title: Local activation of Rap1 contributes to directional vascular endothelial cell migration accompanied by extension of microtubules on which RAPL, a Rap1-associating molecule, localizes.
abstract: Endothelial cell migration is promoted by chemoattractants and is accompanied with microtubule extension toward the leading edge. Cytoskeletal microtubules polarize to function as rails for delivering a variety of molecules by motor proteins during cell migration. It remains, however, unclear how directional migration with polarized extension of microtubules is regulated. Here we report that Rap1 controls the migration of vascular endothelial cells. We found that Rap1-associating molecule, RAPL, which belongs to the Ras association domain family (Rassf), localized on microtubules and that activated Rap1 induced dissociation of RAPL from microtubules. A Rap1 activation-monitoring probe based on the fluorescence resonance energy transfer enabled us to demonstrate that local Rap1 activation occurs at the leading edge of the cells under the two types of cell migration, chemotaxis and wound healing. Time lapse imaging of microtubules marked by enhanced green fluorescent protein-RAPL showed the directional growth of microtubules toward the leading edge of the migrating cells. Using adenovirus, inactivation of Rap1 by expression of rap1GAPII inhibited wound healing. In addition, disconnection of Rap1 and RAPL by expression of a RAPL mutant also perturbed wound healing. Collectively, the locally activated Rap1 and its association with RAPL controls the directional migration of vascular endothelial cells.
authors.affiliation: Department of Structural Analysis, National Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan.
authors.forename: Hisakazu
authors.initials: H
authors.lastname: Fujita
authors.forename: Shigetomo
authors.initials: S
authors.lastname: Fukuhara
authors.forename: Atsuko
authors.initials: A
authors.lastname: Sakurai
authors.forename: Akiko
authors.initials: A
authors.lastname: Yamagishi
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kamioka
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Nakaoka
authors.forename: Michitaka
authors.initials: M
authors.lastname: Masuda
authors.forename: Naoki
authors.initials: N
authors.lastname: Mochizuki
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000256:Adenoviridae / Q000235:genetics; D051017:Apoptosis Regulatory Proteins; D002460:Cell Line; D002465:Cell Movement; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology*; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D008954:Models, Biological; D020559:Monomeric GTP-Binding Proteins; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014945:Wound Healing; D020827:rap1 GTP-Binding Proteins / Q000096:biosynthesis* / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C454042:RASSF5 protein, human; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D020559:Monomeric GTP-Binding Proteins; D020827:rap1 GTP-Binding Proteins