Limits...
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.

Show MeSH

Related in: MedlinePlus

Dissociation of AIP1, but not of cofilin, slows by jaskplakinolide treatment; analogous to Jas-induced stabilization of capping protein.Images of EGFF-AIP1 (A) or XAC2-EGFP (B) speckles in live XTC cells were acquired before and 20–40 s after treatment with 2 µM jasplakinolide. The decay rate of persistent single-molecule AIP1 or cofilin speckles before and 21–34 s after drug perfusion is compared in the same cells (connected by dashed lines) and expressed as half-life (n = 5 cells for each construct). *p = 0.012 (A), n.s.: not significant (B), paired t-test, two-tailed.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2654150&req=5

pone-0004921-g004: Dissociation of AIP1, but not of cofilin, slows by jaskplakinolide treatment; analogous to Jas-induced stabilization of capping protein.Images of EGFF-AIP1 (A) or XAC2-EGFP (B) speckles in live XTC cells were acquired before and 20–40 s after treatment with 2 µM jasplakinolide. The decay rate of persistent single-molecule AIP1 or cofilin speckles before and 21–34 s after drug perfusion is compared in the same cells (connected by dashed lines) and expressed as half-life (n = 5 cells for each construct). *p = 0.012 (A), n.s.: not significant (B), paired t-test, two-tailed.

Mentions: To test this, we acquired single-molecule AIP1 images before and shortly after perfusion of Jas and compared the decay rate of persistent AIP1 speckles. As described above (Fig. 3), Jas eliminated the formation of AIP1 speckles completely ∼1 minute after drug perfusion. However, before their complete loss, AIP1 speckles temporally showed significantly slower dissociation kinetics 20–40 sec after drug administration than prior to the treatment (Fig. 4A). These results indicate that in addition to its strong inhibitory effect on de novo association of AIP1 to F-actin, Jas attenuates dissociation of AIP1 bound to the barbed end. In vitro, AIP1 cosediments with F-actin with low affinity (estimated dissociation constant of 15 µM) [32]. This rapidly dissociating side-binding of AIP1 would not be detected using our current experiment setting. By analogy to the strong inhibitory effect of Jas on dissociation of CP from actin [12], we conclude that AIP1 speckles represent their association with the actin barbed end.


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)

Dissociation of AIP1, but not of cofilin, slows by jaskplakinolide treatment; analogous to Jas-induced stabilization of capping protein.Images of EGFF-AIP1 (A) or XAC2-EGFP (B) speckles in live XTC cells were acquired before and 20–40 s after treatment with 2 µM jasplakinolide. The decay rate of persistent single-molecule AIP1 or cofilin speckles before and 21–34 s after drug perfusion is compared in the same cells (connected by dashed lines) and expressed as half-life (n = 5 cells for each construct). *p = 0.012 (A), n.s.: not significant (B), paired t-test, two-tailed.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004921-g004: Dissociation of AIP1, but not of cofilin, slows by jaskplakinolide treatment; analogous to Jas-induced stabilization of capping protein.Images of EGFF-AIP1 (A) or XAC2-EGFP (B) speckles in live XTC cells were acquired before and 20–40 s after treatment with 2 µM jasplakinolide. The decay rate of persistent single-molecule AIP1 or cofilin speckles before and 21–34 s after drug perfusion is compared in the same cells (connected by dashed lines) and expressed as half-life (n = 5 cells for each construct). *p = 0.012 (A), n.s.: not significant (B), paired t-test, two-tailed.
Mentions: To test this, we acquired single-molecule AIP1 images before and shortly after perfusion of Jas and compared the decay rate of persistent AIP1 speckles. As described above (Fig. 3), Jas eliminated the formation of AIP1 speckles completely ∼1 minute after drug perfusion. However, before their complete loss, AIP1 speckles temporally showed significantly slower dissociation kinetics 20–40 sec after drug administration than prior to the treatment (Fig. 4A). These results indicate that in addition to its strong inhibitory effect on de novo association of AIP1 to F-actin, Jas attenuates dissociation of AIP1 bound to the barbed end. In vitro, AIP1 cosediments with F-actin with low affinity (estimated dissociation constant of 15 µM) [32]. This rapidly dissociating side-binding of AIP1 would not be detected using our current experiment setting. By analogy to the strong inhibitory effect of Jas on dissociation of CP from actin [12], we conclude that AIP1 speckles represent their association with the actin barbed end.

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