Silencing of directional migration in roundabout4 knockdown endothelial cells

<p>Abstract</p> <p>Background</p> <p>Roundabouts are axon guidance molecules that have recently been identified to play a role in vascular guidance as well. In this study, we have investigated gene knockdown analysis of endothelial Robos, in particular <it>roundabout 4 </it>(<it>robo4</it>), the predominant Robo in endothelial cells using small interfering RNA technology <it>in vitro</it>.</p> <p>Results</p> <p><it>Robo1 and Robo4 </it>knockdown cells display distinct activity in endothelial cell migration assay. The knockdown of <it>robo4 </it>abrogated the chemotactic response of endothelial cells to serum but enhanced a chemokinetic response to Slit2, while <it>robo1 </it>knockdown cells do not display chemotactic response to serum or VEGF. <it>Robo4 </it>knockdown endothelial cells unexpectedly show up regulation of Rho GTPases. Zebrafish Robo4 rescues both Rho GTPase homeostasis and serum reduced chemotaxis in <it>robo4 </it>knockdown cells. Robo1 and Robo4 interact and share molecules such as Slit2, Mena and Vilse, a Cdc42-GAP. In addition, this study mechanistically implicates IRSp53 in the signaling nexus between activated Cdc42 and Mena, both of which have previously been shown to be involved with Robo4 signaling in endothelial cells.</p> <p>Conclusion</p> <p>This study identifies specific components of the Robo signaling apparatus that work together to guide directional migration of endothelial cells.</p

Thrombospondin-1 signaling through CD47 inhibits self-renewal by regulating c-myc and other stem cell transcription factors

Signaling through the thrombospondin-1 receptor CD47 broadly limits cell and tissue survival of stress, but the molecular mechanisms are incompletely understood. We now show that loss of CD47 permits sustained proliferation of primary murine endothelial cells, increases asymmetric division, and enables these cells to spontaneously reprogram to form multipotent embryoid body-like clusters. c-Myc, Klf4, Oct4, and Sox2 expression is elevated in CD47-null endothelial cells, in several tissues of CD47- and thrombospondin-1-null mice, and in a human T cell line lacking CD47. CD47 knockdown acutely increases mRNA levels of c-Myc and other stem cell transcription factors in cells and in vivo, whereas CD47 ligation by thrombospondin-1 suppresses c-Myc expression. The inhibitory effects of increasing CD47 levels can be overcome by maintaining c-Myc expression and are absent in cells with dysregulated c-Myc. Thus, CD47 antagonists enable cell self-renewal and reprogramming by overcoming negative regulation of c-Myc and other stem cell transcription factors

Effects of farnesol on embedded growth of an <i>efg1/efg1</i> strain.

<p>Photomicrographs taken <i>in situ</i> of HLC52 cells embedded in YPS agar containing 100 M farnesol after 2 days at 24^°C. <b>(A).</b> Three focal planes illustrating three colony morphologies. Arrow, non-filamentous spindle-shaped colonies in the lower level of the matrix; Arrowhead, upper level colony with compact pseudohyphal filaments that do not produce yeast; Boxed region, a colony expressing arboreal-shaped branching hyphae that develop midlevel in the matrix. Bar = 2 mm. <b>(B).</b> Higher magnification of area such as boxed in (A). Masses of elongated opaque or opaque-like cells (brackets) produced through invasive pseudohyphal migration. Bar = 150 μm. <b>(C).</b> Higher magnification of a leading edge of the pseudohyphal expansion from (B). Bar = 10 μm. <b>(D).</b> An opaque-like cell aggregation illustrating the final maturation step of opaque-like cells from the pseudohyphae. Calcofluor white stain. Bar = 5 μm.</p

Colony phenotypes under various growth conditions.

<p>(†) Per cent filamented colonies;</p><p>(±) Cell separation / cytokinesis defects</p><p>Colony phenotypes under various growth conditions.</p

Mutations in the Hbr1 P-loop define distinct embedded phenotypes.

<p><b>(A).</b> Locations of key predicted Hbr1 structural domains as indicated in the text. Accession numbers: Hbr1 (AF466197_1), AD-004 (NP_057367), Fap7 (NP_010115). Positions of Hbr1 mutated amino acids, (*). <b>(B).</b> Filamentation in the agar matrix can be modulated through HBR1 P-loop mutations. <i>C</i>. <i>albicans</i> cells were cultured within YPS agar at 24°C as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126919#pone.0126919.g001" target="_blank">Fig 1</a>. Photographs were taken through the agar matrix at two depths. Left, G19S mutation and hyphal ‘sprout’ development from yeast colonies at all levels in the matrix except from the lower level spindle-shaped colonies. A yeast mass with developing filaments is indicated by the arrow; Middle, G21S mutation and a uniform production of simple hyphae lacking yeast from spindle-shaped colonies furthest from the agar surface; Right, strain CAMPR8 restored to wild type (Strain R8WT). Arrows indicate sites of yeast production. <b>(C).</b> Manipulation of Hbr1 by increasing copy number using strain R8MET grown without added methionine (left), mutation of solvent-exposed Lys residue using strain R866R (center) and addition of a carboxyl-terminal protein tag using strain R8CTH (right). Arrows indicate sites of yeast production. Bars = 1.5 mm.</p

Hypoxia can rescue the agar invasion defect of the <i>HBR1</i> heterozygous strain.

<p><i>C</i>. <i>albicans</i> strains BWP17 (left column) and CAMPR8 (right column) were cultured for 4 days on the surface of the indicated media under aerobic or hypoxic (5% O₂) conditions at 30°C. Colonies were washed from the agar surfaces before photography through the agar.</p

Attenuated virulence of an <i>HBR1</i> heterozygote in a mouse model of disseminated infection.

<p>Strains DAY185 (<i>HBR1/HBR1</i>) and R8PRO (<i>HBR1/hbr1</i> ARG+ URA+ HIS+ prototroph) were introduced into female Balb/c mice <i>via</i> the tail vein at the indicated doses (n = 12, each).</p

Hbr1 is a negative regulator of hyphae formation under embedded conditions.

<p><i>C</i>. <i>albicans</i> log-phase yeast cells were embedded in molten YPS agar and incubated at 24°C. Photomicrographs were taken through the agar matrix. <b>(A).</b> Wild type strain BWP17 incubated for 4 days illustrating three modes of filamentation or yeast formation from spindle-shaped colonies. Note that these filamented colony types were only infrequently encountered. Panel 1, Pseudohyphal filaments; Panel 2, a single hyphal sprout; Panel 3, Yeast lateral outcropping from a spindle-shaped colony. Bar = 200 μm. <b>(B).</b> A comparison of colony types according to agar depth. Strains BWP17 (WT) and JKC19 (<i>cph1/cph1</i>) were incubated for 4 days; CAMPR8 (<i>HBR1/hbr1</i>) and HLC52 (<i>efg1/efg1</i>) for 2 days. The frequency that each strain formed a colony with at least 1 hypha is indicated (n >125). Strains CAMPR8 and HLC52 formed filaments at all agar depths. Surface colonies from all strains were smooth and lacked filaments. Arrow, spindle-shaped colony; arrow with line, spindle with yeast outgrowth; y, yeast colony. Bar = 2 mm. <b>(C).</b> Strain CAMPR8 cultured as above. Panel 1, simple hyphae with yeast (arrow), 2 day culture; Panel 2, hyphal sprout (arrow), 6 day culture; Bar = 200 μm; Panel 3, lateral yeast formation, Bar = 100 μm; Panel 4, perpendicular lateral branches (L) and yeast0020colonies originating at branch junctions (Y), Bar = 50 μm.</p

Hemoglobin Regulates Expression of an Activator of Mating-Type Locus α Genes in Candida albicans

Phenotypic switching from the white to the opaque phase is a necessary step for mating in the pathogenic fungus Candida albicans. Suppressing switching during vascular dissemination of the organism may be advantageous, because opaque cells are more susceptible to host defenses. A repressor of white-opaque switching, HBR1 (hemoglobin response gene 1), was identified based on its specific induction following growth in the presence of exogenous hemoglobin. Deletion of a single HBR1 allele allowed opaque phase switching and mating competence, accompanied by a lack of detectable MTL α1 and α2 gene expression and enhanced MTLa1 gene expression. Conversely, overexpression of Hbr1p or exposure to hemoglobin increased MTLα gene expression. The a1/α2 repressed target gene CAG1 was derepressed in the same mutant in a hemoglobin-sensitive manner. Regulation of CAG1 by hemoglobin required an intact MTLa1 gene. Several additional Mtlp targets were perturbed in HBR1 mutants in a manner consistent with commitment to an a mating phenotype, including YEL007w, MFα, HST6, and RAM2. Therefore, Hbr1 is part of a host factor-regulated signaling pathway that controls white-opaque switching and mating in the absence of allelic deletion at the MTL locus