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Zika virus (ZIKV) has gained notoriety in recent years because there are no targeted therapies or vaccines available so far. Caveolin-1 (Cav-1) in host cells plays crucial functions in the invasion of many viruses. However, its specific involvement in ZIKV infection has remained unclear. Here, we reveal that depleting Cav-1 leads to a substantial reduction in ZIKV RNA levels, protein expression and viral particle production, indicating that ZIKV exploits Cav-1 for its infection. By dissecting each stage of the viral life cycle, we unveil that, unlike its invasion role in many other viruses, Cav-1 depletion selectively impairs ZIKV replication, resulting in altered replication dynamics and reduced strand-specific RNA levels, but does not affect viral entry, maturation and release. These results reveal an unforeseen function of Cav-1 in facilitating ZIKV replication, which provides new insights into the intricate interaction between Cav-1 and ZIKV and underscores Cav-1 as a potential candidate for anti-ZIKV approaches. [ABSTRACT FROM AUTHOR]

Rab5 is a small GTPase that regulates early endosome trafficking and other cellular processes, including cell adhesion and migration. Specifically, Rab5 promotes Rac1 activation and cancer cell migration, but little is known about the upstream regulators of Rab5. We have previously shown that the scaffolding protein Caveolin-1 (CAV1) promotes Rac1 activation and migration of cancer cells. Here, we hypothesized that CAV1 stimulates Rab5 activation, leading to increased Rac1 activity and cell migration. Expression of CAV1 in B16-F10 mouse melanoma and HT-29(US) human colon adenocarcinoma cells increased the GTP loading of Rab5, whereas shRNA-mediated targeting of endogenous CAV1 in MDA-MB-231 breast cancer cells decreased Rab5-GTP levels. Accordingly, shRNA-mediated downregulation of Rab5 decreased CAV1-mediated Rac1 activation, cell migration and invasion in B16-F10 and HT-29(US) cells. Expression of CAV1 was accompanied by increased recruitment of Tiam1, a Rac1 guanine nucleotide exchange factor (GEF), to Rab5-positive early endosomes. Using the inhibitor NSC23766, Tiam1 was shown to be required for Rac1 activation and cell migration induced by CAV1 and Rab5. Mechanistically, we provide evidence implicating p85α (also known as PIK3R1), a Rab5 GTPase-activating protein (GAP), in CAV1-dependent effects, by showing that CAV1 recruits p85α, precluding p85α-mediated Rab5 inactivation and increasing cell migration. In summary, these studies identify a novel CAV1-Rab5-Rac1 signaling axis, whereby CAV1 prevents Rab5 inactivation, leading to increased Rac1 activity and enhanced tumor cell migration and invasion. [ABSTRACT FROM AUTHOR]

The biology of caveolin-1 (Cav1)/caveolae is intimately linked to actin dynamics and adhesion receptors. Caveolar domains are organized in hierarchical levels of complexity from curved or flattened caveolae to large, higher-order caveolar rosettes. We report that stress fibers controlled by Abl kinases and mDia1 determine the level of caveolar domain organization, which conditions the subsequent inward trafficking of caveolar domains induced upon loss of cell adhesion from the extracellular matrix. Abl-deficient cells have fewer stress fibers, a smaller pool of stress-fiber co-aligned Cav1 and increased clustering of Cav1/caveolae at the cell surface. Defective caveolar linkage to stress fibers prevents the formation of big caveolar rosettes upon loss of cell adhesion, correlating with a lack of inward trafficking. Live imaging of stress fibers and Cav1 showed that the actin-linked Cav1 pool loses its spatial organization in the absence of actin polymerization and is dragged and clustered by depolymerizing filaments. We identified mDia1 as the actin polymerization regulator downstream of Abl kinases that controls the stress-fiber-linked Cav1 pool. mDia1 knockdown results in Cav1/caveolae clustering and defective inward trafficking upon loss of cell adhesion. By contrast, cell elongation imposed by the excess of stress fibers induced by active mDia1 flattens caveolae. Furthermore, active mDia1 rescues the actin co-aligned Cav1 pool and Cav1 inward trafficking upon loss of adhesion in Abl-deficient cells. Thus, caveolar domain organization and trafficking are tightly coupled to adhesive and stress fiber regulatory pathways. [ABSTRACT FROM AUTHOR]

Caveolae form a specialized platform within the plasma membrane that is crucial for an array of important biological functions, ranging from signaling to endocytosis. Using total internal reflection fluorescence (TIRF) and 3D fast spinning-disk confocal imaging to follow caveola dynamics for extended periods, and electron microscopy to obtain high resolution snapshots, we found that the vast majority of caveolae are dynamic with lifetimes ranging from a few seconds to several minutes. Use of these methods revealed a change in the dynamics and localization of caveolae during mitosis. During interphase, the equilibrium between the arrival and departure of caveolae from the cell surface maintains the steady-state distribution of caveolin-1 (Cav1) at the plasma membrane. During mitosis, increased dynamics coupled to an imbalance between the arrival and departure of caveolae from the cell surface induces a redistribution of Cav1 from the plasma membrane to intracellular compartments. These changes are reversed during cytokinesis. The observed redistribution of Cav1 was reproduced by treatment of interphase cells with nocodazole, suggesting that microtubule rearrangements during mitosis can mediate caveolin relocalization. This study provides new insights into the dynamics of caveolae and highlights precise regulation of caveola budding and recycling during mitosis. [ABSTRACT FROM AUTHOR]

Cathepsin B and pro-urokinase plasminogen activator (pro-uPA) localize to the caveolae of HCT 116 human colorectal carcinoma cells, an association mediated by active KRAS. In this study, we established a stable HCT 116 cell line with a gene encoding antisense caveolin-1 (AS-cav-1) to examine the effects of caveolin-1, the main structural protein of caveolae, on the expression and localization of cathepsin B and pro-uPA, and their cell-surface receptors p11 and uPA receptor (uPAR), respectively. AS-cav-1 HCT 116 cells secreted less procathepsin B than control (empty vector) cells as measured by immunoblotting and pepsin activation of the proenzyme. Expression and secretion of pro-uPA was also downregulated in AS-cav-1 HCT 116 cells. Localization of cathepsin B and pro-uPA to caveolae was reduced in AS-cav-1 HCT 116 cells, and these cells expressed less total and caveolae-associated p11 and UPAR compared with control cells. Previous studies have shown that uPAR forms a complex with caveolin-1 and β1-integrin, and we here show that downregulation of caveolin-1 also suppressed the localization of β1-integrin to caveolae of these cells. Finally, downregulation of caveolin-1 in HCT 116 cells inhibited degradation of the extracellular matrix protein collagen IV and the invasion of these cells through Matrigel. Based on these results, we hypothesize that caveolin-1 affects the expression and localization of cathepsin B and pro-uPA, and their receptors, thereby mediating cell-surface proteolytic events associated with invasion of colon cancer cells. [ABSTRACT FROM AUTHOR]

Caveolae are small invaginations of the cell membrane that are thought to play a role in important physiological functions such as cell surface signaling, endocytosis and intracellular cholesterol transport. Caveolin-1 is a key protein in these domains and contributes to the organization of cholesterol and saturated lipids within these vesicular invaginations of the plasma membrane. Caveolae are thought to be involved in the signaling of tyrosine kinase receptors and serine threonine receptors. In this article we focus on the involvement of caveolae in the signal transduction of bone morphogenetic proteins (BMPs). BMPs play important roles during embryonic development and especially in chondrogenesis, osteogenesis, neurogenesis and hematopoiesis. The initiation of the signal tranduction starts by the binding of a BMP to a corresponding set of BMP receptors. Using image cross-correlation spectroscopy, we show that the BMP receptors BRIa and BRII colocalize with caveolin-1 isoforms α and β on the cell surface. BRIa colocalizes predominantly with the caveolin-1 α isoform. Coexpression of BRII leads to a redistribution of BRIa into domains enriched in caveolin-1 β. After stimulation with BMP-2, BRIa moves back into the region with caveolin-1 α. BRII is expressed in regions enriched in caveolin-1 α and β. Stimulation of cells with BMP-2 leads to a redistribution of BRII into domains enriched in caveolin-1 α. Immunoprecipitation studies using transfected COS-7 cells indicate that BRII binds to caveolin-1 α and β. The binding of BRII to caveolin-1 was verified using A431 cells. Stimulation of starved A431 cells with BMP-2 lead to a release of caveolin-1 from the BMP receptors. We show further that the caveolin-1 β isoform inhibits BMP signaling whereas the α isoform does not. [ABSTRACT FROM AUTHOR]

The function of occludin remains elusive. Proposed roles include maintenance of tight junction barriers, signaling and junction remodeling. To investigate a potential role in mediating cytokine-induced changes in barrier properties, we measured barrier responses to interferon-α plus TNF in control, occludin-overexpressing and occludin knockdown MDCK II monolayers. MDCK cells show a complex response to cytokines characterized by a simultaneous increase in the transepithelial electrical resistance and a decrease in the barrier for large solutes. We observed that overexpression of occludin increased and occludin knockdown decreased sensitivity to cytokines as assessed by both these parameters. It is known that caveolin-1 interacts with occludin and is implicated in several models of cytokine-dependent barrier disruption; we found that occludin knockdown altered the subcellular distribution of caveolin-1 and that partitioning of caveolin into detergent-insoluble lipid rafts was influenced by changing occludin levels. Knockdown of caveolin decreased the cytokine-induced flux increase, whereas the increase in the electrical barrier was unaltered; the effect of double knockdown of occludin and caveolin was similar to that of occludin single knockdown, consistent with the possibility that they function in the same pathway. These results demonstrate that occludin is required for cells to transduce cytokine-mediated signals that either increase the electrical barrier or decrease the large solute barrier, possibly by coordinating the functions of caveolin-1. [ABSTRACT FROM AUTHOR]

Directional cell migration is crucially dependent on the spatiotemporal control of intracellular signalling events. These events regulate polarized actin dynamics, resulting in protrusion at the front of the cell and contraction at the rear. The actin cytoskeleton is regulated through signalling by Rho-like GTPases, such as RhoA, which stimulates myosin-based contractility, and CDC42 and Rac1, which promote actin polymerization and protrusion. Here, we show that Rac1 binds the adapter protein caveolin-1 (Cav1) and that Rac1 activity promotes Cav1 accumulation at Rac1-positive peripheral adhesions. Using Cav1-deficient mouse fibroblasts and depletion of Cav1 expression in human epithelial and endothelial cells mediated by small interfering RNA and short hairpin RNA, we show that loss of Cav1 induces an increase in Rac1 protein and its activated, GTP-bound form. Cav1 controls Rac1 protein levels by regulating ubiquitylation and degradation of activated Rac1 in an adhesion-dependent fashion. Finally, we show that Rac1 ubiquitylation is not required for effector binding, but regulates the dynamics of Rac1 at the periphery of the cell. These data extend the canonical model of Rac1 inactivation and uncover Cav1-regulated polyubiquitylation as an additional mechanism to control Rac1 signalling. [ABSTRACT FROM AUTHOR]

The relationship between the plasminogen activator system and integrin function is well documented but incompletely understood. The mechanism of uPAR-mediated signaling across the membrane and the molecular basis of uPAR-dependent activation of integrins remain important issues. The present study was undertaken to identify the molecular intermediates involved in the uPAR signaling pathway controlling α5β1-integrin activation and fibronectin polymerization. Disruption of lipid rafts with MβCD or depletion of caveolin-1 by siRNA led to the inhibition of uPAR-dependent integrin activation and stimulation of fibronectin polymerization in human dermal fibroblasts. The data indicate a dual role for caveolin-1 in the uPAR signaling pathway, leading to integrin activation. Caveolin-1 functions initially as a membrane adaptor or scaffold to mediate uPAR-dependent activation of Src and EGFR. Subsequently, in its phosphorylated form, caveolin-1 acts as an accessory molecule to direct trafficking of activated EGFR to focal adhesions. These studies provide a novel paradigm for the regulation of crosstalk among integrins, growth-factor receptors and uPAR. [ABSTRACT FROM AUTHOR]

Caveolae are membrane domains having caveolin-1 (Cav1) as their main structural component. Here, we determined whether Cav1 affects Ca2+ signaling through the Gαq-phospholipase-Cβ (PLCβ) pathway using Fischer rat thyroid cells that lack Cav1 (FRTcav-) and a sister line that forms caveolae-like domains due to stable transfection with Cav1 (FRTcav+). In the resting state, we found that eCFP-Gβγ and Gαq-eYFP are similarly associated in both cell lines by Forster resonance energy transfer (FRET). Upon stimulation, the amount of FRET between Gαq-eYFP and eCFP-Gβγ remains high in FRTcav- cells, but decreases almost completely in FRTcav+ cells, suggesting that Cav1 is increasing the separation between Gαq-Gβγ subunits. In FRTcav- cells overexpressing PLCβ, a rapid recovery of Ca2+ is observed after stimulation. However, FRTcav+ cells show a sustained level of elevated Ca2+. FRET and colocalization show specific interactions between Gαq and Cav1 that increase upon stimulation. Fluorescence correlation spectroscopy studies show that the mobility of Gαq-eGFP is unaffected by activation in either cell type. The mobility of eGFP-Gβγ remains slow in FRTcav- cells but increases in FRTcav+ cells. Together, our data suggest that, upon stimulation, Gαq(GTP) switches from having strong interactions with Gβγ to Cav1, thereby releasing Gβγ. This prolongs the recombination time for the heterotrimer, thus causing a sustained Ca2+ signal. [ABSTRACT FROM AUTHOR]

Caveolae have been linked to diverse cellular functions and to many disease states. In this study we have used zebrafish to examine the role of caveolin-1 and caveolae during early embryonic development. During development, expression is apparent in a number of tissues including Kupffer's vesicle, tailbud, intersomite boundaries, heart, branchial arches, pronephric ducts and periderm. Particularly strong expression is observed in the sensory organs of the lateral line, the neuromasts and in the notochord where it overlaps with expression of caveolin-3. Morpholino-mediated downregulation of Cav1α caused a dramatic inhibition of neuromast formation. Detailed ultrastructural analysis, including electron tomography of the notochord, revealed that the central regions of the notochord has the highest density of caveolae of any embryonic tissue comparable to the highest density observed in any vertebrate tissue. In addition, Cav1 α downregulation caused disruption of the notochord, an effect that was enhanced further by Cav3 knockdown. These results indicate an essential role for caveolin and caveolae in this vital structural and signalling component of the embryo. [ABSTRACT FROM AUTHOR]

Eph receptor tyrosine kinases are key players during the development of the embryonic vasculature; however, their role and regulation in adult angiogenesis remain to be defined. Caveolae are flask-shaped invaginations of the cell membrane; their major structural protein, caveolin-1, has been shown to regulate signaling molecules localized in these micro-domains. The interaction of caveolin-1 with several of these proteins is mediated by the binding of its scaffolding domain to a region containing hydrophobic amino acids within these proteins. The presence of such a motif within the EphB1 kinase domain prompted us to investigate the caveolar localization and regulation of EphB1 by caveolin-1. We report that EphB1 receptors are localized in caveolae, and directly interact with caveolin-1 upon ligand stimulation. This interaction, as well as EphB1-mediated activation of extracellular-signal-regulated kinase (ERK), was abrogated by overexpression of a caveolin-1 mutant lacking a functional scaffolding domain. Interaction between Ephs and caveolin-1 is not restricted to the B-subclass of receptors, since we show that EphA2 also interacts with caveolin-1. Furthermore, we demonstrate that the caveolin-binding motif within the kinase domain of EphB1 is primordial for its correct membrane targeting. Taken together, our findings establish caveolin-1 as an important regulator of downstream signaling and membrane targeting of EphB1. [ABSTRACT FROM AUTHOR]

Caveolin-1 is suggested to act as a tumor suppressor. We tested the hypothesis that caveolin-1 does so by repression of survivin, an Inhibitor of apoptosis protein that regulates cell-cycle progression as well as apoptosis and is commonly overexpressed in human cancers. Ectopic expression of caveolin-1 in HEK293T and ZR75 cells or siRNA-mediated silencing of caveolin-1 in NIH3T3 cells caused downregulation or upregulation of survivin mRNA and protein, respectively. Survivin downregulation in HEK293T cells was paralleled by reduced cell proliferation, increases in G0-G1 and decreases in G2-M phase of the cell cycle. In addition, apoptosis was evident, as judged by several criteria. Importantly, expression of green fluorescent protein-survivin in caveolin-1-transfected HEK293T cells restored cell proliferation and viability. In addition, expression of caveolin-1 inhibited transcriptional activity of a survivin promoter construct in a β-catenin-Tcf/Lef-dependent manner. Furthermore, in HEK293T cells caveolin-1 associated with β-catenin and inhibited Tcf/Lef-dependent transcription. Similar results were obtained upon caveolin-1 expression in DLD1 cells, where APC mutation leads to constitutive activation of β-catenin-Tcf/Lef-mediated transcription of survivin. Taken together, these results suggest that anti-proliferative and pro-apoptotic properties of caveolin-1 may be attributed to reduced survivin expression via a mechanism involving diminished β-catenin-Tcf/Lef-dependent transcription. [ABSTRACT FROM AUTHOR]