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Contains fulltext : 138984.pdf (Publisher’s version ) (Open Access) Chronic vascular inflammation is driven by interactions between activated leukocytes and the endothelium. Leukocyte beta2-integrins bind to endothelial intercellular adhesion molecule 1 (ICAM-1), which allows leukocyte spreading, crawling and transendothelial migration. Leukocytes scan the vascular endothelium for permissive sites to transmigrate, which suggests that there is apical membrane heterogeneity within the endothelium. However, the molecular basis for this heterogeneity is unknown. Leukocyte adhesion induces ICAM-1 clustering, which promotes its association to the actin-binding proteins filamin B, alpha-actinin-4 and cortactin. We show that these endothelial proteins differentially control adhesion, spreading and transmigration of neutrophils. Loss of filamin B, alpha-actinin-4 and cortactin revealed adaptor-specific effects on a nuclear-to-peripheral gradient of endothelial cell stiffness. By contrast, increasing endothelial cell stiffness stimulates ICAM-1 function. We identify endothelial alpha-actinin-4 as a key regulator of endothelial cell stiffness and of ICAM-1-mediated neutrophil transmigration. Finally, we found that the endothelial lining of human and murine atherosclerotic plaques shows elevated levels of alpha-actinin-4. These results identify endothelial cell stiffness as an important regulator of endothelial surface heterogeneity and of ICAM-1 function, which in turn controls the adhesion and transmigration of neutrophils.

Signaling via growth factor receptors, including the epidermal growth factor (EGF) receptor, is key to various cellular processes, such as proliferation, cell survival, and cell migration. In a variety of human diseases such as cancer, aberrant expression and activation of growth factor receptors can lead to disturbed signaling. Intracellular trafficking is crucial for proper signaling of growth factor receptors. As a result, the level of cell surface expression of growth factor receptors is an important determinant for the outcome of downstream signaling. BAR domain-containing proteins represent an important family of proteins that regulate membrane dynamics. In this study, we identify a novel role for the F-BAR protein PACSIN2 in the regulation of EGF receptor signaling. We show that internalized EGF as well as the (activated) EGF receptor translocated to PACSIN2-positive endosomes. Furthermore, loss of PACSIN2 increased plasma membrane expression of the EGF receptor in resting cells and increased EGF-induced phosphorylation of the EGF receptor. As a consequence, EGF-induced activation of Erk and Akt as well as cell proliferation were enhanced in PACSIN2-depleted cells. In conclusion, this study identifies a novel role for the F-BAR-domain protein PACSIN2 in regulating EGF receptor surface levels and EGF-induced downstream signaling.

Endothelial cells line the vasculature and are important for the regulation of blood pressure, vascular permeability, clotting and transendothelial migration of leukocytes and tumor cells. A group of proteins that that control the endothelial barrier function are the RhoGTPases. This study focuses on three homologous (>88%) RhoGTPases: RhoA, RhoB, RhoC of which RhoB and RhoC have been poorly characterized. Using a RhoGTPase mRNA expression analysis we identified RhoC as the highest expressed in primary human endothelial cells. Based on an existing RhoA FRET sensor we developed new RhoB/C FRET sensors to characterize their spatiotemporal activation properties. We found all these RhoGTPase sensors to respond to physiologically relevant agonists (e.g. Thrombin), reaching transient, localized FRET ratio changes up to 200%. These RhoA/B/C FRET sensors show localized GEF and GAP activity and reveal spatial activation differences between RhoA/C and RhoB. Finally, we used these sensors to monitor GEF-specific differential activation of RhoA/B/C. In summary, this study adds high-contrast RhoB/C FRET sensors to the currently available FRET sensor toolkit and uncover new insights in endothelial and RhoGTPase cell biology. This allows us to study activation and signaling by these closely related RhoGTPases with high spatiotemporal resolution in primary human cells.

In this study, the translocation of the NADPH oxidase components p67(phox) and Rac2 was studied during phagocytosis in living cells. For this purpose, green fluorescent protein (GFP)-tagged versions of these proteins were expressed in the myeloid cell line PLB-985. First, the correct localization of p67GFP and GFP-Rac2 was shown during phagocytosis of serum-treated zymosan by wild-type PLB-985 cells and PLB-985 X-CGD (chronic granulomatous disease) cells, which lack expression of flavocytochrome b(558). Subsequently, these constructs were used for fluorescence recovery after photobleaching studies to elucidate the turnover of these proteins on the phagosomal membrane. The turnover of p67GFP and GFP-Rac2 proved to be very high, indicating a continuous exchange of flavocytochrome b(558)-bound p67GFP and GFP-Rac2 for cytosolic, free p67GFP and GFP-Rac2. Furthermore, the importance of an intact actin cytoskeleton for correct localization of these proteins was investigated by disrupting the actin cytoskeleton with cytochalasin B. However, cytochalasin B treatment of PLB-985 cells did not alter the localization of p67GFP and GFP-Rac2 once phagocytosis was initiated. In addition, the continuous exchange of flavocytochrome b(558)-bound p67GFP and GFP-Rac2 for cytosolic p67GFP and GFP-Rac2 was still intact in cytochalasin B-treated cells, indicating that the translocation of these proteins does not depend on a rearrangement of the actin cytoskeleton.

The RhoGTPase Rac1 is activated in a polarised fashion and controls cell motility. We previously showed that Rac1 binds the PP2A inhibitor SET and recruits nuclear SET to the cytosol. We show that a SET mutant, lacking a nuclear localization signal, SET(ΔNLS), promotes cell spreading and motility. This was accompanied by an increase in the number and frequency of membrane ruffles. Pharmacological inhibition of PP2A did not mimic the effects of SET(ΔNLS), however, we found that expression of SET and SET(ΔNLS) increases the levels of the MAP kinases ERK1 and ERK2. Structured summary of protein interactions: RAC1 physically interacts with SET by pull down (View interaction).

The Rho-GTPase Rac1 promotes actin polymerization and membrane protrusion that mediate initial contact and subsequent maturation of cell-cell junctions. Here we report that Rac1 associates to the ubiquitin-protein ligase neural precursor cell expressed developmentally down-regulated 4 (Nedd4). This interaction requires the hypervariable C-terminal domain of Rac1 and the WW domains of Nedd4. Activated Rac1 co-localizes with endogenous Nedd4 at epithelial cell-cell contacts. Reduction of Nedd4 expression by shRNA results in reduced transepithelial electrical resistance (TER) and concomitant changes in the distribution of adherens and tight junction markers. Conversely, expression of Nedd4 promotes TER, suggesting that Nedd4 cooperates with Rac1 in the induction of junctional maturation. We found that Nedd4, but not Nedd4-2, mediates the ubiquitylation and degradation of the adapter protein dishevelled-1 (Dvl1), the expression of which negatively regulates cell-cell contact. Nedd4-mediated ubiquitylation requires its binding to the C-terminal domain of Dvl1, comprising the DEP domain, and targets a N-terminal lysine-rich region upstream of the Dvl1 DIX domain. We found that endogenous Rac1 co-localizes with endogenous Dvl1 in intracellular puncta as well as on cell-cell junctions. Finally, activated Rac1 was found to stimulate Nedd4 activity, resulting in increased ubiquitylation of Dvl1. Together, these data reveal a novel Rac1-dependent signalling pathway which, through Nedd4-mediated ubiquitylation of Dvl1, stimulates the maturation of epithelial cell-cell contacts.

SET/I2PP2A is a nuclear protein that was initially identified as an oncogene in human undifferentiated acute myeloid leukemia, fused to the nuclear porin Nup-214. In addition, SET is a potent inhibitior of the phosphatase PP2A. Previously, we proposed a model in which the small GTPase Rac1 recruits SET from the nucleus to the plasma membrane to promote cell migration. This event represents an entirely novel concept in the field of cell migration. Now, fluorescent versions of the SET protein are generated to analyze its nucleo-cytoplasmic shuttling in live cells. Our studies showed that under steady-state conditions a fraction of the SET protein, which is primarily localized in the nucleus, translocates to the cytosol in an apparently random fashion. SET exiting the nucleus was also seen in spreading as well as dividing cells. We designed an image analysis method to quantify the frequency of nuclear exit of the SET proteins, based on 4D confocal imaging. This straightforward method was validated by analysis of SET wild-type and mutant proteins. This showed that the frequency of nuclear exit of a Ser-9 phosphomimetic mutant (S9E) is enhanced compared to wild-type SET or a S9A mutant. Thus, we have developed a novel method to analyze the nucleo-cytoplasmic shuttling of the proto-oncogene SET dynamics in live cells. This method will also be applicable to monitor dynamic localization of other nuclear and/or cytoplasmic signaling proteins. (C) 2011 International Society for Advancement of Cytometry

The Rac1 GTPase controls cytoskeletal dynamics and is a key regulator of cell spreading and migration mediated by signaling through effector proteins, such as the PAK kinases and the Scar and WAVE proteins. We previously identified a series of regulatory proteins that associate with Rac1 through its hypervariable C-terminal domain, including the Rac1 activator β-Pix (also known as Rho guanine-nucleotide-exchange factor 7) and the membrane adapter caveolin-1. Here, we show that Rac1 associates, through its C-terminus, with the F-BAR domain protein PACSIN2, an inducer of membrane tubulation and a regulator of endocytosis. We show that Rac1 localizes with PACSIN2 at intracellular tubular structures and on early endosomes. Active Rac1 induces a loss of PACSIN2-positive tubular structures. By contrast, Rac1 inhibition results in an accumulation of PACSIN2-positive tubules. In addition, PACSIN2 appears to regulate Rac1 signaling; siRNA-mediated loss of PACSIN2 increases the levels of Rac1-GTP and promotes cell spreading and migration in a wound healing assay. Moreover, ectopic expression of PACSIN2 reduces Rac1-GTP levels in a fashion that is dependent on the PACSIN2–Rac1 interaction, on the membrane-tubulating capacity of PACSIN2 and on dynamin. These data identify the BAR-domain protein PACSIN2 as a Rac1 interactor that regulates Rac1-mediated cell spreading and migration.

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.

Rac1 is a member of the Rho family of small GTPases, which regulate cell adhesion and migration through their control of the actin cytoskeleton. Rho-GTPases are structurally very similar, with the exception of a hypervariable domain in the C terminus. Using peptide-based pulldown assays in combination with mass spectrometry, we previously showed that the hyper-variable domain in Rac1 mediates specific protein-protein interactions. Most recently, we found that the Rac1 C terminus associates to the ubiquitously expressed adapter protein CMS/ CD2AP.CD2APis critical for the formation and maintenance of a specialized cell-cell contact between kidney podocyte foot processes, the slit diaphragm. Here, CD2AP links the cell adhesion protein nephrin to the actin cytoskeleton. In addition, CMS/ CD2AP binds actin-regulating proteins, such as CAPZ and cortactin, and has been implicated in the internalization of growth factor receptors. We found that CD2AP specifically interacts with the C-terminal domain of Rac1 but not with that of other Rho family members. Efficient interaction between Rac1 and CD2AP requires both the proline-rich domain and the polybasic region in the Rac1 C terminus, and at least two of the three N-terminal SH3 domains of CD2AP. CD2AP co-localizes with Rac1 to membrane ruffles, and small interfering RNA-based experiments showed that CD2AP links Rac1 to CAPZ and cortactin. Finally, expression of constitutive active Rac1 recruits CD2AP to cell-cell contacts in epithelial cells, where we found CD2AP to participate in the control of the epithelial barrier function. These data identify CD2AP as a novel Rac1-associated adapter protein that participates in the regulation of epithelial cell-cell contact.

The Rho GTPase Rac1 controls cell adhesion and motility. The effector loop of Rac1 mediates interactions with downstream effectors, whereas its C-terminus binds the exchange factor β-Pix, which mediates Rac1 targeting and activation. Here, we report that Rac1, through its C-terminus, also binds the nuclear oncogene SET/I2PP2A, an inhibitor of the serine/threonine phosphatase PP2A. We found that SET translocates to the plasma membrane in cells that express active Rac1 as well as in migrating cells. Membrane targeting of SET stimulates cell migration in a Rac1-dependent manner. Conversely, reduction of SET expression inhibits Rac1-induced migration, indicating that efficient Rac1 signalling requires membrane recruitment of SET. The recruitment of the SET oncogene to the plasma membrane represents a new feature of Rac1 signalling. Our results suggest a model in which Rac1-stimulated cell motility requires both effector loop-based downstream signalling and recruitment of a signalling amplifier, that is, SET, through the hypervariable C-terminus.

Vascular endothelial-cadherin (VE-cadherin) controls endothelial cell-cell adhesion and preserves endothelial integrity. In order to maintain endothelial barrier function, VE-cadherin function is tightly regulated through mechanisms that involve protein phosphorylation and cytoskeletal dynamics. Here, we show that loss of VE-cadherin function results in intercellular gap formation and a drop in electrical resistance of monolayers of primary human endothelial cells. Detailed analysis revealed that loss of endothelial cell-cell adhesion, induced by VE-cadherin-blocking antibodies, is preceded by and dependent on a rapid activation of Rac1 and increased production of reactive oxygen species. Moreover, VE-cadherin-associated beta-catenin is tyrosine-phosphorylated upon loss of cell-cell contact. Finally, the redox-sensitive proline-rich tyrosine kinase 2 (Pyk2) is activated and recruited to cell-cell junctions following the loss of VE-cadherin homotypic adhesion. Conversely, the inhibition of Pyk2 activity in endothelial cells by the expression of CRNK (CADTK/CAKbeta-related non-kinase), an N-terminal deletion mutant that acts in a dominant negative fashion, not only abolishes the increase in beta-catenin tyrosine phosphorylation but also prevents the loss of endothelial cell-cell contact. These results implicate Pyk2 in the reduced cell-cell adhesion induced by the Rac-mediated production of ROS through the tyrosine phosphorylation of beta-catenin. This signaling is initiated upon loss of VE-cadherin function and is important for our insight in the modulation of endothelial integrity.

Reactive oxygen species (ROS) control the integrity of the vascular endothelium. Our laboratory has recently shown that transduction of human umbilical vein endothelial cells (HUVECs) with an active variant of the small GTPase Rac promotes the production of ROS, ROS-dependent activation of p38 mitogen-activated protein kinase, and loss of vascular/endothelial-cadherin- mediated cell-cell adhesion. Here we show that HUVECs express mRNAs for NOX2 as well as NOX4 mRNA, but not for NOX1 or NOX3. Interestingly, NOX4 was expressed at ∼100-fold higher levels compared with NOX2. NOX4-green fluorescent protein largely localizes to an intracellular compartment that costained with a marker for the endoplasmic reticulum, and its distribution did not overlap with lysosomes, Weibel-Palade bodies, or mitochondria. The NOX2-regulatory proteins p47phox and p67phox associated with the actin cytoskeleton and were found in cell protrusions and membrane ruffles, colocalizing with Rac1. This translocation to the cell periphery was promoted by tumor necrosis factor (TNF)-α. Finally, scavenging of ROS was found to impair TNF-α-induced cytoskeletal rearrangements and the formation of a confluent endothelial monolayer. Together, these data prove the differential mRNA expression of NOX family members in human endothelium and indicate that these NOX proteins and their regulators may be involved in the control of endothelial cell spreading, motility, and cell-cell adhesion.

Chemokine-driven migration is accompanied by polarization of the cell body and of the intracellular signaling machinery. The extent to which chemokine receptors polarize during chemotaxis is currently unclear. To analyze the distribution of the chemokine receptor CXCR4 during SDF-1 (CXCL12)-induced chemotaxis, we retrovirally expressed a CXCR4-GFP fusion protein in the CXCR4-deficient human hematopoietic progenitor cell line KG1a. This KG1a CXCR4-GFP cell line showed full restoration of SDF-1 responsiveness in assays detecting activation of ERK1/2 phosphorylation, actin polymerization, adhesion to endothelium under conditions of physiological flow, and (transendothelial) chemotaxis. When adhered to cytokine-activated endothelium in the absence of SDF-1, CXCR4 did not localize to the leading edge of the cell but was uniformly distributed over the plasma membrane. In contrast, when SDF-1 was immobilized on cytokine-activated endothelium, the CXCR4-GFP receptors that were present on the cell surface markedly redistributed to the leading edge of migrating cells. In addition, CXCR4-GFP co-localized with lipid rafts in the leading edge of SDF-1-stimulated cells, at the sites of contact with the endothelial surface. Inhibition of lipid raft formation prevents SDF-1-dependent migration, internalization of CXCR4, and polarization to the leading edge of CXCR4, indicating that CXCR4 surface expression and signaling requires lipid rafts. These data show that SDF-1, immobilized on activated human endothelium, induces polarization of CXCR4 to the leading edge of migrating cells, revealing co-operativity between chemokine and substrate in the control of cell migration.

Rho-like GTPases control a wide range of cellular functions such as integrin- and cadherin-mediated adhesion, cell motility, and gene expression. The hypervariable C-terminal domain of these GTPases has been implicated in membrane association and effector binding. We found that cell-permeable peptides, encoding the C termini of Rac1, Rac2, RhoA, and Cdc42, interfere with GTPase signaling in a specific fashion in a variety of cellular models. Pull-down assays showed that the C terminus of Rac1 does not associate to either RhoGDI or to Pak. In contrast, the C terminus of Rac1 (but not Rac2 or Cdc42) binds to phosphatidylinositol 4,5-phosphate kinase (PIPSK) via amino acids 185-187 (RKR). Moreover, Rac1 associates to the adapter protein Crk via the N-terminal Src homology 3 (SH3) domain of Crk and the proline-rich stretch in the Rac1 C terminus. These differential interactions mediate Rac1 localization, as well as Rac1 signaling, toward membrane ruffling, cell-cell adhesion, and migration. These data show that the C-terminal, hypervariable domain of Rac1 encodes two distinct binding motifs for signaling proteins and regulates intracellular targeting and differential signaling in a unique and non-redundant fashion.

The integrity of the endothelium is dependent on cell-cell adhesion, which is mediated by vascular-endothelial (VE)-cadherin. Proper VE-cadherin-mediated homotypic adhesion is, in turn, dependent on the connection between VE-cadherin and the cortical actin cytoskeleton. Rho-like small GTPases are key molecular switches that control cytoskeletal dynamics and cadherin function in epithelial as well as endothelial cells. We show here that a cell-penetrating, constitutively active form of Rac (Tat-RacV12) induces a rapid loss of VE-cadherin-mediated cell-cell adhesion in endothelial cells from primary human umbilical veins (pHUVEC). This effect is accompanied by the formation of actin stress fibers and is dependent on Rho activity. However,transduction of pHUVEC with Tat-RhoV14, which induces pronounced stress fiber and focal adhesion formation, did not result in a redistribution of VE-cadherin or an overall loss of cell-cell adhesion. In line with this observation, endothelial permeability was more efficiently increased by Tat-RacV12 than by Tat-RhoV14. The loss of cell-cell adhesion, which is induced by Tat-RacV12, occurred in parallel to and was dependent upon the intracellular production of reactive oxygen species (ROS). Moreover, Tat-RacV12 induced an increase in tyrosine phosphorylation of a component the VE-cadherin-catenin complex, which was identified as α-catenin. The functional relevance of this signaling pathway was further underscored by the observation that endothelial cell migration, which requires a transient reduction of cell-cell adhesion, was blocked when signaling through ROS was inhibited. In conclusion, Rac-mediated production of ROS represents a previously unrecognized means of regulating VE-cadherin function and may play an important role in the (patho)physiology associated with inflammation and endothelial damage as well as with endothelial cell migration and angiogenesis.

The success of stem cell transplantation depends on the ability of i.v. infused stem cells to engraft the bone marrow, a process referred to as homing. Efficient homing requires migration of CD34+ cells across the bone marrow endothelium, most likely through the intercellular junctions. In this study, we show that loss of vascular endothelial (VE)-cadherin-mediated endothelial cell-cell adhesion increases the permeability of monolayers of human bone marrow endothelial cells (HBMECs) and stimulates the transendothelial migration of CD34+ cells in response to stromal cell-derived factor-1α. Stromal cell-derived factor-1α-induced migration was dependent on VCAM-1 and ICAM-1, even in the absence of VE-cadherin function. Cross-linking of ICAM-1 to mimic the leukocyte-endothelium interaction induced actin stress fiber formation but did not induce loss of endothelial integrity, whereas cross-linking of VCAM-1 increased the HBMEC permeability and induced gaps in the monolayer. In addition, VCAM-1-mediated gap formation in HBMEC was accompanied by and dependent on the production of reactive oxygen species. These data suggest that modulation of VE-cadherin function directly affects the efficiency of transendothelial migration of CD34+ cells and that activation of ICAM-1 and, in particular, VCAM-1 plays an important role in this process through reorganization of the endothelial actin cytoskeleton and by modulating the integrity of the bone marrow endothelium through the production of reactive oxygen species.

Objective: The capacity of hematopoietic progenitor cells (HPCs; CD34+ cells) to respond to chemotactic stimulation is essential for their homing efficiency, e.g., during stem cell transplantation. Previous studies established that stromal cell-derived factor-1 (SDF-1) and its receptor CXCR-4 play an important role in the homing of HPCs. The aim of the present study was to analyze SDF-1-induced actin polymerization and migration of HL-60 cells and primary human CD34+ cells.Materials and MethodsSDF-1-induced migration of CD34+ cells from cord blood (CB) and peripheral blood (PB) across fibronectin-coated filters was measured in a Transwell assay. Actin polymerization was detected using fluorescent phalloidin and analyzed by confocal microscopy and FACS analysis.ResultsSDF-1 induced a rapid and transient increase in actin polymerization and in polarization of the actin cytoskeleton in primary CD34+ cells and HL-60 cells. SDF-1 was found to induce significantly more actin polymerization in CB CD34+ cells that show fast migration in vitro compared to slow migrating PB CD34+ cells. Moreover, CB CD34+ cells that had migrated toward SDF-1 showed an elevated and prolonged rise in F-actin upon second exposure to SDF-1 compared to nonmigrated cells, although both cell types expressed equal levels of the SDF-1 receptor CXCR-4.ConclusionsThe relatively high migratory capacity of CB-derived human HPCs is not related to cellular polarization or high expression of the SDF-1 receptor but is largely determined by their capacity to efficiently polymerize F-actin in response to SDF-1.

Vascular endothelial (VE)-cadherin is the endothelium-specific member of the cadherin family of homotypic cell adhesion molecules. VE-cadherin, but not the cell adhesion molecule platelet/endothelial cell adhesion molecule (PECAM-1), markedly colocalizes with actin stress fibers at cell-cell junctions between human umbilical vein endothelial cells. Inhibition of VE-cadherin-mediated, but not PECAM-1-mediated, adhesion induced reorganization of the actin cytoskeleton, loss of junctional VE-cadherin staining and loss of cell-cell adhesion. In functional assays, inhibition of VE-cadherin caused increased monolayer permeability and enhanced neutrophil transendothelial migration. In a complementary set of experiments, modulation of the actin cytoskeleton was found to strongly affect VE-cadherin distribution. Brief stimulation of the beta2-adrenergic receptor with isoproterenol induced a loss of actin stress fibers resulting in a linear, rather than ‘jagged’, VE-cadherin distribution. The concomitant, isoproterenol-induced, reduction in monolayer permeability was alleviated by a VE-cadherin-blocking antibody. Finally, cytoskeletal reorganization resulting from the inactivation of p21Rho caused a diffuse localization of VE-cadherin, which was accompanied by reduced cell-cell adhesion. Together, these data show that monolayer permeability and neutrophil transendothelial migration are modulated by VE-cadherin-mediated cell-cell adhesion, which is in turn controlled by the dynamics of the actin cytoskeleton.