Abstract
Lysophosphatidic acid (LPA) stimulates sphingosine-1-phosphate (S1P)-sensitive motility in NIH3T3 clone7 cells. S1P inhibits motility only when added to the bottom well of the Boyden chamber, suggesting that pseudopodia can respond to their microenvironment. In order to study and localize this effect, we utilized a Transwell insert system to isolate pseudopodia. LPA stimulates protrusion of pseudopodia that are enriched in RhoA compared to cell bodies. Removal of LPA results in slow retraction with loss of vinculin-rich adhesion complexes and prolonged activation of RhoA. However, RhoA, ROCK and mDia are not required for this process. In contrast, rapid retraction, induced by adding S1P to the bottom well, is associated with a quick spike of activated RhoA and coalescence of adhesion complexes that colocalize with the ends of stress fibers. S1P-induced retraction requires RhoA and ROCK but is only delayed by inhibition of mDia. These data indicate that pseudopodia sense and integrate signals initiated by localized bioactive lipids, affecting both cellular polarity and their own function in motility.
Publication types
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Research Support, N.I.H., Intramural
MeSH terms
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Actins / metabolism
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Animals
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Carrier Proteins / antagonists & inhibitors
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Carrier Proteins / metabolism
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Cell Adhesion / drug effects
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Cell Line
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Cell Movement / drug effects
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Cell Polarity / drug effects
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Enzyme Activation / drug effects
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Formins
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Humans
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Intracellular Signaling Peptides and Proteins / antagonists & inhibitors
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Intracellular Signaling Peptides and Proteins / metabolism
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Lysophospholipids / pharmacology*
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Mice
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Microtubules / drug effects
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Microtubules / metabolism
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NIH 3T3 Cells
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Protein Serine-Threonine Kinases / antagonists & inhibitors
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Protein Serine-Threonine Kinases / metabolism
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Protein Transport / drug effects
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Pseudopodia / drug effects*
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Pseudopodia / enzymology*
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Sphingosine / analogs & derivatives*
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Sphingosine / pharmacology
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Vinculin / metabolism
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rac GTP-Binding Proteins / metabolism
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rho-Associated Kinases
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rhoA GTP-Binding Protein / antagonists & inhibitors
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rhoA GTP-Binding Protein / metabolism*
Substances
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Actins
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Carrier Proteins
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Diap1 protein, mouse
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Formins
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Intracellular Signaling Peptides and Proteins
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Lysophospholipids
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Vinculin
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sphingosine 1-phosphate
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Protein Serine-Threonine Kinases
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rho-Associated Kinases
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rac GTP-Binding Proteins
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rhoA GTP-Binding Protein
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Sphingosine
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lysophosphatidic acid