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Published 18 December 2006. doi:10.1083/jcb.200604176
The Rockefeller University Press, 0021-9525 $8.00
JCB, Volume 175, Number 6, 947-955
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Actin turnover–dependent fast dissociation of capping protein in the dendritic nucleation actin network: evidence of frequent filament severing



Takushi Miyoshi1, Takahiro Tsuji1, Chiharu Higashida1, Maud Hertzog2,3, Akiko Fujita1, Shuh Narumiya1, Giorgio Scita2,3, and Naoki Watanabe1

1 Department of Pharmacology, Kyoto University Faculty of Medicine, Sakyo-ku, Kyoto 606-8501, Japan
2 Fondazione Italiana per la Ricerca sul Cancro Istituto di Oncologia Molecolare, 20139 Milan, Italy
3 Department of Experimental Oncology, Istituto Europeo di Oncologia, 20141 Milan, Italy

Correspondence to Naoki Watanabe: naoki-w{at}mfour.med.kyoto-u.ac.jp

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.

Abbreviations used in this paper: CMV, cytomegalovirus; CP, capping protein; F-actin, filamentous actin; Jas, jasplakinolide; LIMK, LIM kinase; mRFP, monomeric red fluorescent protein; PLL, poly-L-lysine; SF, stress fiber.


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