The Cdc42 and Rac1 GTPases are required for capillary lumen formation in three-dimensional extracellular matrices.
Here we show a requirement for the Cdc42 and Rac1 GTPases in endothelial cell (EC) morphogenesis in three-dimensional extracellular matrices. Cdc42 and Rac1 specifically regulate EC intracellular vacuole and lumen formation in both collagen and fibrin matrices. Clostridium difficile toxin B (which blocks all three Rho GTPases) completely inhibited the ability of ECs to form both vacuoles and lumens, whereas C3 transferase, a selective inhibitor of Rho, did not. Expression of either dominant-negative (N17) or constitutively active (V12) Cdc42 using recombinant adenoviruses dramatically inhibited EC vacuole and lumen formation in both collagen and fibrin matrices. Both vacuole and lumen formation initiated in ECs expressing dominant-negative (N17) Rac1 but later collapsed, indicating a role for Rac1 during later stages of vessel development. Analysis of cultures using confocal microscopy revealed green fluorescent protein-V12Rac1, -Rac1 wild-type and -Cdc42 wild-type chimeric proteins targeted to intracellular vacuole membranes during the lumen formation process. Also, expression of the verprolin-cofilin-acidic domain of N-WASP, a downstream Cdc42 effector, in ECs completely interfered with vacuole and lumen formation. These results collectively reveal a novel role for Cdc42 and Rac1 in the process of EC vacuole and lumen formation in three-dimensional extracellular matrices.
Bayless
Kayla J
KJ
Department of Pathology and Laboratory Medicine, Texas A&M University System Health Science Center, College Station, Texas 77843-1114, USA.
Davis
George E
GE
D051339:Actin Depolymerizing Factors; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000494:pharmacology; D002196:Capillaries / Q000033:anatomy & histology / Q000201:enzymology / Q000254:growth & development*; D004730:Endothelium, Vascular / Q000033:anatomy & histology / Q000201:enzymology* / Q000254:growth & development*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005109:Extracellular Matrix / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D006801:Humans; D021621:Imaging, Three-Dimensional; D008840:Microfilament Proteins / Q000378:metabolism; D009024:Morphogenesis; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010873:Pinocytosis; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins; D014617:Vacuoles / Q000648:ultrastructure; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D020764:cdc42 GTP-Binding Protein / Q000502:physiology*; D020830:rac1 GTP-Binding Protein / Q000502:physiology*; D020741:rho GTP-Binding Proteins / Q000037:antagonists & inhibitors
D051339:Actin Depolymerizing Factors; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D029701:Saccharomyces cerevisiae Proteins; C090350:VRP1 protein, S cerevisiae; C495771:WASL protein, human; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; C031815:toxB protein, Clostridium difficile; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins