Shear stress induces caveolin-1 translocation in cultured endothelial cells

Considering that vascular endothelial caveolae could be flow sensors converting mechanical stimuli into chemical signals transmitted into the cell, this work studied, in vitro, the change of caveolin-1 expression and distribution of cultured endothelial cells exposed to laminar flows. Experimental results showed that, in control cells, caveolin-1 were primarily localized on the cell surface, and presented some local concentrations. In cells exposed to laminar flows, caveolin-1 distribution showed a time-dependent variation. After 24 h of shear (1.0 Pa), the expression of caveolin-1 increased and a local caveolin-1 concentration was found, in most cells, at the upstream side of the cell body where the hydrostatic pressure and the spatial gradient of shear stress were at a maximum. As a comparison, tumor necrosis factor-a induced a decrease of caveolin-1 in the cells.     

Contribution of caveolin-1 alpha and Akt to TNF-alpha-induced cell death

We used retrovirus insertion-mediated random mutagenesis to generate tumor necrosis factor-alpha (TNF-alpha)-resistant lines from L929 cells. Using this approach, we discovered that caveolin-1 alpha is required for TNF-alpha-induced cell death in L929 cells. The need for caveolin-1 alpha in TNF-alpha-induced cell death was confirmed by the restoration of sensitivity to TNF-alpha after ectopic reconstitution of caveolin-1 alpha/beta expression. This caveolin-1 alpha-mutated line was also resistant to H(2)O(2) and staurosporine, but not to lonidamine. HepG2 cells are known to lack endogenous caveolins. HepG2 cells stably transfected with caveolin-1 alpha/beta were found to be much more sensitive to TNF-alpha than either parental cells transfected with caveolin-1 beta or parental cells transfected with an empty vector. In contrast to its extensively documented antiapoptotic effect, the elevated activity of Akt appears to be important in sensitizing caveolin-1-expressing cells to TNF-alpha, since pretreatment of cells with the phosphatidylinositide 3-kinase (PI3K) inhibitor LY-294002 or wortmannin completely blocked PI3K activation and markedly improved the survival of TNF-alpha-treated L929 cells. The survival rates of caveolin-1 alpha-normal and caveolin-1 alpha-deficient L929 cells were comparable after treatment with PI3K inhibitor and TNF-alpha. Similar results were obtained with HepG2 cells that stably expressed caveolin-1 alpha/beta or -beta and parental cells transfected with an empty vector. In summary, our results indicate that caveolin-1 alpha preferentially sensitizes L929 cells to TNF-alpha through the activation of a PI3K/Akt signaling pathway.     

Role of the cytoskeleton in extracellular calcium-regulated PTH release

The calcium-sensing receptor (CaR) mediates the effects of extracellular calcium ([Ca(2+)](o)) on PTH release, such that increasing levels of [Ca(2+)](o) inhibit PTH secretion through poorly defined mechanisms. In the present studies, immunocytochemical analysis demonstrated that F-actin, PTH, CaR, and caveolin-1 are colocalized at the apical secretory pole of PT cells, and subcellular fractionation of PT cells showed these proteins to be present within the secretory granule fraction. High [Ca(2+)](o) caused F-actin, PTH, and caveolin-1 to move to the apical pole of the cells. Depolymerization of F-actin by cytochalasin reduced the actin network and induced redistribution of actin/caveolin-1 to a dispersed pattern within the cell. The F-actin-severing compounds, latrunculin and cytochalasin, significantly increased PTH secretion, while the actin polymerizing agent, jasplakinolide, substantially inhibited PTH secretion. We have demonstrated that in polarized PT cells, the F-actin cytoskeleton is involved in the regulation of PTH secretion and is critical for inhibition of PTH secretion by high calcium.     

Caveolae are an essential component of the pathway for endothelial cell signaling associated with abrupt reduction of shear stress

Abrupt cessation of flow representing the acute loss of shear stress (simulated ischemia) to flow-adapted pulmonary microvascular endothelial cells (PMVEC) leads to reactive oxygen species (ROS) generation that signals for EC proliferation. We evaluated the role of caveolin-1 on this cellular response with mouse PMVEC that were preconditioned for 72 h to laminar flow at 5 dyn/cm(2) followed by stop of flow ("ischemia"). Preconditioning resulted in a 2.7-fold increase in cellular expression of K(ATP) (K(IR) 6.2) channels but no change in expression level of caveolin-1, gp91(phox), or MAP kinases. The initial response to ischemia in wild type cells was cell membrane depolarization that was abolished by gene targeting of K(IR) 6.2. The subsequent response was increased ROS production associated with activation of NADPH oxidase (NOX2) and then phosphorylation of MAP kinases (Erk, JNK). After 24 h of ischemia in wild type cells, the cell proliferation index increased 2.5 fold and the % of cells in S+G(2)/M phases increased 6-fold. This signaling cascade (cell membrane depolarization, ROS production, MAP kinase activation and cell proliferation) was abrogated in caveolin-1 null PMVEC or by treatment of wild type cells with filipin. These studies indicate that caveolin-1 functions as a shear sensor in flow-adapted EC resulting in ROS-mediated cell signaling and endothelial cell proliferation following the abrupt reduction in flow.     

Recruitment of endothelial caveolae into mechanotransduction pathways by flow conditioning in vitro

The luminal surface of rat lung microvascular endothelial cells in situ is sensitive to changing hemodynamic parameters. Acute mechanosignaling events initiated in response to flow changes in perfused lung microvessels are localized within specialized invaginated microdomains called caveolae. Here we report that chronic exposure to shear stress alters caveolin expression and distribution, increases caveolae density, and leads to enhanced mechanosensitivity to subsequent changes in hemodynamic forces within cultured endothelial cells. Flow-preconditioned cells expressed a fivefold increase in caveolin (and other caveolar-residing proteins) at the luminal surface compared with no-flow controls. The density of morphologically identifiable caveolae was enhanced sixfold at the luminal cell surface of flow-conditioned cells. Laminar shear stress applied to static endothelial cultures (flow step of 5 dyn/cm2), enhanced the tyrosine phosphorylation of luminal surface proteins by 1.7-fold, including caveolin-1 by 1.3-fold, increased Ser1179 phosphorylation of endothelial nitric oxide synthase (eNOS) by 2.6-fold, and induced a 1.4-fold activation of mitogen-activated protein kinases (ERK1/2) over no-flow controls. The same shear step applied to endothelial cells preconditioned under 10 dyn/cm2 of laminar shear stress for 6 h and induced a sevenfold increase of total phosphotyrosine signal at the luminal endothelial cell surface enhanced caveolin-1 tyrosine phosphorylation 5.8-fold and eNOS phosphorylation by 3.3-fold over static control values. In addition, phosphorylated caveolin-1 and eNOS proteins were preferentially localized to caveolar microdomains. In contrast, ERK1/2 activation was not detected in conditioned cells after acute shear challenge. These data suggest that cultured endothelial cells respond to a sustained flow environment by directing caveolae to the cell surface where they serve to mediate, at least in part, mechanotransduction responses.     

Biomechanics of Schlemm's canal endothelial cells: influence on F-actin architecture

Aqueous humor drains from the eye through Schlemm's canal, a small endothelial-lined collecting duct. Schlemm's canal endothelial cells may be important in controlling the pressure within the eye (and hence are of interest in glaucoma), and are subject to an unusual combination of shear stress and a basal-to-apical pressure gradient. We sought to characterize this biomechanical environment and determine its effects on F-actin architecture in situ. A theoretical model of flow in Schlemm's canal was used to estimate shear stresses applied to endothelial cells by flowing aqueous humor. Alignment of Schlemm's canal endothelial cells in human eyes was quantified by scanning electron microscopy. F-actin architecture was visualized by fluorescent labeling and compared for closely adjacent cells exposed to different biomechanical environments. We found that, despite the relatively low flow rate of aqueous humor, shear stresses experienced by Schlemm's canal endothelial cells could reach those in the arterial system. Schlemm's canal endothelial cells showed a statistically significant preferential alignment, consistent with a shear-mediated effect. Schlemm's canal endothelial cells subjected to a basal-to-apical pressure gradient due to transendothelial flow showed a prominent marginal band of F-actin with relatively few cytoplasmic filaments. Adjacent cells not subject to this gradient showed little marginal F-actin, with a denser cytoplasmic random network. We conclude that Schlemm's canal endothelial cells experience physiologically significant levels of shear stress, promoting cell alignment. We speculate that this may help control the calibre of Schlemm's canal. F-actin distribution depends critically on the presence or absence of transendothelial flow and its associated pressure gradient. In the case of this pressure gradient, mechanical reinforcement around the cell periphery by F-actin seems to be critical.     

Chronic shear induces caveolae formation and alters ERK and Akt responses in endothelial cells

Caveolae are plasmalemmal domains enriched with cholesterol, caveolins, and signaling molecules. Endothelial cells in vivo are continuously exposed to shear conditions, and their caveolae density and location may be different from that of static cultured cells. Here, we show that chronic shear exposure regulates formation and localization of caveolae and caveolin-1 in bovine aortic endothelial cells (BAEC). Chronic exposure (1 or 3 days) of BAEC to laminar shear increased the total number of caveolae by 45-48% above static control. This increase was due to a rise in the luminal caveolae density without changing abluminal caveolae numbers or increasing caveolin-1 mRNA and protein levels. Whereas some caveolin-1 was found in the plasma membrane in static-cultured cells, it was predominantly localized in the Golgi. In contrast, chronic shear-exposed cells showed intense caveolin-1 staining in the luminal plasma membrane with minimum Golgi association. The preferential luminal localization of caveolae may play an important role in endothelial mechanosensing. Indeed, we found that chronic shear exposure (preconditioning) altered activation patterns of two well-known shear-sensitive signaling molecules (ERK and Akt) in response to a step increase in shear stress. ERK activation was blunted in shear preconditioned cells, whereas the Akt response was accelerated. These results suggest that chronic shear stimulates caveolae formation by translocating caveolin-1 from the Golgi to the luminal plasma membrane and alters cell signaling responses.     

Adhesion of L1210 cells to sulfonated styrene copolymer surfaces: imaging of F-actin AND alpha-actinin

The static adhesion of living L1210 cells to sulfonated copolymer surfaces of different sulfonic group content and the actin cytoskeleton organization in the adhering cells were studied. The strength of the cell-substratum interaction was estimated by determining the relative number of cells remaining adherent despite experiencing a shearing force equal to 1.25 x 10(-11) N caused by the laminar flow of the medium. The cell-substratum interaction took place in a medium with or without serum. The distribution of F-actin and alpha-actinin in the adhering cells was determined in sequences of fluorescent images of cell optical slices with the use of a computer method of cell image analysis. It was shown that the surface sulfonic groups affect not only the rate and strength of cell-substratum adhesion but also the F-actin and alpha-actinin distribution (in the cell regions near the substratum surface) in cells adhering in the medium containing serum. These proteins, concentrated in the tips of microvilli, were observed as dots. The distinctness (discernibleness) and sizes of these dots depend on the surface content of sulfonic groups. F-actin is located at the periphery of the cells in cells adhering in the medium without serum and alpha-actinin is concentrated in small dots at the periphery and in the central part of the cells.     

Involvement of cell surface ATP synthase in flow-induced ATP release by vascular endothelial cells

Endothelial cells (ECs) release ATP in response to shear stress, a mechanical force generated by blood flow, and the ATP released modulates EC functions through activation of purinoceptors. The molecular mechanism of the shear stress-induced ATP release, however, has not been fully elucidated. In this study, we have demonstrated that cell surface ATP synthase is involved in shear stress-induced ATP release. Immunofluorescence staining of human pulmonary arterial ECs (HPAECs) showed that cell surface ATP synthase is distributed in lipid rafts and co-localized with caveolin-1, a marker protein of caveolae. Immunoprecipitation indicated that cell surface ATP synthase and caveolin-1 are physically associated. Measurement of the extracellular metabolism of [(3)H]ADP confirmed that cell surface ATP synthase is active in ATP generation. When exposed to shear stress, HPAECs released ATP in a dose-dependent manner, and the ATP release was markedly suppressed by the membrane-impermeable ATP synthase inhibitors angiostatin and piceatannol and by an anti-ATP synthase antibody. Depletion of plasma membrane cholesterol with methyl-beta-cyclodextrin (MbetaCD) disrupted lipid rafts and abolished co-localization of ATP synthase with caveolin-1, which resulted in a marked reduction in shear stress-induced ATP release. Pretreatment of the cells with cholesterol prevented these effects of MbetaCD. Downregulation of caveolin-1 expression by transfection of caveolin-1 siRNA also markedly suppressed ATP-releasing responses to shear stress. Neither MbetaCD, MbetaCD plus cholesterol, nor caveolin-1 siRNA had any effect on the amount of cell surface ATP synthase. These results suggest that the localization and targeting of ATP synthase to caveolae/lipid rafts is critical for shear stress-induced ATP release by HPAECs.     

KLF2-dependent, shear stress-induced expression of CD59: a novel cytoprotective mechanism against complement-mediated injury in the vasculature

Complement activation may predispose to vascular injury and atherogenesis. The atheroprotective actions of unidirectional laminar shear stress led us to explore its influence on endothelial cell expression of complement inhibitory proteins CD59 and decay-accelerating factor. Human umbilical vein and aortic endothelial cells were exposed to laminar shear stress (12 dynes/cm(2)) or disturbed flow (+/- 5 dynes/cm(2) at 1Hz) in a parallel plate flow chamber. Laminar shear induced a flow rate-dependent increase in steady-state CD59 mRNA, reaching 4-fold at 12 dynes/cm(2). Following 24-48 h of laminar shear stress, cell surface expression of CD59 was up-regulated by 100%, whereas decay-accelerating factor expression was unchanged. The increase in CD59 following laminar shear was functionally significant, reducing C9 deposition and complement-mediated lysis of flow-conditioned endothelial cells by 50%. Although CD59 induction was independent of PI3-K, ERK1/2 and nitric oxide, an RNA interference approach demonstrated dependence upon an ERK5/KLF2 signaling pathway. In contrast to laminar shear stress, disturbed flow failed to induce endothelial cell CD59 protein expression. Likewise, CD59 expression on vascular endothelium was significantly higher in atheroresistant regions of the murine aorta exposed to unidirectional laminar shear stress, when compared with atheroprone areas exposed to disturbed flow. We propose that up-regulation of CD59 via ERK5/KLF2 activation leads to endothelial resistance to complement-mediated injury and protects from atherogenesis in regions of laminar shear stress.     

Identification of filamin as a novel ligand for caveolin-1: evidence for the organization of caveolin-1-associated membrane domains by the actin cytoskeleton

Reports on the ultrastructure of cells as well as biochemical data have, for several years, been indicating a connection between caveolae and the actin cytoskeleton. Here, using a yeast two-hybrid approach, we have identified the F-actin cross-linking protein filamin as a ligand for the caveolae-associated protein caveolin-1. Binding of caveolin-1 to filamin involved the N-terminal region of caveolin-1 and the C terminus of filamin close to the filamin-dimerization domain. In in vitro binding assays, recombinant caveolin-1 bound to both nonmuscle and muscle filamin, indicating that the interaction might not be cell type specific. With the use of confocal microscopy, colocalization of caveolin-1 and filamin was observed in elongated patches at the plasma membrane. Remarkably, when stress fiber formation was induced with Rho-stimulating Escherichia coli cytotoxic necrotizing factor 1, the caveolin-1-positive structures became coaligned with stress fibers, indicating that there was a physical link connecting them. Immunogold double-labeling electron microscopy confirmed that caveolin-1-labeled racemose caveolae clusters were positive for filamin. The actin network, therefore, seems to be directly involved in the spatial organization of caveolin-1-associated membrane domains.     

ACTH-induced caveolin-1 tyrosine phosphorylation is related to podosome assembly in Y1 adrenal cells

Y1 adrenocortical cells respond to ACTH with a characteristic rounding-up that facilitates cAMP signaling, critical for transport of cholesterol to the mitochondria and increase in steroid secretion. We here demonstrate that caveolin-1 participates in coupling activation of protein kinase A (PKA) to the control of cell shape. ACTH/8-Br-cAMP induced reorganization of caveolin-1-positive structures in correlation with the cellular rounding-up. Concomitant with this change, there was an increase in the phosphorylation of caveolin-1 (Tyr-14) localized at focal adhesions (FA) with reorganization of FA to rounded, ringlike structures. Colocalization with phalloidin showed that phosphocaveolin is present at the edge of actin filaments and that after ACTH stimulation F-actin dots at the cell periphery become surrounded by phosphocaveolin-1. These observations along with electron microscopy studies revealed these structures as podosomes. Podosome assembly was dependent on both PKA and tyrosine kinase activities because their formation was impaired after treatment with specific inhibitors [myristoylated PKI (mPKI) or PP2, respectively] previous to ACTH/8-Br-cAMP stimulation. These results show for the first time that ACTH induces caveolin-1 phosphorylation and podosome assembly in Y1 cells and support the view that the morphological and functional responses to PKA activation in steroidogenic cells are related to cytoskeleton dynamics.     

Loss of caveolin-1 polarity impedes endothelial cell polarization and directional movement

The ability of a cell to move requires the asymmetrical organization of cellular activities. To investigate polarized cellular activity in moving endothelial cells, human endothelial cells were incubated in a Dunn chamber to allow migration toward vascular endothelial growth factor. Immunofluorescent staining with a specific antibody against caveolin-1 revealed that caveolin-1 was concentrated at the rear of moving cells. Similarly, monolayer scraping to induce random cell walk resulted in relocation of caveolin-1 to the cell rear. These results suggest that posterior polarization of caveolin-1 is a common feature both for chemotaxis and chemokinesis. Dual immunofluorescent labeling showed that, during cell spreading, caveolin-1 was compacted in the cell center and excluded from nascent focal contacts along the circular lamellipodium, as revealed by integrin beta1 and FAK staining. When cells were migrating, integrin beta1 and FAK appeared at polarized lamellipodia, whereas caveolin-1 was found at the posterior of moving cells. Notably, wherever caveolin-1 was polarized, there was a conspicuous absence of lamellipod protrusion. Transmission electron microscopy showed that caveolae, similar to their marker caveolin-1, were located at the cell center during cell spreading or at the cell rear during cell migration. In contrast to its unphosphorylated form, tyrosine-phosphorylated caveolin-1, upon fibronectin stimulation, was associated with the focal complex molecule phosphopaxillin along the lamellipodia of moving cells. Thus, unphosphorylated and phosphorylated caveolin-1 were located at opposite poles during cell migration. Importantly, loss of caveolin-1 polarity by targeted down-regulation of the protein prevented cell polarization and directional movement. Our present results suggest a potential role of caveolin polarity in lamellipod extension and cell migration.     

Caveolin-1抑制胰腺癌细胞PANC1的体内外生长和增殖

窖蛋白-1(caveolin-1)是胞膜窖(caveolae)中重要的结构和功能蛋白.Caveolin-1参与细胞的多种生命活动并与恶性肿瘤的发生相关.为探讨caveolin-1对胰腺癌细胞PANC1的体外增殖、迁移、侵袭以及裸鼠体内成瘤能力的影响,通过基因转染技术培育caveolin-1过表达细胞株PANC1/cav-1作为实验组,转染空载体细胞株PANC1/vector作为对照组,采用RT-PCR及Western blot方法检测caveolin-1的表达量,流式细胞术分析细胞周期,软琼脂细胞克隆实验检测细胞增殖能力,侵袭小室实验检测癌细胞迁移和侵袭的能力,建立裸鼠皮下种植瘤模型并检测肿瘤组织的增殖与凋亡.PANC1/cav-1中的caveolin-1表达稳定,表达量明显高于对照组细胞株和亲本细胞株(P<0.01),细胞周期检测显示大量PANC1/cav-1细胞被抑制于G0/G1期,caveolin-1抑制PANC1的增殖,迁移和侵袭能力.在裸鼠的体内实验中,caveolin-1显著抑制PANC1细胞在裸鼠体内的生长,Ki-67染色和TUNEL染色表明在PANC1细胞中过表达caveolin-1,可以抑制肿瘤增殖并诱导肿瘤凋亡.上述结果表明,caveolin-1可能通过对胰腺癌细胞周期的影响(抑制于G0/G1期),抑制胰腺癌PANC1细胞在体内外的增殖、迁移和侵袭,并导致肿瘤凋亡.     

Activation of nuclear factor kappaB and induction of apoptosis by tumor necrosis factor-alpha in the mouse uterine epithelial WEG-1 cell line

In order to better understand how tumor necrosis factor (TNF)-alpha may contribute to the local regulation of uterine cell death, cultures of mouse uterine epithelial WEG-1 cells were exposed to TNF-alpha and observed at different time intervals. Earliest decrease in cell viability was observed after 31 h of exposure to 50 ng/ml mouse TNF-alpha and was associated with the expression of several markers of apoptosis. Treatment with human TNF-alpha or addition of a neutralizing antibody against TNF-alpha receptor protein 80 to mouse TNF-alpha resulted in attenuated induction of apoptosis, suggesting that coengagement of the two TNF-alpha receptor types is required for maximal impact. Ceramide analogs failed to replicate the effect of TNF-alpha and the stress-activated protein kinase signaling pathway was not activated by the cytokine. Treatment with mouse TNF-alpha resulted in an increase in nuclear factor (NF)kappaB activity that receded after 24 h. The impact of human TNF-alpha on NFkappaB activation was more moderate. Addition of either one of three different inhibitors of NFkappaB (SN50, PDTC, and A771726) to mouse TNF-alpha sensitized WEG-1 cells to the toxicity of the cytokine. Our data suggest that WEG-1 cells initiate their response to TNF-alpha with an increase in NFkappaB activation that may have transiently biased these cells toward cell death resistance.     

NrCAM coupling to the cytoskeleton depends on multiple protein domains and partitioning into lipid rafts

NrCAM is a cell adhesion molecule of the L1 family that is implicated in the control of axonal growth. Adhesive contacts may promote advance of the growth cone by triggering the coupling of membrane receptors with the F-actin retrograde flow. We sought to understand the mechanisms leading to clutching the F-actin at the site of ligand-mediated clustering of NrCAM. Using optical tweezers and single particle tracking of beads coated with the ligand TAG-1, we analyzed the mobility of NrCAM-deletion mutants transfected in a neuroblastoma cell line. Deletion of the cytoplasmic tail did not prevent the coupling of NrCAM to the actin flow. An additional deletion of the FNIII domains to remove cis-interactions, was necessary to abolish the rearward movement of TAG-1 beads, which instead switched to a stationary behavior. Next, we showed that the actin-dependent retrograde movement of NrCAM required partitioning into lipid rafts as indicated by cholesterol depletion experiments using methyl-beta-cyclodextrin. Recruitment of the raft component caveolin-1 was induced at the adhesive contact between the cell surface and TAG-1 beads, indicating that enlarged rafts were generated. Photobleaching experiments showed that the lateral mobility of NrCAM increased with raft dispersion in these contact areas, further suggesting that TAG-1-coated beads induced the coalescence of lipid rafts. In conclusion, we propose that anchoring of NrCAM with the retrograde actin flow can be triggered by adhesive contacts via cooperative processes including interactions with the cytoplasmic tail, formation of cis-complex via the FNIII repeats, and lipid raft aggregation.     

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 ∼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.     

Lymphocyte transcellular migration occurs through recruitment of endothelial ICAM-1 to caveola- and F-actin-rich domains

During inflammation, leukocytes bind to the adhesion receptors ICAM-1 and VCAM-1 on the endothelial surface before undergoing transendothelial migration, also called diapedesis. ICAM-1 is also involved in transendothelial migration, independently of its role in adhesion, but the molecular basis of this function is poorly understood. Here we demonstrate that, following clustering, apical ICAM-1 translocated to caveolin-rich membrane domains close to the ends of actin stress fibres. In these F-actin-rich areas, ICAM-1 was internalized and transcytosed to the basal plasma membrane through caveolae. Human T-lymphocytes extended pseudopodia into endothelial cells in caveolin- and F-actin-enriched areas, induced local translocation of ICAM-1 and caveolin-1 to the endothelial basal membrane and transmigrated through transcellular passages formed by a ring of F-actin and caveolae. Reduction of caveolin-1 levels using RNA interference (RNAi) specifically decreased lymphocyte transcellular transmigration. We propose that the translocation of ICAM-1 to caveola- and F-actin-rich domains links the sequential steps of lymphocyte adhesion and transendothelial migration and facilitates lymphocyte migration through endothelial cells from capillaries into surrounding tissue.     

Cytoskeleton proteins F-actin and tubulin distribution and interaction with mitochondria in the granulosa cells surrounding stage III zebrafish (Danio rerio) oocytes

The distributional arrangement of mitochondria in the granulosa cells surrounding stage III zebrafish oocyte has been reported as a contiguous aggregation of mitochondria at the margin of the each granulosa cell. The aim of the present study was to further investigate the mitochondrial distribution in the granulosa cell layer in stage III ovarian follicles and the interaction between mitochondria and cytoskeleton elements actin and tubulin. To determine mitochondrial distribution/transport, immunocytochemistry analysis of tubulin and mitochondrial COX-I was carried out along with phalloidin staining of polymerised F-actin. The follicles were also exposed to a range of conditions that are known to affect mitochondria and the cytoskeleton proteins actin and tubulin. The mitochondrial inhibitor FCCP, the anti-mitotic drug nocodazole, and actin polymerisation inhibitor cytochalasin B were used. Levels of ATP, mtDNA copy number, and viability assessed by Trypan blue were also studied after exposure to inhibitors in order to determine the relationship between mitochondrial distribution/activity and ATP production. F-actin showed a hexagonal-polygonal distribution surrounding the mitochondria in granulosa cells, with the F-actin network adjacent to the plasma membrane of each granulosa cell. Tubulin structure presented a less organised distribution than F-actin, it was sparse in the cytosol. Interaction between mitochondria and tubulin was found indicating that mitochondria and tubulin are colocalised in zebrafish ovarian follicles. The exposure of ovarian follicles to inhibitors induced the loss of mitochondrial structural integrity showing that mitochondria distribution in granulosa cells of stage III zebrafish ovarian follicles is determined by the microtubules network.