ABSTRACT
Controlled regulation of Rho GTPase activity is an essential component mediating growth factor-stimulated migration. We have previously shown that angiomotin (Amot), a membrane-associated scaffold protein, plays a critical role during vascular patterning and endothelial migration during embryogenesis. However, the signaling pathways by which Amot controls directional migration are not known. Here we have used peptide pull-down and yeast 2-hybrid (Y2H) screening to identify proteins that interact with the C-terminal PDZ-binding motifs of Amot and its related proteins AmotL1 and 2. We report that Amot and its related proteins bind to the RhoA GTPase exchange factor (RhoGEF) protein Syx. We show that Amot forms a ternary complex together with Patj (or its paralogue Mupp1) and Syx. Using FRET analysis, we provide evidence that Amot controls targeting of RhoA activity to lamellipodia in vitro. We also report that, similar to Amot, morpholino knockdown of Syx in zebrafish results in inhibition of migration of intersegmental arteries. Taken together, our results indicate that the directional migration of capillaries in the embryo is governed by the Amot:Patj/Mupp1:Syx signaling that controls local GTPase activity.
Subject(s)
Capillaries/embryology , Endothelial Cells/physiology , Guanine Nucleotide Exchange Factors/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , rhoA GTP-Binding Protein/metabolism , Angiomotins , Animals , Animals, Genetically Modified , Aorta/cytology , Capillaries/cytology , Capillaries/metabolism , Carrier Proteins/metabolism , Cell Line, Transformed , Cell Movement/physiology , Endothelial Cells/cytology , Guanine Nucleotide Exchange Factors/genetics , Humans , Intercellular Signaling Peptides and Proteins/genetics , Kidney/cytology , Membrane Proteins/genetics , Mice , Microfilament Proteins , Neovascularization, Physiologic/physiology , PDZ Domains/physiology , Rho Guanine Nucleotide Exchange Factors , Tight Junction Proteins , Zebrafish , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
We have previously shown that angiomotin (Amot) plays an important role in growth factor-induced migration of endothelial cells in vitro. Genetic knock-down of Amot in zebrafish also results in inhibition of migration of intersegmental vessels in vivo. Amot is expressed as two different isoforms, p80-Amot and p130-Amot. Here we have analyzed the expression of the two Amot isoforms during retinal angiogenesis in vivo and demonstrate that p80-Amot is expressed during the migratory phase. In contrast, p130-Amot is expressed during the period of blood vessel stabilization and maturation. We also show that the N-terminal domain of p130-Amot serves as a targeting domain responsible for localization of p130-Amot to actin and tight junctions. We further show that the relative expression levels of p80-Amot and p130-Amot regulate a switch between a migratory and a non-migratory cell phenotype where the migratory function of p80-Amot is dominant over the stabilization and maturation function of p130-Amot. Our data indicates that homo-oligomerization of p80-Amot and hetero-oligomerization of both isoforms are critical for this regulation.
Subject(s)
Cell Adhesion/physiology , Cell Movement/physiology , Endothelial Cells/physiology , Intercellular Signaling Peptides and Proteins/physiology , Microfilament Proteins/physiology , Angiomotins , Animals , Animals, Newborn , CHO Cells , Cell Communication/physiology , Cells, Cultured , Cricetinae , Cricetulus , Dimerization , Dogs , Endothelial Cells/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Mice , Microfilament Proteins/metabolism , Neovascularization, Physiologic/physiology , Protein Binding , Protein Isoforms/metabolism , Protein Isoforms/physiology , Protein Transport , Retinal Vessels/growth & development , Retinal Vessels/metabolismABSTRACT
The development of the embryonic vascular system into a highly ordered network requires precise control over the migration and branching of endothelial cells (ECs). We have previously identified angiomotin (Amot) as a receptor for the angiogenesis inhibitor angiostatin. Furthermore, DNA vaccination targeting Amot inhibits angiogenesis and tumor growth. However, little is known regarding the role of Amot in physiological angiogenesis. We therefore investigated the role of Amot in embryonic neovascularization during zebrafish and mouse embryogenesis. Here we report that knockdown of Amot in zebrafish reduced the number of filopodia of endothelial tip cells and severely impaired the migration of intersegmental vessels. We further show that 75% of Amot knockout mice die between embryonic day 11 (E11) and E11.5 and exhibit severe vascular insufficiency in the intersomitic region as well as dilated vessels in the brain. Furthermore, using ECs differentiated from embryonic stem (ES) cells, we demonstrate that Amot-deficient cells have intact response to vascular endothelial growth factor (VEGF) in regard to differentiation and proliferation. However, the chemotactic response to VEGF was abolished in Amot-deficient cells. We provide evidence that Amot is important for endothelial polarization during migration and that Amot controls Rac1 activity in endothelial and epithelial cells. Our data demonstrate a critical role for Amot during vascular patterning and endothelial polarization.
Subject(s)
Intercellular Signaling Peptides and Proteins/physiology , Membrane Proteins/physiology , Microfilament Proteins/physiology , Neovascularization, Physiologic/physiology , Zebrafish Proteins/physiology , Zebrafish/embryology , Zebrafish/physiology , Angiomotins , Animals , Base Sequence , Body Patterning/genetics , Body Patterning/physiology , Cell Line , Cell Movement/genetics , Cell Movement/physiology , DNA Primers/genetics , Endothelial Cells/cytology , Endothelial Cells/physiology , Female , Gene Deletion , Gene Silencing , Humans , Intercellular Signaling Peptides and Proteins/deficiency , Intercellular Signaling Peptides and Proteins/genetics , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Microfilament Proteins/deficiency , Microfilament Proteins/genetics , Neovascularization, Physiologic/genetics , Phenotype , Pregnancy , Pseudopodia/ultrastructure , Zebrafish/genetics , Zebrafish Proteins/deficiency , Zebrafish Proteins/genetics , rac1 GTP-Binding Protein/metabolismABSTRACT
Angiomotin, an 80 kDa protein expressed in endothelial cells, promotes cell migration and invasion, and stabilizes tube formation in vitro. Angiomotin belongs to a new protein family with two additional members, Amotl-1 and Amotl-2, which are characterized by conserved coiled-coil domains and C-terminal PDZ binding motifs. Here, we report the identification of a 130 kDa splice isoform of angiomotin that is expressed in different cell types including vascular endothelial cells, as well as cytotrophoblasts of the placenta. p130-Angiomotin consists of a cytoplasmic N-terminal extension that mediates its association with F-actin. Transfection of p130-angiomotin into endothelial cells induces actin fiber formation and changes cell shape. The p130-angiomotin protein remained associated with actin after destabilization of actin fibers with cytochalasin B. In contrast to p80-angiomotin, p130-angiomotin does not promote cell migration and did not respond to angiostatin. We propose that p80- and p130-angiomotin play coordinating roles in tube formation by affecting cell migration and cell shape, respectively.
Subject(s)
Actins/metabolism , Cell Shape/physiology , Endothelial Cells/cytology , Endothelial Cells/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Alternative Splicing , Amino Acid Sequence , Angiomotins , Angiostatins/physiology , Animals , Cell Line , Cell Line, Transformed , Cell Movement/physiology , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , HeLa Cells , Humans , Intercellular Signaling Peptides and Proteins/biosynthesis , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/physiology , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Membrane Proteins/physiology , Mice , Mice, Knockout , Microfilament Proteins/biosynthesis , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Microfilament Proteins/physiology , Molecular Sequence Data , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Isoforms/physiologyABSTRACT
We have previously identified angiomotin by its ability to bind to and mediate the anti-angiogenic properties of angiostatin. In vivo and in vitro data indicate an essential role of angiomotin in endothelial cell motility. Here we show that angiostatin binds angiomotin on the cell surface and provide evidence for a transmembrane model for the topology of both p80 and p130 angiomotin isoforms. Immunofluorescence analysis shows that angiomotin co-localized with ZO-1 in cell-cell contacts in endothelial cells in vitro and in angiogenic blood vessels of the postnatal mouse retina in vivo. Transfection of p80 as well as p130 angiomotin in Chinese hamster ovary cells resulted in junctional localization of both isoforms. Furthermore, p130 angiomotin could recruit ZO-1 to actin stress fibers. The p130 but not p80 isoform could be coprecipitated with MAGI-1b, a component of endothelial tight junctions. Paracellular permeability, as measured by diffusion of fluorescein isothiocyanate-dextran, was reduced by p80 and p130 angiomotin expression with 70 and 88%, respectively, compared with control. Angiostatin did not have any effect on cell permeability but inhibited the migration of angiomotin-expressing cells in the Boyden chamber assay. We conclude that angiomotin, in addition to controlling cell motility, may play a role in the assembly of endothelial cell-cell junctions.
