Journal Article

Cdc42 and k-Ras Control Endothelial Tubulogenesis through Apical Membrane and Cytoskeletal Polarization: Novel Stimulatory Roles for GTPase Effectors, the Small GTPases, Rac2 and Rap1b, and Inhibitory Influence of Arhgap31 and Rasa1

Barry, David M., Norden, Pieter R., Cleaver, Ondine B., Kim, Dae Joong, Davis, George E.

2016

Overview

A critical and understudied property of endothelial cells is their ability to form lumens and tube networks. Although considerable information has been obtained concerning these issues, including the role of Cdc42 and Rac1 and their effectors such as Pak2, Pak4, Par6b, and co-regulators such as integrins, MT1-MMP and Par3; many key questions remain that are necessary to elucidate molecular and signaling requirements for this fundamental process. In this work, we identify new small GTPase regulators of EC tubulogenesis including k-Ras, Rac2 and Rap1b that act in conjunction with Cdc42 as well as the key downstream effectors, IQGAP1, MRCKβ, beta-Pix, GIT1, and Rasip1 (which can assemble into multiprotein complexes with key regulators including α2β1 integrin and MT1-MMP). In addition, we identify the negative regulators, Arhgap31 (by inactivating Cdc42 and Rac) and Rasa1 (by inactivating k-Ras) and the positive regulator, Arhgap29 (by inactivating RhoA) which play a major functional role during the EC tubulogenic process. Human EC siRNA suppression or mouse knockout of Rasip1 leads to identical phenotypes where ECs form extensive cord networks, but cannot generate lumens or tubes. Essential roles for these molecules during EC tubulogenesis include; i) establishment of asymmetric EC cytoskeletal polarization (subapical distribution of acetylated tubulin and basal membrane distribution of F-actin); and ii) directed membrane trafficking of pinocytic vacuoles or other intracellular vesicles along acetylated tubulin tracks to the developing apical membrane surface. Cdc42 co-localizes subapically with acetylated tubulin, while Rac1 and k-Ras strongly label vacuole/ vesicle membranes which accumulate and fuse together in a polarized, perinuclear manner. We observe polarized apical membrane and subapical accumulation of key GTPases and effectors regulating EC lumen formation including Cdc42, Rac1, Rac2, k-Ras, Rap1b, activated c-Raf and Rasip1 to control EC tube network assembly. Overall, this work defines novel key regulators and their functional roles during human EC tubulogenesis.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Actin / Animals / Biology and life sciences / cdc42 GTP-Binding Protein - antagonists & inhibitors / cdc42 GTP-Binding Protein - genetics / cdc42 GTP-Binding Protein - metabolism / Cdc42 protein / Cell Line / Cellular signal transduction / Cytoskeleton / Cytoskeleton - metabolism / Effectors / Endothelial cells / Endothelial Cells - cytology / Endothelial Cells - drug effects / Endothelial Cells - metabolism / Endothelium / Female / Genetic aspects / Growth factors / GTPase-Activating Proteins - antagonists & inhibitors / GTPase-Activating Proteins - genetics / GTPase-Activating Proteins - metabolism / GTPases / Guanosine triphosphatases / Guanosinetriphosphatase / Humans / Integrins / IQGAP1 protein / K-Ras protein / Lumens / Male / Membrane trafficking / Membranes / Mice / Microscopy, Fluorescence / Molecular biology / Morphogenesis / p120 GTPase Activating Protein - antagonists & inhibitors / p120 GTPase Activating Protein - genetics / p120 GTPase Activating Protein - metabolism / Pharmacology / Phorbol Esters - pharmacology / Phosphoproteins - antagonists & inhibitors / Phosphoproteins - genetics / Phosphoproteins - metabolism / Physiological aspects / Physiology / Polarization / Proteins / rac GTP-Binding Proteins - antagonists & inhibitors / rac GTP-Binding Proteins - genetics / rac GTP-Binding Proteins - metabolism / Rac1 protein / RAC2 GTP-Binding Protein / Rac2 protein / Raf protein / rap GTP-Binding Proteins - antagonists & inhibitors / rap GTP-Binding Proteins - genetics / rap GTP-Binding Proteins - metabolism / ras Proteins - antagonists & inhibitors / ras Proteins - genetics / ras Proteins - metabolism / Regulation / RhoA protein / RNA Interference / Signal Transduction - drug effects / siRNA / Skewed distributions / Tubes / Tubulin / Tubulin - metabolism / Vacuoles / Vacuoles - metabolism