Actin turnover-dependent fast dissociation of capping protein in the dendritic nucleation actin network: evidence of frequent filament severing.
Actin forms the dendritic nucleation network and undergoes rapid polymerization-depolymerization cycles in lamellipodia. To elucidate the mechanism of actin disassembly, we characterized molecular kinetics of the major filament end-binding proteins Arp2/3 complex and capping protein (CP) using single-molecule speckle microscopy. We have determined the dissociation rates of Arp2/3 and CP as 0.048 and 0.58 s(-1), respectively, in lamellipodia of live XTC fibroblasts. This CP dissociation rate is three orders of magnitude faster than in vitro. CP dissociates slower from actin stress fibers than from the lamellipodial actin network, suggesting that CP dissociation correlates with actin filament dynamics. We found that jasplakinolide, an actin depolymerization inhibitor, rapidly blocked the fast CP dissociation in cells. Consistently, the coexpression of LIM kinase prolonged CP speckle lifetime in lamellipodia. These results suggest that cofilin-mediated actin disassembly triggers CP dissociation from actin filaments. We predict that filament severing and end-to-end annealing might take place fairly frequently in the dendritic nucleation actin arrays.
Miyoshi
Takushi
T
Department of Pharmacology, Kyoto University Faculty of Medicine, Sakyo-ku, Kyoto 606-8501, Japan.
Tsuji
Takahiro
T
Higashida
Chiharu
C
Hertzog
Maud
M
Fujita
Akiko
A
Narumiya
Shuh
S
Scita
Giorgio
G
Watanabe
Naoki
N
D051344:Actin Capping Proteins / Q000235:genetics / Q000378:metabolism*; D008841:Actin Cytoskeleton / Q000378:metabolism*; D051377:Actin-Related Protein 2 / Q000378:metabolism; D051378:Actin-Related Protein 3 / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D051338:Cofilin 1 / Q000378:metabolism; D003598:Cytoskeletal Proteins; D003599:Cytoskeleton / Q000378:metabolism; D019281:Dimerization; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D007700:Kinetics; D054461:Lim Kinases; D010750:Phosphoproteins / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D011554:Pseudopodia / Q000378:metabolism; D029867:Xenopus Proteins / Q000378:metabolism; D014982:Xenopus laevis / Q000378:metabolism
D051344:Actin Capping Proteins; D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins; D051338:Cofilin 1; D003598:Cytoskeletal Proteins; D010750:Phosphoproteins; C098933:XAC1 protein, Xenopus; D029867:Xenopus Proteins; D011494:Protein Kinases; D054461:Lim Kinases