Epidermal growth factor-induced contraction regulates paxillin phosphorylation to temporally separate traction generation from de-adhesion.
Directed cell migration is mediated by cycles of protrusion, adhesion, traction generation on the extracellular matrix and retraction. However, how the events after protrusion are timed, and what dictates their temporal order is completely unknown. We used acute epidermal growth factor (EGF) stimulation of epidermal keratinocytes to initiate the cell migration cycle to study the mechanism of the timing of adhesion, traction generation, and de-adhesion. Using microscopic and biochemical assays, we surprisingly found that at approximately 2 min after EGF stimulation protrusion, activation of myosin-II, traction generation, adhesion assembly, and paxillin phosphorylation occurred nearly simultaneously, followed by a 10-min delay during which paxillin became dephosphorylated before cell retraction. Inhibition of myosin-II blocked both the EGF-stimulated paxillin phosphorylation and cell retraction, and a paxillin phosphomimic blocked retraction. These results suggest that EGF-mediated activation of myosin-II acts as a mechanical signal to promote a cycle of paxillin phosphorylation/dephosphorylation that mediates a cycle of adhesion strengthening and weakening that delays cell retraction. Thus, we reveal for the first time a mechanism by which cells may temporally segregate protrusion, adhesion, and traction generation from retraction during EGF-stimulated cell migration.
Schneider
Ian C
IC
Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. ians@iastate.edu
Hays
Cristen K
CK
Waterman
Clare M
CM
D001381:Azepines / Q000494:pharmacology; D015153:Blotting, Western; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects*; D048430:Cell Shape / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D018994:Myosin Light Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors; D009281:Naphthalenes / Q000494:pharmacology; D051419:Paxillin / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D054460:rho-Associated Kinases
D001381:Azepines; C466107:MRLC2 protein, human; D018994:Myosin Light Chains; D009281:Naphthalenes; D051419:Paxillin; D011993:Recombinant Fusion Proteins; C070571:ML 7; D049452:Green Fluorescent Proteins; D004815:Epidermal Growth Factor; D054460:rho-Associated Kinases; D009219:Myosin-Light-Chain Kinase