The N-Terminal DH-PH Domain of Trio Induces Cell Spreading and Migration by Regulating Lamellipodia Dynamics in a Rac1-Dependent Fashion

The guanine-nucleotide exchange factor Trio encodes two DH-PH domains that catalyze nucleotide exchange on Rac1, RhoG and RhoA. The N-terminal DH-PH domain is known to activate Rac1 and RhoG, whereas the C-terminal DH-PH domain can activate RhoA. The current study shows that the N-terminal DH-PH domain, upon expression in HeLa cells, activates Rac1 and RhoG independently from each other. In addition, we show that the flanking SH3 domain binds to the proline-rich region of the C-terminus of Rac1, but not of RhoG. However, this SH3 domain is not required for Rac1 or RhoG GDP-GTP exchange. Rescue experiments in Trio-shRNA-expressing cells showed that the N-terminal DH-PH domain of Trio, but not the C-terminal DH-PH domain, restored fibronectin-mediated cell spreading and migration defects that are observed in Trio-silenced cells. Kymograph analysis revealed that the N-terminal DH-PH domain, independent of its SH3 domain, controls the dynamics of lamellipodia. Using siRNA against Rac1 or RhoG, we found that Trio-D1-induced lamellipodia formation required Rac1 but not RhoG expression. Together, we conclude that the GEF Trio is responsible for lamellipodia formation through its N-terminal DH-PH domain in a Rac1-dependent manner during fibronectin-mediated spreading and migration

Inside-Out Regulation of ICAM-1 Dynamics in TNF-α-Activated Endothelium

Background: During transendothelial migration, leukocytes use adhesion molecules, such as ICAM-1, to adhere to the endothelium. ICAM-1 is a dynamic molecule that is localized in the apical membrane of the endothelium and clusters upon binding to leukocytes. However, not much is known about the regulation of ICAM-1 clustering and whether membrane dynamics are linked to the ability of ICAM-1 to cluster and bind leukocyte integrins. Therefore, we studied the dynamics of endothelial ICAM-1 under non-clustered and clustered conditions. Principal Findings: Detailed scanning electron and fluorescent microscopy showed that the apical surface of endothelial cells constitutively forms small filopodia-like protrusions that are positive for ICAM-1 and freely move within the lateral plane of the membrane. Clustering of ICAM-1, using anti-ICAM-1 antibody-coated beads, efficiently and rapidly recruits ICAM-1. Using fluorescence recovery after photo-bleaching (FRAP), we found that clustering increased the immobile fraction of ICAM-1, compared to non-clustered ICAM-1. This shift required the intracellular portion of ICAM-1. Moreover, biochemical assays showed that ICAM-1 clustering recruited beta-actin and filamin. Cytochalasin B, which interferes with actin polymerization, delayed the clustering of ICAM-1. In addition, we could show that cytochalasin B decreased the immobile fraction of clustered ICAM-1-GFP, but had no effect on non-clustered ICAM-1. Also, the motor protein myosin-II is recruited to ICAM-1 adhesion sites and its inhibition increased the immobile fraction of both non-clustered and clustered ICAM-1. Finally, blocking Rac1 activation, the formation of lipid rafts, myosin-II activity or actin polymerization, but not Src, reduced the adhesive function of ICAM-1, tested under physiological flow conditions. Conclusions: Together, these findings indicate that ICAM-1 clustering is regulated in an inside-out fashion through the actin cytoskeleton. Overall, these data indicate that signaling events within the endothelium are required for efficient ICAM-1-mediated leukocyte adhesio

Repercussion of megakaryocyte-specific Gata1 Loss on megakaryopoiesis and the hematopoietic precursor compartment

During hematopoiesis, transcriptional programs are essential for the commitment and differentiation of progenitors into the different blood lineages. GATA1 is a transcription factor expressed in several hematopoietic lineages and essential for proper erythropoiesis and megakaryopoiesis. Megakaryocyte-specific genes, such as GP1BA, are known to be directly regulated by GATA1. Mutations in GATA1 can lead to dyserythropoietic anemia and pseudo gray-platelet syndrome. Selective loss of Gata1 expression in adult mice results in macrothrombocytopenia with platelet dysfunction, characterized by an excess of immature megakaryocytes. To specifically analyze the impact of Gata1 loss in mature committed megakaryocytes, we generated Gata1-Lox|Pf4-Cre mice (Gata1cKOMK). Consistent with previous findings, Gata1cKOMK mice are macrothrombocytopenic with platelet dysfunction. Supporting this notion we demonstrate that Gata1 regulates directly the transcription of Syk, a tyrosine kinase that functions downstream of Clec2 and GPVI receptors in megakaryocytes and platelets. Furthermore, we show that Gata1cKOMK mice display an additional aberrant megakaryocyte differentiation stage. Interestingly, these mice present a misbalance of the multipotent progenitor compartment and the erythroid lineage, which translates into compensatory stress erythropoiesis and splenomegaly. Despite the severe thrombocytopenia, Gata1cKOMK mice display a mild reduction of TPO plasma levels, and Gata1cK-OMK megakaryocytes show a mild increase in Pf4 mRNA levels; such a misbalance might be behind the general hematopoietic defects observed, affecting locally normal TPO and Pf4 levels at hematopoietic stem cell niches. © 2016 Meinders et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

SIRP alpha on Mouse B1 Cells Restricts Lymphoid Tissue Migration and Natural Antibody Production

The inhibitory immunoreceptor SIRPα is expressed on myeloid and neuronal cells and interacts with the broadly expressed CD47. CD47-SIRPα interactions form an innate immune checkpoint and its targeting has shown promising results in cancer patients. Here, we report expression of SIRPα on B1 lymphocytes, a subpopulation of murine B cells responsible for the production of natural antibodies. Mice defective in SIRPα signaling (SIRPαΔCYT mice) displayed an enhanced CD11b/CD18 integrin-dependent B1 cell migration from the peritoneal cavity to the spleen, local B1 cell accumulation, and enhanced circulating natural antibody levels, which was further amplified upon immunization with T-independent type 2 antigen. As natural antibodies are atheroprotective, we investigated the involvement of SIRPα signaling in atherosclerosis development. Bone marrow (SIRPαΔCYT>LDLR−/−) chimaeric mice developed reduced atherosclerosis accompanied by increased natural antibody production. Collectively, our data identify SIRPα as a unique B1 cell inhibitory receptor acting to control B1 cell migration, and imply SIRPα as a potential therapeutic target in atherosclerosis

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para-and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para-and transcellular migration routes of the leukocytes across the endotheliu

The tyrosine phosphatase SHP2 regulates recovery of endothelial adherens junctions through control of beta-catenin phosphorylation

Impaired endothelial barrier function results in a persistent increase in endothelial permeability and vascular leakage. Repair of a dysfunctional endothelial barrier requires controlled restoration of adherens junctions, comprising vascular endothelial (VE)-cadherin and associated beta-, gamma-, alpha-, and p120-catenins. Little is known about the mechanisms by which recovery of VE-cadherin-mediated cell-cell junctions is regulated. Using the inflammatory mediator thrombin, we demonstrate an important role for the Src homology 2-domain containing tyrosine phosphatase (SHP2) in mediating recovery of the VE-cadherin-controlled endothelial barrier. Using SHP2 substrate-trapping mutants and an in vitro phosphatase activity assay, we validate beta-catenin as a bona fide SHP2 substrate. SHP2 silencing and SHP2 inhibition both result in delayed recovery of endothelial barrier function after thrombin stimulation. Moreover, on thrombin challenge, we find prolonged elevation in tyrosine phosphorylation levels of VE-cadherin-associated beta-catenin in SHP2-depleted cells. No disassembly of the VE-cadherin complex is observed throughout the thrombin response. Using fluorescence recovery after photobleaching, we show that loss of SHP2 reduces the mobility of VE-cadherin at recovered cell-cell junctions. In conclusion, our data show that the SHP2 phosphatase plays an important role in the recovery of disrupted endothelial cell-cell junctions by dephosphorylating VE-cadherin-associated beta-catenin and promoting the mobility of VE-cadherin at the plasma membran

Flow-induced Reorganization of Laminin-integrin Networks Within the Endothelial Basement Membrane Uncovered by Proteomics

The vessel wall is continuously exposed to hemodynamic forces generated by blood flow. Endothelial mechanosensors perceive and translate mechanical signals via cellular signaling pathways into biological processes that control endothelial development, phenotype and function. To assess the hemodynamic effects on the endothelium on a system-wide level, we applied a quantitative mass spectrometry approach combined with cell surface chemical footprinting. SILAC-labeled endothelial cells were subjected to flow-induced shear stress for 0, 24 or 48 h, followed by chemical labeling of surface proteins using a non-membrane permeable biotin label, and analysis of the whole proteome and the cell surface proteome by LC-MS/MS analysis. These studies revealed that of the >5000 quantified proteins 104 were altered, which were highly enriched for extracellular matrix proteins and proteins involved in cell-matrix adhesion. Cell surface proteomics indicated that LAMA4 was proteolytically processed upon flow-exposure, which corresponded to the decreased LAMA4 mass observed on immunoblot. Immunofluorescence microscopy studies highlighted that the endothelial basement membrane was drastically remodeled upon flow exposure. We observed a network-like pattern of LAMA4 and LAMA5, which corresponded to the localization of laminin-adhesion molecules ITGA6 and ITGB4. Furthermore, the adaptation to flow-exposure did not affect the inflammatory response to tumor necrosis factor α, indicating that inflammation and flow trigger fundamentally distinct endothelial signaling pathways with limited reciprocity and synergy. Taken together, this study uncovers the blood flow-induced remodeling of the basement membrane and stresses the importance of the subendothelial basement membrane in vascular homeostasis

Trio-D1 binds to the C-terminus of Rac1 but not of RhoG and activates Rac1 independent of its SH3 domain.

<p>(<b>A</b>) HeLa cells were transfected with Myc-Trio-Full-length (FL) and a pull-down experiment with biotin-tagged peptides that encode for the last 10 amino-acids of the C-terminus of Rac1, RhoG and RhoA was performed, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#s4" target="_blank">Materials and Methods</a>. Western blot analysis showed that Trio-FL binds to the Rac1 C-terminus peptide, but not to RhoA or RhoG C-termini. As a control, β-Pix binding to the C-terminus of Rac1, but not of RhoA or RhoG is shown. (<b>B</b>) Myc-Trio-D1 was transfected into HeLa cells, and a peptide pull down was performed as described under A. Western blot analysis showed that Trio-D1 associates with the C-terminus of Rac1, but not with the CTRL or RhoG peptide. Left lane shows Myc-Trio-D1 input. (<b>C</b>) HeLa cells were transfected with GFP-Trio-D1ΔSH3 or GFP-Trio-D1+SH3 constructs and a Rac1 C-terminal peptide pull down was performed. Western blot analysis showed that the C-terminus of Rac1 required the SH3 domain of Trio-D1 to interact. Blots were incubated with an Ab against GFP to stain for Trio constructs. (<b>D</b>) HeLa cells were transfected with GFP-Trio-D1+SH3 constructs and a peptide pull down was performed with biotinylated peptides encoding control sequence, the Rac1 C-terminal domain or the Rac1 C-terminal domains in which the proline stretch had been mutated to alanines (P/A) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#pone.0029912-Nethe1" target="_blank">[10]</a>. Western blot analysis showed that Trio-D1+SH3 required the proline-rich stretch in the Rac1 C-terminus to bind. Blots were incubated with an Ab against GFP to stain for Trio constructs. (<b>E</b>) HeLa cells were transfected with GFP-CAAX, GFP-Trio-D1ΔSH3 or GFP-Trio-D1+SH3 constructs, and Rac1-GTP activity assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#s4" target="_blank">Materials and Methods</a>. Western blot analysis showed that Rac1 is activated by Trio-D1, independent of the SH3 domain (upper panel). (<b>F</b>) HeLa cells were transfected with GFP-CAAX, GFP-Trio-D1ΔSH3 or GFP-Trio-D1+SH3 constructs and a pull-down assay using glutathione-beads to precipitate GST-Rac1-G15A mutants was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#s4" target="_blank">Materials and Methods</a>. Western blot analysis showed that Rac1 needed the SH3 domain of Trio-D1 to interact (upper panel), because the binding was less efficient when Trio-D1 lacked the SH3 domain. Lower panel shows GST-Rac1-G15A input. Lower unidentified band in upper panel is due to GST isolation and a-specific staining of the antibody. Graph below shows the quantification of the binding of GST-Rac1-G15A to Trio-D1ΔSH3 and Trio-D1+SH3. No significant difference was found for the presence of the SH3 domain in the binding to GST-Rac1-G15A. Experiment was carried out three times, independently from each other. Data are mean ± SEM. ns: not significant.</p

Trio induces membrane ruffles.

<p>(<b>A</b>) Schematic overview of the Trio protein (3097 amino acids, molecular weight approximately 350 kDa), indicating in green the N-terminal DH-PH unit including an SH3 domain and in red the C-terminal DH-PH unit. The third catalytic domain of Trio is a kinase domain (yellow). At the N-terminus, a Sec14 domain and spectrin repeats are present. Below the GFP/Myc-tagged constructs used in this manuscript are depicted: Trio-D1 encodes for the N-terminal DH-PH domain including the flanking SH3 domain, Trio-D1ΔSH3 domain represents the N-terminal DH-PH domain lacking the SH3 domain, and Trio-D2 representing the C-terminal DH-PH domain. (<b>B</b>) HeLa cells were cultured on glass cover slips and transfected as indicated with GFP-tagged constructs. Immunofluorescent imaging showed that GFP did not affect the morphology of the cells. GFP-Trio full length (FL) and GFP-Trio-D1 induced lamellipodia (arrowheads) and co-localized with F-actin (red), as is shown in the merge images. For the Trio-FL, 68%±7 of the transfected cells induced lamellipodia as illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#pone-0029912-g001" target="_blank">figure 1B</a>. For Trio-D1, 79%±4 of the transfected cells induced lamellipodia. GFP-Trio-D2 in green induced stress fibers (arrowheads), shown by F-actin staining in red. 46%±12 of these transfected cells i9nduced stress fibers, as shown. Data are mean ± SEM. Bar, 20 µm. Images at the right show merged magnification of F-actin in red and GFP-tagged protein in green. Bar, 10 µm. (<b>C</b>) Changes in morphology analyzed by scanning electron microscopy. No change in morphology is observed at the periphery or surface of GFP-expressing HeLa cells (arrowheads), whereas Trio-D1 induced large dorsal and lateral lamellipodia (arrowheads). Bar, 50 µm. Image on the right shows a magnification (Zoom) of Trio-D1-induced dorsal lamellipodia (arrowheads). Bar, 5 µm. Two lower images show lamellipodia (arrowheads), induced by a constitutively active form of RhoG (Q61L) (left image) and Rac1 (Q61L) (right image), both comparable with the lamellipodia induced by Trio-D1. Bar, 10 µm.</p