An order of magnitude faster AIP1-associated actin disruption than nucleation by the Arp2/3 complex in lamellipodia

Takahiro Tsuji, Takushi Miyoshi, Chiharu Higashida, Shuh Narumiya, Naoki Watanabe

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    32 Citations (Scopus)

    Abstract

    The mechanism of lamellipod actin turnover is still under debate. To clarify the intracellular behavior of the recently-identified actin disruption mechanism, we examined kinetics of AIP1 using fluorescent single-molecule speckle microscopy. AIP1 is thought to cap cofilin-generated actin barbed ends. Here we demonstrate a reduction in actin-associated AIP1 in lamellipodia of cells overexpressing LIM-kinase. Moreover, actin-associated AIP1 was rapidly abolished by jasplakinolide, which concurrently blocked the F-actin-cofilin interaction. Jasplakinolide also slowed dissociation of AIP1, which is analogous to the effect of this drug on capping protein. These findings provide in vivo evidence of the association of AIP1 with barbed ends generated by cofilin-catalyzed filament disruption. Single-molecule observation found distribution of F-actin-associated AIP1 throughout lamellipodia, and revealed even faster dissociation of AIP1 than capping protein. The estimated overall AIP1-associated actin disruption rate, 1.8 μM/s, was one order of magnitude faster than Arp2/3 complex-catalyzed actin nucleation in lamellipodia. This rate does not suffice the filament severing rate predicted in our previous high frequency filament severing-annealing hypothesis. Our data together with recent biochemical studies imply barbed end-preferred frequent filament disruption. Frequent generation of AIP1-associated barbed ends and subsequent release of AIP1 may be the mechanism that facilitates previously observed ubiquitous actin polymerization throughout lamellipodia.

    Original languageEnglish
    Article numbere4921
    JournalPloS one
    Volume4
    Issue number3
    DOIs
    Publication statusPublished - 2009 Mar 17

    ASJC Scopus subject areas

    • Biochemistry, Genetics and Molecular Biology(all)
    • Agricultural and Biological Sciences(all)
    • General

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