G Proteins G12 and G13 Control the Dynamic Turnover of Growth Factor-induced Dorsal Ruffles

Abstract

Growth factors induce massive actin cytoskeletal remodeling in cells. These reorganization events underlie various cellular responses such as cell migration and morphological changes. One major form of actin reorganization is the formation and disassembly of dorsal ruffles (also named waves, dorsal rings, or circular ruffles). Dorsal ruffles are involved in physiological functions including cell migration, invasion, macropinocytosis, plasma membrane recycling, and others. Growth factors initiate rapid formation (within 5 min) of circular membrane ruffles, and these ruffles move along the dorsal side of the cells, constrict, close, and eventually disassemble (ϳ20 min). Considerable attention has been devoted to the mechanism by which growth factors induce the formation of dorsal ruffles. However, little is known of the mechanism by which these ruffles are disassembled. Here we have shown that G proteins G 12 and G 13 control the rate of disassembly of dorsal ruffles. In G␣ 12 ؊/؊ G␣ 13 ؊/؊ fibroblast cells, dorsal ruffles induced by growth factor treatment remain visible substantially longer (ϳ60 min) than in wild-type cells, whereas the rate of formation of these ruffles was the same with or without G␣ 12 and G␣ 13 . Thus, G␣ 12 /G␣ 13 critically regulate dorsal ruffle turnover.The earliest ultrastructural changes of cells treated with growth factors are the intensive bursts of ruffling of the dorsal surface plasma membranes as seen under the phase-contrast microscope (1-6). The physiological functions of dorsal ruffles, including macropinocytosis, cell migration, and invasion, are continually expanding (7-10). It has been suggested that one major function of dorsal ruffles is to reorganize the actin cytoskeleton to prepare a static cell for motility (11).In serum-starved fibroblasts, platelet-derived growth factor (PDGF) 2 induces at least two types of membrane ruffles, peripheral membrane ruffles (or lamellipodia) and dorsal ruffles (12). In addition to fibroblast cells, dorsal ruffle formation in response to growth factors has been reported in glial cells, endothelial cells, hippocampal neurons, kidney epithelial cells, and tumor cells (1,2,11,13,14). In addition, other stimuli can induce dorsal ruffles such as activation of IgE receptors in mast cells (15). Dorsal ruffles are dynamic structures. They form and disassemble rapidly (11,16,17 EXPERIMENTAL PROCEDURESReagents-Anti-HA monoclonal antibody and anti-Myc monoclonal antibody were purchased from Santa Cruz Biotechnology, Inc. PDGF-BB was purchased from Oncogene. Texas Red Phalloidin and Alexa Fluor 488 F(abЈ) 2 fragment of goat anti-mouse IgG(HϩL) were purchased from Molecular Probes. ROCK inhibitor Y-27632 and Src family inhibitor PP2 were purchased from BD Biosciences. The PCR products of chicken Src constitutively active mutant SrcE378G and SrcY527F cDNAs were subcloned into pcDNA3.1/hyg/myc. HA-Rac1G12V and HA-Rac1T14N were obtained from Guthrie Research Institute.Cell Culture and Transfection-G␣ 12 Ϫ/Ϫ G␣ 13 Ϫ/Ϫ MEF cells were provided by J. Gu and...

Resistance to Inhibitors of Cholinesterase-8A (Ric-8A) Is Critical for Growth Factor Receptor-induced Actin Cytoskeletal Reorganization

“…Dorsal ruffles formed only one time after PDGF-BB stimulation. After the disassembly of these dorsal ruffles, protrusion of large peripheral membrane ruffles was observed (33). Without PDGF-BB treatment, actin filaments were uniformly distributed in MEF cells (Fig.…”

“…Previously, we have shown that, in wild-type fibroblast cells, dorsal ruffles form and disappear quickly (33). However, in the absence of G␣ 13 , the dorsal ruffles stay much longer (33).…”

“…Previously, we have shown that, in wild-type fibroblast cells, dorsal ruffles form and disappear quickly (33). However, in the absence of G␣ 13 , the dorsal ruffles stay much longer (33). Because dorsal ruffles take up much of the actin polymers, a slow disassembly of dorsal ruffles would slow down the formation of other actin polymer-based structures such as peripheral membrane ruffles that are required for cell migra- 13 .…”

“…They form and disassemble rapidly (47,48). Previously, we have reported that, in MEF cells, dorsal ruffles formed within ϳ5 min after PDGF-BB (20 ng/ml) treatment (33). These dorsal ruffles were disassembled ϳ10 min after PDGF-BB treatment.…”

“…Boyden Chamber Cell Migration Assay-Cell migration was assayed using Boyden chambers (8.0-m pore size polyethylene terephthalate membrane, FALCON cell culture insert (BD Biosciences)) (31)(32)(33)(34). Cells were trypsinized and counted.…”