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

Tsuji T, Miyoshi T, Higashida C, Narumiya S, Watanabe N - PLoS ONE (2009)

Bottom Line: Here we demonstrate a reduction in actin-associated AIP1 in lamellipodia of cells overexpressing LIM-kinase.This rate does not suffice the filament severing rate predicted in our previous high frequency filament severing-annealing hypothesis.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto, Japan.

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 microM/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.

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Jasplikinolide rapidly blocks appearance of cofilin and AIP1 speckles.(A) Disappearance of XAC2-EGFP speckles induced by perfusion of 4 µM jasplakinolide (Jas). (B) XAC2-EGFP speckles disappear quickly after 4 µM Jas treatment (left panels), whereas actin speckles and F-actin structures are well preserved. (C) Rapid disappearance of EGFP-AIP1 speckles after perfusion of 1 µM Jas. (D) AIP1 speckles disappear quickly after the treatment with 4 µM Jas (left panels), whereas capping protein (CP) speckles remain persistently (right panels). As shown in the previous study (Miyoshi et al., 2006), a marked prolongation of retention time of CP speckles is observed. The loss in EGFP and mPlum signals due to photobleaching is less than 15% and 50%, respectively. Bars, 5 µm.
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pone-0004921-g003: Jasplikinolide rapidly blocks appearance of cofilin and AIP1 speckles.(A) Disappearance of XAC2-EGFP speckles induced by perfusion of 4 µM jasplakinolide (Jas). (B) XAC2-EGFP speckles disappear quickly after 4 µM Jas treatment (left panels), whereas actin speckles and F-actin structures are well preserved. (C) Rapid disappearance of EGFP-AIP1 speckles after perfusion of 1 µM Jas. (D) AIP1 speckles disappear quickly after the treatment with 4 µM Jas (left panels), whereas capping protein (CP) speckles remain persistently (right panels). As shown in the previous study (Miyoshi et al., 2006), a marked prolongation of retention time of CP speckles is observed. The loss in EGFP and mPlum signals due to photobleaching is less than 15% and 50%, respectively. Bars, 5 µm.

Mentions: Here we demonstrate direct evidence that Jas interferes with the cofilin-actin interaction in cells. We generated a GFP-tagged cofilin probe of XAC2 (XAC2-EGFP), one of two closely-related cofilin isoforms in Xenopus laevis [40]. We tagged the C-terminus of XAC2 with EGFP. Interestingly, XAC2-EGFP appeared as speckles in lamellipodia when expressed at a low level (Fig. 3A and B). Cofilin speckles showed similar localization with AIP1, distributed throughout the lamellipodial F-actin network. Upon perfusion of Jas, we observed rapid disappearance of cofilin speckles within a minute (Fig. 3A). Cofilin speckles disappeared before collapse of the F-actin network as judged by persistent speckles of coexpressed mPlum-actin (Fig. 3B). Although the precise function of F-actin-attached cofilin visualized as speckle images is unknown, our data clearly show that Jas interferes with the interaction of cofilin with F-actin in cells.


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

Tsuji T, Miyoshi T, Higashida C, Narumiya S, Watanabe N - PLoS ONE (2009)

Jasplikinolide rapidly blocks appearance of cofilin and AIP1 speckles.(A) Disappearance of XAC2-EGFP speckles induced by perfusion of 4 µM jasplakinolide (Jas). (B) XAC2-EGFP speckles disappear quickly after 4 µM Jas treatment (left panels), whereas actin speckles and F-actin structures are well preserved. (C) Rapid disappearance of EGFP-AIP1 speckles after perfusion of 1 µM Jas. (D) AIP1 speckles disappear quickly after the treatment with 4 µM Jas (left panels), whereas capping protein (CP) speckles remain persistently (right panels). As shown in the previous study (Miyoshi et al., 2006), a marked prolongation of retention time of CP speckles is observed. The loss in EGFP and mPlum signals due to photobleaching is less than 15% and 50%, respectively. Bars, 5 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654150&req=5

pone-0004921-g003: Jasplikinolide rapidly blocks appearance of cofilin and AIP1 speckles.(A) Disappearance of XAC2-EGFP speckles induced by perfusion of 4 µM jasplakinolide (Jas). (B) XAC2-EGFP speckles disappear quickly after 4 µM Jas treatment (left panels), whereas actin speckles and F-actin structures are well preserved. (C) Rapid disappearance of EGFP-AIP1 speckles after perfusion of 1 µM Jas. (D) AIP1 speckles disappear quickly after the treatment with 4 µM Jas (left panels), whereas capping protein (CP) speckles remain persistently (right panels). As shown in the previous study (Miyoshi et al., 2006), a marked prolongation of retention time of CP speckles is observed. The loss in EGFP and mPlum signals due to photobleaching is less than 15% and 50%, respectively. Bars, 5 µm.
Mentions: Here we demonstrate direct evidence that Jas interferes with the cofilin-actin interaction in cells. We generated a GFP-tagged cofilin probe of XAC2 (XAC2-EGFP), one of two closely-related cofilin isoforms in Xenopus laevis [40]. We tagged the C-terminus of XAC2 with EGFP. Interestingly, XAC2-EGFP appeared as speckles in lamellipodia when expressed at a low level (Fig. 3A and B). Cofilin speckles showed similar localization with AIP1, distributed throughout the lamellipodial F-actin network. Upon perfusion of Jas, we observed rapid disappearance of cofilin speckles within a minute (Fig. 3A). Cofilin speckles disappeared before collapse of the F-actin network as judged by persistent speckles of coexpressed mPlum-actin (Fig. 3B). Although the precise function of F-actin-attached cofilin visualized as speckle images is unknown, our data clearly show that Jas interferes with the interaction of cofilin with F-actin in cells.

Bottom Line: Here we demonstrate a reduction in actin-associated AIP1 in lamellipodia of cells overexpressing LIM-kinase.This rate does not suffice the filament severing rate predicted in our previous high frequency filament severing-annealing hypothesis.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto, Japan.

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 microM/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.

Show MeSH
Related in: MedlinePlus