title: Rap1 can bypass the FAK-Src-Paxillin cascade to induce cell spreading and focal adhesion formation.
abstract: We developed new image analysis tools to analyse quantitatively the extracellular-matrix-dependent cell spreading process imaged by live-cell epifluorescence microscopy. Using these tools, we investigated cell spreading induced by activation of the small GTPase, Rap1. After replating and initial adhesion, unstimulated cells exhibited extensive protrusion and retraction as their spread area increased, and displayed an angular shape that was remodelled over time. In contrast, activation of endogenous Rap1, via 007-mediated stimulation of Epac1, induced protrusion along the entire cell periphery, resulting in a rounder spread surface, an accelerated spreading rate and an increased spread area compared to control cells. Whereas basal, anisotropic, spreading was completely dependent on Src activity, Rap1-induced spreading was refractory to Src inhibition. Under Src inhibited conditions, the characteristic Src-induced tyrosine phosphorylations of FAK and paxillin did not occur, but Rap1 could induce the formation of actomyosin-connected adhesions, which contained vinculin at levels comparable to that found in unperturbed focal adhesions. From these results, we conclude that Rap1 can induce cell adhesion and stimulate an accelerated rate of cell spreading through mechanisms that bypass the canonical FAK-Src-Paxillin signalling cascade.
authors.affiliation: Department of Molecular Cancer Research, Centre for Biomedical Genetics and Cancer Genomics Centre, University Medical Center, Utrecht, The Netherlands. s.h.ross@outlook.com
authors.forename: Sarah H
authors.initials: SH
authors.lastname: Ross
authors.forename: Emma
authors.initials: E
authors.lastname: Spanjaard
authors.forename: Anneke
authors.initials: A
authors.lastname: Post
authors.forename: Marjolein J
authors.initials: MJ
authors.lastname: Vliem
authors.forename: Hendy
authors.initials: H
authors.lastname: Kristyanto
authors.forename: Johannes L
authors.initials: JL
authors.lastname: Bos
authors.forename: Johan
authors.initials: J
authors.lastname: de Rooij
mesh_terms: D000205:Actomyosin / Q000494:pharmacology; D016880:Anisotropy; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D005109:Extracellular Matrix / Q000378:metabolism; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D051419:Paxillin / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D000089804:Shelterin Complex; D015398:Signal Transduction; D034501:Telomere-Binding Proteins / Q000378:metabolism*; D016596:Vinculin / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: C473217:8-(4-chloro-phenylthio)-2'-O-methyladenosine-3'-5'-cyclic monophosphate; D020662:Guanine Nucleotide Exchange Factors; D051419:Paxillin; C116122:RAPGEF3 protein, human; D034741:RNA, Small Interfering; D000089804:Shelterin Complex; C440809:TERF2IP protein, human; D034501:Telomere-Binding Proteins; D016596:Vinculin; D006160:Guanosine Triphosphate; D000205:Actomyosin; D000242:Cyclic AMP; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019061:src-Family Kinases