphorylation. Abolished Y949 signalling in the mouse mutant Vegfr2(Y949F/Y949F) leads to VEGFA-resistant endothelial adherens junctions and a block in molecular extravasation. Vessels in Vegfr2(Y949F/Y949F) mice remain sensitive to inflammatory cytokines, and vascular morphology, blood pressure and flow parameters are normal. Tumour-bearing Vegfr2(Y949F/Y949F) mice display reduced vascular leakage and oedema, improved response to chemotherapy and, importantly, reduced metastatic spread. The inflammatory infiltration in the tumour micro-environment is unaffected. Blocking VEGFA-induced disassembly of endothelial junctions, thereby suppressing tumour oedema and metastatic spread, may be preferable to full vascula...
Caveolin-1 negatively regulates eNOS derived NO production and this has been mapped to several re... more Caveolin-1 negatively regulates eNOS derived NO production and this has been mapped to several residues on Cav-1 including F92. Herein, we reasoned that endothelial expression of an F92ACav-1 transgene would let us decipher the mechanisms and relationships between caveolae structure and intracellular signaling. This study was designed to separate caveolae formation from its downstream signaling effects. An endothelial specific doxycycline-regulated mouse model for the expression of Cav 1 F92A was developed. Blood pressure by telemetry and nitric oxide bioavailability by electron paramagnetic resonance and phosphorylation of VASP were determined. Caveolae integrity in the presence of Cav-1-F92A was measured by stabilization of Cav 2, sucrose gradient and electron microscopy. Histological analysis of heart and lung, echocardiography and signaling were performed. This study shows that mutant Cav-1-F92A forms caveolae structures similar to WT but leads to increases in NO bioavailability...
CMGH Cellular and Molecular Gastroenterology and Hepatology, 2015
Background & Aims, Methods: Severe intestinal diseases observed in very young children are often ... more Background & Aims, Methods: Severe intestinal diseases observed in very young children are often the result of monogenic defects. We used whole exome sequencing (WES) to examine the genetic cause in a patient with a distinct severe form of protein losing enteropathy (PLE) characterized by hypoproteinemia, hypoalbuminemia, and hypertriglyceridemia.
Page 48. 2 Anatomy of the Pulmonary Endothelium Radu V. Stan Departments of Pathology and Microbi... more Page 48. 2 Anatomy of the Pulmonary Endothelium Radu V. Stan Departments of Pathology and Microbiology & Immunology, Dartmouth Medical School, Lebanon, NH, USA INTRODUCTION Vascular endothelium is a highly ...
ABSTRACT Tissues with high light absorption, such as melanomas, present a significant challenge t... more ABSTRACT Tissues with high light absorption, such as melanomas, present a significant challenge to fluorescence imaging approaches that seek to estimate molecular expression in vivo, since any fluorescence originating in the tissue will suffer substantial attenuation prior to detection. This can lead to sizable underestimations in estimated fluorescent tracer concentration in these tissues using conventional fluorescence imaging. In this study, a dual-tracer fluorescence imaging approach was employed to correct for severe tissue absorption by 1) using simultaneous injection and imaging of an untargeted tracer to normalize tissue absorption effects on the targeted tracer, and 2) using kinetic modeling that capitalizes on subtle differences in the dynamics of targeted and untargeted tracer uptake to quantify targeted molecule concentrations in the high absorbing tissue. Monte Carlo simulation and kinetic models demonstrated that the effect of optical properties on the approach could be eliminated by a pixel-by-pixel normalization of the targeted and untargeted tracer uptakes prior to 5 min post-tracer injection for fluorescence planar dynamic imaging.
American journal of physiology. Lung cellular and molecular physiology, 2003
Caveolae are the sites in the cell membrane responsible for concentrating an array of signaling m... more Caveolae are the sites in the cell membrane responsible for concentrating an array of signaling molecules critical for cell function. Recent studies have begun to identify the functions of caveolin-1, the 22-kDa caveolar protein that oligomerizes and inserts into the cytoplasmic face of the plasma membrane. Caveolin-1 appears to regulate caveolar internalization by stabilizing caveolae at the plasma membrane rather than controlling the shape of the membrane invagination. Because caveolin-1 is a scaffolding protein, it has also been hypothesized to function as a "master regulator" of signaling molecules in caveolae. Deletion of the caveolin-1 gene in mice resulted in cardiac hypertrophy and lung fibrosis, indicating its importance in cardiac and lung development. In the endothelium, caveolin-1 regulates nitric oxide signaling by binding to and inhibiting endothelial nitric oxide synthase (eNOS). Increased cytosolic Ca2+ or activation of the kinase Akt leads to eNOS activati...
Proceedings of the National Academy of Sciences, 2002
Caveolins are important components of caveolae, which have been implicated in vesicular trafficki... more Caveolins are important components of caveolae, which have been implicated in vesicular trafficking and signal transduction. To investigate the in vivo significance of Caveolins in mammals, we generated mice deficient in the caveolin-1 (cav-1) gene and have shown that, in the absence of Cav-1, no caveolae structures were observed in several nonmuscle cell types. Although cav-1 ؊/؊ mice are viable, histological examination and echocardiography identified a spectrum of characteristics of dilated cardiomyopathy in the left ventricular chamber of the cav-1-deficient hearts, including an enlarged ventricular chamber diameter, thin posterior wall, and decreased contractility. These animals also have marked right ventricular hypertrophy, suggesting a chronic increase in pulmonary artery pressure. Direct measurement of pulmonary artery pressure and histological analysis revealed that the cav-1 ؊/؊ mice exhibit pulmonary hypertension, which may contribute to the right ventricle hypertrophy. In addition, the loss of Cav-1 leads to a dramatic increase in systemic NO levels. Our studies provided in vivo evidence that cav-1 is essential for the control of systemic NO levels and normal cardiopulmonary function.
Caveolae are spherical invaginations of the plasma membrane and associated vesicles that are foun... more Caveolae are spherical invaginations of the plasma membrane and associated vesicles that are found at high surface densities in most cells, endothelia included. Their structural framework has been shown to consist of oligomerized caveolin molecules interacting with cholesterol and sphingolipids. Caveolae have been involved in many cellular functions such as endocytosis, signal transduction, mechano-transduction, potocytosis, and cholesterol trafficking. Some confusion still persists in the field with respect to the relationship between caveolae and the lipid rafts, which have been involved in many of the above functions. In addition to all these, endothelial caveolae have been involved in capillary permeability by their participation in the process of transcytosis. This short review will focus on their structure and components, methods used to determine these components, and the role of caveolae in the transendothelial exchanges between blood plasma and the interstitial fluid.
Caveolin is the principal component of caveolae in vivo. In addition to a structural role, it is ... more Caveolin is the principal component of caveolae in vivo. In addition to a structural role, it is believed to play a scaffolding function to organize and inactivate signaling molecules that are concentrated on the cytoplasmic surface of caveolar membranes. The large GTPase dynamin has been shown to mediate the scission of caveolae from the plasma membrane, although it is unclear if dynamin interacts directly with caveolin or via accessory proteins. Therefore, the goal of this study was to test whether dynamin associates with caveolae via a direct binding to the caveolin 1 (Cav1) protein. Immunoelectron microscopy of lung endothelium or a cultured hepatocyte cell line stained with antibodies for Dyn2 and Cav1 shows that these proteins co-localize to caveolae. To further define this interaction biochemically, in vitro experiments were performed using glutathione-S-transferase (GST)-Dyn2 and GST-Cav1 fusion proteins, which demonstrated a direct interaction between these proteins. This interaction appears to be mediated by the proline-arginine-rich domain (PRD) of Dyn2, as a GST-PRD fragment binds Cav1 while GST-Dyn2DPRD does not. Further, in vitro binding studies using two Dyn2 spliced forms and Cav1 peptides immobilized on paper identify specific domains of Cav1 that bind Dyn2. Interestingly, these Cav1-binding domains differ markedly between two spliced variant forms of Dyn2. In support of these distinctive physical interactions, we find that the different Dyn2 forms, when expressed as GTPase-defective mutants, exert markedly different inhibitory effects on caveolae internalization, as assayed by cholera toxin uptake. These studies provide the first evidence for a direct interaction between dynamin and the caveolin coat, and demonstrate a selectivity of one Dyn2 form toward the caveolae-mediated endocytosis.
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