Caveolae Trafficking Research Articles

Caveolin-1: A Promising Therapeutic Target for Diverse Diseases.

The plasma membrane of eukaryotic cells contains small flask-shaped invaginations known as caveolae that are involved in the regulation of cellular signaling. Caveolin-1 is a 21-24k- Da protein localized in the caveolar membrane. Caveolin-1 (Cav-1) has been considered as a master regulator among the various signaling molecules. It has been emerging as a chief protein regulating cellular events associated with homeostasis, caveolae formation, and caveolae trafficking. In addition to the physiological role of cav-1, it has a complex role in the progression of various diseases. Caveolin-1 has been identified as a prognosticator in patients with cancer and has a dual role in tumorigenesis. The expression of Cav-1 in hippocampal neurons and synapses is related to neurodegeneration, cognitive decline, and aging. Despite the ubiquitous association of caveolin-1 in various pathological processes, the mechanisms associated with these events are still unclear. Caveolin- 1 has a significant role in various events of the viral cycle, such as viral entry. This review will summarize the role of cav-1 in the development of cancer, neurodegeneration, glaucoma, cardiovascular diseases, and infectious diseases. The therapeutic perspectives involving clinical applications of Caveolin-1 have also been discussed. The understanding of the involvement of caveolin-1 in various diseased states provides insights into how it can be explored as a novel therapeutic target.

Caveolae mediated lipid uptake and trafficking in health and disease

Caveolae are 70-100 nm sized plasma membrane invaginations creating a unique lipid environment that are particularly abundant in adipocytes, myocytes and endothelial cells. Structurally, caveolae are formed by a protein coat of Caveolins and Cavins. Additionally, several membrane binding proteins such as Syndapin2, EHD2 and dynamin are important regulators of caveolae function and behavior. In the past, caveolae were found to be involved in the cellular lipid uptake. Mice lacking caveolae revealed an impaired lipid metabolism resulting in reduced weight gain and lipodystrophy. Contrary, raised numbers of caveolae resulted in increased cellular lipid uptake leading to large adipose tissue depots. As caveolae are mobile membrane domains which can get endocytosed as small vesicles, it was proposed that caveolae are able to migrate to lipid droplets to facilitate lipid trafficking. However, up to date it is not understood how caveolae migrate from the plasma membrane, what triggers the trafficking and what happens mechanistically at the lipid droplet coat. By using genetically modified mouse embryonic fibroblasts, 3T3-L1 adipocytes and mouse models we investigate the involvement of caveolae trafficking in cellular lipid metabolism. We showed that changing the rate of caveolar endocytosis in vivo results in altered lipid uptake and consequently lipid droplet sizes. Thereby, either an increased caveolae internalization or raised numbers of caveolae lead to an increased fatty acid uptake and larger lipid droplets. Furthermore, platinum replica electron microscopy revealed that the fatty acid treatment itself triggers caveolae detachment from the plasma membrane. Detailed confocal microscopy and structured illumination microscopy (SIM) in live cells showed that caveolae re-locate to lipid droplets after fatty acid treatment. Importantly, this migration can be prevented when caveolae endocytosis is inhibited either by overexpressing the caveolae stabilizer EHD2 or a non-hydrolyzing dynamin mutant. The classic endocytic pathway involves endosomal trafficking followed by lysosomal degradation. However, caveolae trafficking to lipid droplets was not impaired in cells treated with endo- or lysosomal inhibitors indicating a rather direct trafficking from the plasma membrane to lipid droplets. Additionally, we showed the involvement of the fatty acid binding receptor CD36 in this process. Loss of CD36 completely abolished the caveolae dependent fatty acid uptake. Taken together, here we present novel insights in caveolae mediated lipid uptake and trafficking. Notably, this precisely regulated pathway is impaired in obesity. By analyzing adipose tissue obtained from obese patients or mouse models we observed an impaired caveolae plasma membrane stabilization. Detailed electron microscopy investigation of murine obese tissue showed increased numbers of caveolae detached from the plasma membrane indicating enhanced caveolar endocytosis and consequently raised lipid uptake. These results give novel insights in the underlying molecular changes helping to better understand obesity. Future studies are essential to investigate in detail the involvement of caveolae mediated lipid metabolism in obesity and other metabolic challenging diseases.

Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation

Caveolae are spherically shaped nanodomains of the plasma membrane, generated by cooperative assembly of caveolin and cavin proteins. Cavins are cytosolic peripheral membrane proteins with negatively charged intrinsically disordered regions that flank positively charged α-helical regions. Here, we show that the three disordered domains of Cavin1 are essential for caveola formation and dynamic trafficking of caveolae. Electrostatic interactions between disordered regions and α-helical regions promote liquid-liquid phase separation behaviour of Cavin1 in vitro, assembly of Cavin1 oligomers in solution, generation of membrane curvature, association with caveolin-1, and Cavin1 recruitment to caveolae in cells. Removal of the first disordered region causes irreversible gel formation in vitro and results in aberrant caveola trafficking through the endosomal system. We propose a model for caveola assembly whereby fuzzy electrostatic interactions between Cavin1 and caveolin-1 proteins, combined with membrane lipid interactions, are required to generate membrane curvature and a metastable caveola coat.

Energy and Dynamics of Caveolae Trafficking.

Caveolae are 70–100 nm diameter plasma membrane invaginations found in abundance in adipocytes, endothelial cells, myocytes, and fibroblasts. Their bulb-shaped membrane domain is characterized and formed by specific lipid binding proteins including Caveolins, Cavins, Pacsin2, and EHD2. Likewise, an enrichment of cholesterol and other lipids makes caveolae a distinct membrane environment that supports proteins involved in cell-type specific signaling pathways. Their ability to detach from the plasma membrane and move through the cytosol has been shown to be important for lipid trafficking and metabolism. Here, we review recent concepts in caveolae trafficking and dynamics. Second, we discuss how ATP and GTP-regulated proteins including dynamin and EHD2 control caveolae behavior. Throughout, we summarize the potential physiological and cell biological roles of caveolae internalization and trafficking and highlight open questions in the field and future directions for study.

Abstract 2635: ROR1-CAVIN3 interaction required for caveolae-dependent endocytosis and pro-survival signaling in lung adenocarcinoma

Abstract TTF-1/NKX2-1, a homeobox-containing transcription factor indispensable for lung morphogenesis, was previously identified by four independent groups including us, as a “lineage-survival” oncogene involved in the pathogenesis of lung adenocarcinoma. We have further shown that TTF-1 induces expression of the receptor tyrosine kinase-like orphan receptor 1 (ROR1), which in turn sustains a favorable balance between pro-survival PI3K-AKT and pro-apoptotic ASK1-p38 signaling in both ROR1 kinase activity-dependent and -independent manner. It is interesting to note that while alterations in EGFR signaling are involved as a “driver” in lung adenocarcinoma pathogenesis, ROR1 appears to play a “sustainer role” for the EGFR-mediated signaling. Previously, we have also found an unanticipated function of this ROR1 receptor tyrosine kinase (RTK) as a scaffold of CAVIN1 and caveolin-1 (CAV1), two essential structural components of caveolae. This kinase-independent function of ROR1 facilitates the interactions of CAVIN1 and CAV1 at the plasma membrane, thereby preventing the lysosomal degradation of CAV1. Caveolae structures and pro-survival signaling towards AKT through multiple RTKs, including EGFR, MET, and IGF-IR are consequently sustained. Therefore, ROR1 is an attractive target for overcoming EGFT-TKI resistance due to various mechanisms such as EGFR T790M double mutation and bypass signaling from other RTKs. Caveolae function in vesicular and cholesterol trafficking, and have roles in internalization of cholera toxins and the SV40 virus, as well as glycosylphosphatidylinositol (GPI)-anchored proteins and various RTKs. Endocytosis supports various cellular functions, such as nutrient uptake, intracellular signaling, morphogenesis, and defense against pathogens, and is tightly regulated, while it also ensures an appropriate amplitude of growth factor signaling by recycling RTKs back to the cell surface or sorting them to lysosomes for degradation. Here we report that ROR1 possesses a novel scaffold function indispensable for efficient caveolae-dependent endocytosis. CAVIN3 was found to bind with ROR1 at a site distinct from sites for CAV1 and CAVIN1, a novel function required for proper CAVIN3 subcellular localization and caveolae-dependent endocytosis, but not caveolae formation itself. Furthermore, evidence of a mechanistic link between ROR1-CAVIN3 interaction and consequential caveolae trafficking, which was found to utilize a binding site distinct from those for ROR1 interactions with CAV1 and CAVIN1, with RTK-mediated pro-survival signaling towards AKT in early endosomes in lung adenocarcinoma cells was also obtained. The present findings warrant future study to enable development of novel therapeutic strategies for inhibiting the multifaceted scaffold functions of ROR1 in order to reduce the intolerable death toll from this devastating cancer. Citation Format: Tomoya Yamaguchi, Miyu Hayashi, Lisa Ida, Masatoshi Yamamoto, Can Lu, Taisuke Kajino, Jinglei Cheng, Masahiro Nakatochi, Hisanori Isomura, Motoshi Suzuki, Toyoshi Fujimoto, Takashi Takahashi. ROR1-CAVIN3 interaction required for caveolae-dependent endocytosis and pro-survival signaling in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2635.

ROR1-CAVIN3 interaction required for caveolae-dependent endocytosis and pro-survival signaling in lung adenocarcinoma.

The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a transcriptional target of the lineage-survival oncogene NKX2-1/TTF-1 in lung adenocarcinomas. In addition to its kinase-dependent role, ROR1 functions as a scaffold protein to facilitate interaction between caveolin-1 (CAV1) and CAVIN1, and consequently maintains caveolae formation, which in turn sustains pro-survival signaling toward AKT from multiple receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR), MET (proto-oncogene, receptor tyrosine kinase), and IGF-IR (insulin-like growth factor receptor 1). Therefore, ROR1 is an attractive target for overcoming EGFR-TKI resistance due to various mechanisms such as EGFR T790M double mutation and bypass signaling from other RTKs. Here, we report that ROR1 possesses a novel scaffold function indispensable for efficient caveolae-dependent endocytosis. CAVIN3 was found to bind with ROR1 at a site distinct from sites for CAV1 and CAVIN1, a novel function required for proper CAVIN3 subcellular localization and caveolae-dependent endocytosis, but not caveolae formation itself. Furthermore, evidence of a mechanistic link between ROR1-CAVIN3 interaction and consequential caveolae trafficking, which was found to utilize a binding site distinct from those for ROR1 interactions with CAV1 and CAVIN1, with RTK-mediated pro-survival signaling towards AKT in early endosomes in lung adenocarcinoma cells was also obtained. The present findings warrant future study to enable development of novel therapeutic strategies for inhibiting the multifaceted scaffold functions of ROR1 in order to reduce the intolerable death toll from this devastating cancer.

Inhibition of c-Src protects paraquat induced microvascular endothelial injury by modulating caveolin-1 phosphorylation and caveolae mediated transcellular permeability.

The mechanisms underlying paraquat induced acute lung injury (ALI) is still not clear. C-Src plays an important role in the regulation of microvascular endothelial barrier function and the pathogenesis of ALI. In the present study, we found that paraquat induced cell toxicity and an increase of reactive oxygen species (ROS) in endothelium. Paraquat exposure also induced significant increase of caveolin-1 phosphorylation, caveolae trafficking and albumin permeability in endothelial monolayers. C-Src depletion by siRNA significantly attenuate paraquat induced cell toxicity, caveolin-1 phosphorylation, caveolae formation and endothelial hyperpermeability. N-acetylcysteine (NAC) failed to protect endothelial monolayers against paraquat induced toxicity. Thus, our findings suggest that paraquat exposure increases paracellular endothelial permeability by increasing caveolin-1 phosphorylation in a c-Src dependant manner. The depletion of c-Src might protect microvascular endothelial function by regulating caveolin-1 phosphorylation and caveolae trafficking during paraquat exposure, and might have potential therapeutic effects on paraquat induced ALI.

Phosphatidic Acid Produced by RalA-activated PLD2 Stimulates Caveolae-mediated Endocytosis and Trafficking in Endothelial Cells

Caveolae are the primary route for internalization and transendothelial transport of macromolecules, such as insulin and albumin. Caveolae-mediated endocytosis is activated by Src-dependent caveolin-1 (Cav-1) phosphorylation and subsequent recruitment of dynamin-2 and filamin A (FilA), which facilitate vesicle fission and trafficking, respectively. Here, we tested the role of RalA and phospholipase D (PLD) signaling in the regulation of caveolae-mediated endocytosis and trafficking. The addition of albumin to human lung microvascular endothelial cells induced the activation of RalA within minutes, and siRNA-mediated down-regulation of RalA abolished fluorescent BSA uptake. Co-immunoprecipitation studies revealed that albumin induced the association between RalA, Cav-1, and FilA; however, RalA knockdown with siRNA did not affect FilA recruitment to Cav-1, suggesting that RalA was not required for FilA and Cav-1 complex formation. Rather, RalA probably facilitates caveolae-mediated endocytosis by activating downstream effectors. PLD2 was shown to be activated by RalA, and inhibition of PLD2 abolished Alexa-488-BSA uptake, indicating that phosphatidic acid (PA) generated by PLD2 may facilitate caveolae-mediated endocytosis. Furthermore, using a PA biosensor, GFP-PASS, we observed that BSA induced an increase in PA co-localization with Cav-1-RFP, which could be blocked by a dominant negative PLD2 mutant. Total internal reflection fluorescence microscopy studies of Cav-1-RFP also showed that fusion of caveolae with the basal plasma membrane was dependent on PLD2 activity. Thus, our results suggest that the small GTPase RalA plays an important role in promoting invagination and trafficking of caveolae, not by potentiating the association between Cav-1 and FilA but by stimulating PLD2-mediated generation of phosphatidic acid.

Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae.

Caveolin 1 (Cav1) is a required structural component of caveolae, and its phosphorylation by Src is associated with an increase in caveolae-mediated endocytosis. Here we demonstrate, using quantitative live-cell 4D, TIRF, and FRET imaging, that endocytosis and trafficking of caveolae are associated with a Cav1 Tyr-14 phosphorylation-dependent conformational change, which spatially separates, or loosens, Cav1 molecules within the oligomeric caveolar coat. When tracked by TIRF and spinning-disk microscopy, cells expressing phosphomimicking Cav1 (Y14D) mutant formed vesicles that were greater in number and volume than with Y14F-Cav1-GFP. Furthermore, we observed in HEK cells cotransfected with wild-type, Y14D, or Y14F Cav1-CFP and -YFP constructs that FRET efficiency was greater with Y14F pairs than with Y14D, indicating that pY14-Cav1 regulates the spatial organization of Cav1 molecules within the oligomer. In addition, albumin-induced Src activation or direct activation of Src using a rapamycin-inducible Src construct (RapR-Src) led to an increase in monomeric Cav1 in Western blots, as well as a simultaneous increase in vesicle number and decrease in FRET intensity, indicative of a Src-mediated conformational change in CFP/YFP-tagged WT-Cav1 pairs. We conclude that phosphorylation of Cav1 leads to separation or "spreading" of neighboring negatively charged N-terminal phosphotyrosine residues, promoting swelling of caveolae, followed by their release from the plasma membrane.

Role of Albumin‐induced RalA Activation and PLD2‐mediated Phosphatidic Acid Production in Caveolae‐mediated Endocytosis and Trafficking in Endothelial Cells

Caveolae are the primary route for internalization and transcellular transport of macromolecules through the endothelium. Caveolae-mediated endocytosis is activated by Src-dependent caveolin-1 (Cav-1) phosphorylation and recruitment of dynamin-2 and filamin A (FilA). Here, we test the hypothesis that activation of caveolae-mediated endocytosis requires RalA. Addition of albumin to human lung microvascular endothelial cells (HLMVECs) induced the activation RalA within minutes and siRNA-mediated downregulation of RalA abolished fluorescent BSA uptake. Co-IP studies revealed albumin induced the association of RalA, Cav-1 and FilA, however, RalA siRNA did not affect FilA recruitment to Cav-1 suggesting RalA was not required for complex formation. PLD is known to be activated by RalA, and PLD inhibitor 1-butanol abolished Alexa 488-BSA uptake. Using the PA biosensor mRFP-PASS, studies will determine whether the generation PA in Cav-1-YFP or PLD2-GFP positive membrane microdomains and whether PA production and trafficking of Cav-1-YFP coated vesicles can be blocked by RalA siRNA, 1-butanol, and PLD2 siRNA. Results thus far suggest the small GTPase RalA is important in promoting invagination and trafficking of caveolae, not by potentiating the association between Cav-1 and FilA, but rather by promoting PLD2-mediated generation of PA. Supported by NIH P01 HL60678 (RM), P01 HL98050 (VN), and the China Scholarship Council (YJ, YL)

Endothelin Induces Rapid, Dynamin-mediated Budding of Endothelial Caveolae Rich in ET-B

Clathrin-independent trafficking pathways for internalizing G protein-coupled receptors (GPCRs) remain undefined. Clathrin-mediated endocytosis of receptors including ligand-engaged GPCRs can be very rapid and comprehensive (<10 min). Caveolae-mediated endocytosis of ligands and antibodies has been reported to be much slower in cell culture (≫10 min). Little is known about the role of physiological ligands and specific GPCRs in regulating caveolae trafficking. Here, we find that one receptor for endothelin, ET-B but not ET-A, resides on endothelial cell surfaces in both tissue and cell culture primarily concentrated within caveolae. Reconstituted cell-free budding assays show that endothelins (ETs) induce the fission of caveolae from endothelial plasma membranes purified from rat lungs. Electron microcopy of lung tissue sections and tissue subcellular fractionation both show that endothelin administered intravascularly in rats also induces a significant loss of caveolae at the luminal surface of lung vascular endothelium. Endothelial cells in culture show that ET stimulates very rapid internalization of caveolae and cargo including caveolin, caveolae-targeting antibody, and itself. The ET-B inhibitor BQ788, but not the ET-A inhibitor BQ123, blocks the ET-induced budding of caveolae. Both the pharmacological inhibitor Dynasore and the genetic dominant negative K44A mutant of dynamin prevent this induced budding and internalization of caveolae. Also shRNA lentivirus knockdown of caveolin-1 expression prevents rapid internalization of ET and ET-B. It appears that endothelin can engage ET-B already highly concentrated in caveolae of endothelial cells to induce very rapid caveolae fission and endocytosis. This transport requires active dynamin function. Caveolae trafficking may occur more rapidly than previously documented when it is stimulated by a specific ligand to signaling receptors already located in caveolae before ligand engagement.

S‐Nitrosylation of Caveolin‐1 Cys156 Stimulates Src‐dependent Caveolin‐1 Tyr14 Phosphorylation Required for Caveolae‐mediated Endocytosis in Endothelial Cells

Membrane‐associated Caveolin‐1 (Cav‐1) oligomers are required for caveolae formation. We previously showed that during an inflammatory response, Src‐dependent Cav‐1 phosphorylation increases transendothelial transport via caveolae and breakdown of the endothelial barrier, both of which were abolished in Cav‐1−/− lung microvessels. Thus, in this study we tested the hypothesis that TNFα‐induced NO production induces Cav‐1 S‐nitrosylation (SNO) resulting in Src displacement and activation which increases caveolae trafficking. Endothelial cells (ECs) stimulated with TNFα or NO donor showed reduced Cav‐1 oligomerization (MW &gt; 250 kDa) as measured by immunoblot analysis of non‐reduced SDS PAGE gels and also increased Cav‐1 Tyr14 phosphorylation. Of the 3 Cys residues in the C‐terminus (Cys 133, 143, and 156), we observed S‐nitrosylation of Cys156by biotin switch assay. eNOS dependent increase in Cav‐1 Tyr14 phosphorylation and Cav‐1‐GFP labeled caveolae trafficking were blocked by L‐NAME and PP2. Cav1−/− ECs expressing C156S Cav‐1 mutants did not form stable oligomers and showed increased Src activity but were unable to form caveolae. When expressed in normal endothelial cells, C156S‐Cav‐1‐GFP and Y14D‐Cav‐1‐GFP had a dominant positive effect whereas Y14F‐Cav‐1‐GFP reduced caveolae‐mediated uptake and trafficking. These data suggest that Cav‐1 Cys156 SNO leads to Src activation and phosphorylation of Cav‐1 Tyr14 which increases caveolae trafficking dynamics in endothelial cells.

Leukocyte rolling and adhesion via ICAM-1 signals to endothelial permeability. Focus on “Leukocyte rolling and adhesion both contribute to regulation of microvascular permeability to albumin via ligation of ICAM-1”

THE VASCULAR ENDOTHELIUM plays a vital role in the inflammatory response by induction and surface expression of adhesion molecules and chemokines. At sites of acute inflammation, blood flow is increased and postcapillary venules exhibit increased permeability (leakiness) and support the influx of blood leukocytes, primarily neutrophils. The relationship between neutrophil influx and increased vessel permeability has been a topic of investigation for decades. Many investigations have reported a direct neutrophil-dependent increase in vessel permeability during inflammation using in vivo and in vitro models (reviewed in Ref. 4). One aspect that has received increased attention is whether endothelial cell adhesion molecules contribute to the regulation of vessel permeability, in addition to their well-characterized role in neutrophil rolling and adhesion. Intercellular adhesion molecule-1 (ICAM-1, also known as CD54) is an immunoglobulin superfamily member that is abundantly expressed on the endothelial cell surface and is enriched at endothelial cell borders in vivo and in vitro (2, 12, 14, 17). ICAM-1 surface expression is upregulated in endothelial cells by proinflammatory cytokines like TNF-, IL-1, IFN-, or bacterial endotoxins, and ICAM-1 serves as a receptor for leukocyte 2-integrins (LFA-1 and Mac-1) (reviewed in Ref. 15). In an experiment of nature, patients with leukocyte adhesion deficiency-1 (LAD-1) have a severe primary immune deficiency in which blood neutrophils fail to localize to sites of inflammation or injury. This defect occurs because patients’ neutrophils either lack 2-integrins or contain mutations in these molecules. Studies using neutrophils from LAD-1 patients or 2-integrin-deficient mice clearly demonstrate that these cells have normal selectin-dependent rolling on activated endothelium but fail to stably arrest and transmigrate. Additional studies have demonstrated that endothelial-expressed ICAM-1 contributes to both kinetics of leukocyte rolling and arrest in vivo as determined by intravital microscopy of the cremaster muscle microcirculation in ICAM-1 / mice (16). In this issue of American Journal of Physiology-Cell Physiology, Sumagin and colleagues (18) use elegant in vivo studies to demonstrate that microvascular permeability in the cremaster muscle model is regulated by leukocyte engagement of ICAM-1. The authors monitored permeability (Ps) in cremaster microcirculation by efflux of luminal fluorescent-tagged albumin by fluorescence confocal intravital microscopy. Prior studies by Sarelius and colleagues (19) in the same model demonstrated that ICAM-1 engagement induced a localized increase in vessel permeability in unstimulated arterioles and in TNF-stimulated venules. In agreement with this finding ,4ho f TNF- stimulation of arterioles and venules in ICAM-1 knockout (KO) animals did not result in increased vessel permeability as compared with wild-type (WT) mice under identical conditions. Similar results were seen with 2-integrin KO mice. In the current study, the authors provide further evidence linking ICAM-1 expression to regulation of endothelial permeability. Comparisons between unstimulated venules and TNF--treated venules reveal that neutrophil rolling regulates venule permeability in unstimulated venules while leukocyte adhesion regulates venule permeability in TNF--stimulated venules. The 2-integrin-blocking antibodies, which inhibit neutrophil arrest on endothelium, reduced the vessel permeability, and this was further reduced by treatment with a combination of 2-integrin and P-selectin blocking antibodies that inhibits both rolling and arrest. Interestingly, the reduction in venule permeability observed by blockade of P-selectin and 2-integrin function were suggested to be due to insufficient ICAM-1 engagement due to the absence of rolling leukocytes. To examine the role that neutrophils play, circulating neutrophils were depleted by antineutrophil GR-1 antibody treatment. Notably, depletion of neutrophils caused a marked decrease in

S‐nitrosylation of Caveolin‐1 Cys156 Regulates Oligomer Stability and Caveolae Trafficking

Caveolin‐1 (Cav‐1) is required for caveolae formation but it also binds to signaling proteins such as endothelial nitric oxide synthase (eNOS), which generally negatively regulates their activity. Interestingly, activation of caveolae trafficking is associated with an increase in NO production. Here, we test the hypothesis that a NO feedback mechanism directly modifies the assembly and mobility of caveolae. We determined whether eNOS‐derived NO affects Cav‐1 oligomer stability by S‐nitrosylation (SNO) of Cys156 and thereby regulates vesicular trafficking. Endothelial cells stimulated with TNFα for up to 4 hr were lysed and used to measure Cav‐1 SNO by biotin‐switch assay as well as Cav‐1 oligomer stability in non‐reduced SDS PAGE gels. TNFα increased NO production and Cav‐1 SNO and also decreased Cav‐1 oligomerization, both of which were blocked by L‐NAME. We next generated Cav‐1−/− cells expressing GFP‐tagged wild type Cav‐1 or Cys156Ser‐Cav‐1 mutant to determine the role of Cys156 in caveolin oligomerization and vesicle trafficking as characterized by immunoblot analysis and live‐cell spinning disc confocal imaging. These studies revealed that Cys156Ser‐Cav‐1 mutant‐labeled caveolae were less mobile and that oligomers were less stable compared to WT Cav‐1. Thus, S‐nitrosylation of Cav‐1 may be an important regulatory mechanism of caveolae trafficking in endothelial cells. Supported by R01 HL071626, P01 HL060678, and T32 HL007829.

Filamin A Regulates Caveolae Internalization and Trafficking in Endothelial Cells

Transcytosis via caveolae is critical for maintaining vascular homeostasis by regulating the tissue delivery of macromolecules, hormones, and lipids. In the present study, we test the hypothesis that interactions between F-actin cross-linking protein filamin A and caveolin-1 facilitate the internalization and trafficking of caveolae. Small interfering RNA-mediated knockdown of filamin A, but not filamin B, reduced the uptake and transcytosis of albumin by approximately 35 and 60%, respectively, without altering the actin cytoskeletal structure or cell-cell adherens junctions. Mobility of both intracellular caveolin-1-green fluorescent protein (GFP)-labeled vesicles measured by fluorescence recovery after photobleaching and membrane-associated vesicles measured by total internal reflection-fluorescence microscopy was decreased in cells with reduced filamin A expression. In addition, in melanoma cells that lack filamin A (M2 cells), the majority of caveolin-1-GFP was localized on the plasma membrane, whereas in cells in which filamin A expression was reconstituted (A7 cells and M2 cells transfected with filamin A-RFP), caveolin-1-GFP was concentrated in intracellular vesicles. Filamin A association with caveolin-1 in endothelial cells was confirmed by cofractionation of these proteins in density gradients, as well as by coimmunoprecipitation. Moreover, this interaction was enhanced by Src activation, associated with increased caveolin-1 phosphorylation, and blocked by Src inhibition. Taken together, these data suggest that filamin A association with caveolin-1 promotes caveolae-mediated transport by regulating vesicle internalization, clustering, and trafficking.

Integrin regulation of caveolin function

Caveolae are unique organelles that are found in the plasma membrane of many cell types. They participate in various processes such as lipid recycling, cellular signalling and endocytosis. A variety of signalling molecules localize to caveolae in response to various stimuli, providing a potential mechanism for the spatial regulation of signal transduction pathways. Caveolin-1, a constitutive protein of caveolae, has been implicated in the regulation of cell growth, lipid trafficking, endocytosis and cell migration. Phosphorylation of caveolin-1 on Tyr 14 is involved in integrin-regulated caveolae trafficking and also in signalling at focal adhesions in migrating cells. In this review, we focus on recent studies that describe the role of caveolin-1 in integrin signal transduction, and how this interplay links extracellular matrix anchorage to cell proliferation, polarity and directional migration.

Caveolin‐1 Phosphorylation Regulates Vesicle Trafficking

Caveolae function as signaling hubs for vesicular trafficking. Caveolin-1−/−mouse lung endothelial cells (ECs) were transduced with caveolin-1-GFP to generate ECs with fluorescently-labeled caveolae. Caveolae were then tracked by spinning disc confocal microscopy to investigate the effect of caveolin-1 phosphorylation on caveolae trafficking. Images were acquired in 0.2 μm step increments; 4D data sets (x, y, z, time) were analyzed with particle-tracking software to quantify number, volume, and velocity. Caveolae trafficking was measured in ECs expressing wild-type (WT)-Cav-1-GFP, phosphorlation-defective mutant Y14F-Cav-1-GFP, phospho-mimicking mutant Y14D-Cav-1-GFP, and non-oligomerizing mutant C156S-Cav-1-GFP at 37°C in serum-free medium. WT-Cav-1-GFP labeled vesicles had an average volume of 0.25 ± 0.06 μm3and average velocity of 0.18 ± 0.04 μm/sec. Y14F-Cav-1 showed a 48% decrease in volume (0.13 ± 0.03 μm3) and 63% decrease in velocity (0.07 ± 0.02 μm/sec), Y14D-Cav-1 labeled vesicles showed a 61% increase in volume (0.41 ± 0.07 μm3) and 20% increase in velocity (0.22 ± 0.04 μm/sec) and C156S-Cav-1 vesicles showed a 34% decrease in volume (0.16 ± 0.03 μm3) and no effect on velocity (0.20 ± 0.04 μm/sec) compared to WT-Cav-1. The addition of albumin to serum-deprived WT-Cav-1-GFP expressing ECs to stimulate Cav-1 Y14 phoshorylation increased the number of vesicles by 53% (150 ± 28 vs 232 ± 41 vesicles) and increased vesicle volume by 30%. Thus, phosphorylation of caveolin-1 at Tyr 14 is a critical determinant of caveolae number, size, and trafficking velocity. Supported by NIH HL060678, HL071626, and the Illinois Research Initiative in Biotechnology.

Real‐time confocal imaging of caveolae trafficking in endothelial cells

Caveolae are cholesterol-rich membrane domains that function as signaling hubs and vesicular transporters. Caveolin-1-GFP was expressed in rat or CAV1−/ −mouse lung endothelial cells to label caveolae, which were then tracked by spinning disc confocal microscopy to investigate caveolae trafficking. Images were acquired in 0.2 μm step increments and 4D data sets (x, y, z, time) were analyzed with particle-tracking software to quantify the number, size, velocity, and directional movement of vesicles. At 25°C, 15 ± 5 % of GFP-caveolin-1 labeled vesicles (250 ± 80 vesicles) were mobile. Caveolae that were <0.1 μm3 (85%) and 0.1-1.0 μm3 (15%) in volume moved with an average velocity of 0.30 ± 0.005 and 0.21 ± 0.01 μm/sec, respectively. 10 ± 2 % of vesicles moved directly to the opposite (basal or apical) membrane (0.55 ± 0.098 μm/sec), 65 ± 15 % made intermediate stops (0.35 ± 0.07 μm/sec), and 10 ± 3% did not move in the z direction (0.32 ± 0.05 μm/sec). Direct routes of caveolae trafficking between apical and basal membranes may represent the transcytosis pathway involved in the regulation of transendothelial albumin permeability, while other trafficking pathways may reflect the processing of constituents carried in caveolae. At 37°C, phospho-mimicking (Y14D)-caveolin-1-GFP labeled vesicles were twice as mobile (59±5%) as wild-type caveolin-1-GFP vesicles (31±8%). The phosphorylation dependence of plasmalemma vesicle trafficking is consistent with the hypothesis that tyrosine phosphorylation of caveolin-1 regulates caveolae-mediated endocytosis and transcellular transport in endothelial cells. Supported by NIH R01 HL71626, P01 HL60678, and The University of Illinois Intercampus Research Initiative in Biotechnology.

Structural insights into the function of human caveolin 1

Caveolin-1 (Cav-1) is emerging as the central protein controlling caveolae formation, caveolae trafficking, and cellular signalling. In particular, it is known that Cav-1 interacts and modulates the activity of several signalling proteins through the so-called caveolin scaffolding domain. In this paper, we used a bioinformatics approach to assess the validity of some long-standing structural features of Cav-1. We could confirm the existence of a membrane spanning region of Cav-1 and highlight an interesting pattern of palmitoylated cysteine residues explaining the structural features of the Cav-1 C-terminal region. Moreover, the scaffolding domain is predicted to have a different structure than previously reported.

Kinase-regulated quantal assemblies and kiss-and-run recycling of caveolae

A functional genomics approach has revealed that caveolae/raft-mediated endocytosis is subject to regulation by a large number of kinases. Here we explore the role of some of these kinases in caveolae dynamics. We discover that caveolae operate using principles different from classical membrane trafficking. First, each caveolar coat contains a set number (one 'quantum') of caveolin-1 molecules. Second, caveolae are either stored as in stationary multi-caveolar structures at the plasma membrane, or undergo continuous cycles of fission and fusion with the plasma membrane in a small volume beneath the surface, without disassembling the caveolar coat. Third, a switch mechanism shifts caveolae from this localized cycle to long-range cytoplasmic transport. We have identified six kinases that regulate different steps of the caveolar cycle. Our observations reveal new principles in caveolae trafficking and suggest that the dynamic properties of caveolae and their transport competence are regulated by different kinases operating at several levels.