title: Coronin localizes to leading edges and is involved in cell spreading and lamellipodium extension in vertebrate cells.
abstract: Coronin is a WD repeat-containing actin-binding protein, which was originally identified in the cellular slime mold Dictyostelium. Coronin-null Dictyostelium cells show defects in cytokinesis, cell motility and phagocytosis. Although the existence of coronin in higher eukaryotes has been reported, its function in vertebrate cells has not been elucidated. We cloned a Xenopus homolog of coronin (Xcoronin) and examined its actin-binding properties, subcellular localization and possible functions. Xcoronin consists of 480 amino acids and is 63% identical to human coronin (p57). Bacterially expressed recombinant Xcoronin co-sedimented with F-actin in vitro. The WD repeat domain (residues 64-299) alone did not have any affinity for F-actin. Anti-Xcoronin antibodies reacted specifically with a single 57 kDa protein present in an extract of the Xenopus A6 cell line. Indirect immunofluorescent staining of A6 cells revealed that Xcoronin is present in the cytoplasm and concentrated in the cell periphery in membrane ruffles. During spreading after replating or wound healing after scratching a confluent monolayer, Xcoronin became concentrated in the leading edges of lamellipodia. A GFP-fusion protein of Xcoronin showed a subcellular distribution essentially identical to endogenous Xcoronin. The localization of Xcoronin to the cell periphery was resistant to treatment with 0.1% Triton X-100. The deletion of 63 N-terminal amino acids or of 65 C-terminal amino acids abolished the localization of Xcoronin to the cell periphery. Xcoronin expressed in 3T3 fibroblasts was concentrated to the leading edges of lamellipodia induced by active Rac. Remarkably, expression of a truncated form of Xcoronin (64-299), but not of full-length Xcoronin, significantly decreased the rate of cell spreading after replating and markedly inhibited lamellipodium extension induced by active Rac. These results suggest that Xcoronin plays an important role in lamellipodium extension and cell spreading.
authors.affiliation: Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. L50174@sakura.kudpc.kyoto-u.ac.jp
authors.forename: M
authors.initials: M
authors.lastname: Mishima
authors.forename: E
authors.initials: E
authors.lastname: Nishida
mesh_terms: D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002465:Cell Movement; D016764:Cell Polarity; D003902:Detergents / Q000494:pharmacology; D004023:Dictyostelium / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007668:Kidney; D008840:Microfilament Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D017830:Octoxynol / Q000494:pharmacology; D015800:Protozoan Proteins / Q000378:metabolism; D011554:Pseudopodia / Q000378:metabolism* / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014982:Xenopus laevis / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000502:physiology
chemical_list: D000199:Actins; D003902:Detergents; D008840:Microfilament Proteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C072061:coronin proteins; D017830:Octoxynol; D020744:rac GTP-Binding Proteins