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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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Models for mode and function of AIP1-associated filament disruption.(A) AIP1 may be associated with only a fraction of filament severing events within the network. (B) CP may preferentially associate with the dynamic filaments prone to AIP-mediated filament severing/disruption. We estimate that roughly a half of F-actin is stabilized in lamellipodia of XTC cells. (C) Alternatively, AIP1-associate filament severing/disruption may occur preferentially at the proximity of the barbed end within a single filament. (D) Cooperation of nucleation and filament severing/disruption to facilitate leading edge protrusion. Release of free barbed ends from AIP1-associated filament disruption processes may amplify the Arp2/3 complex-initiated actin nucleation increase at the leading edge by repeatedly acting along the retrograde flow. The severing and end-to-end annealing may also be relevant to actin remodeling activities that confer filament angle and length alternation along the aging of network seen in published electron micrographs [10], [48].
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pone-0004921-g008: Models for mode and function of AIP1-associated filament disruption.(A) AIP1 may be associated with only a fraction of filament severing events within the network. (B) CP may preferentially associate with the dynamic filaments prone to AIP-mediated filament severing/disruption. We estimate that roughly a half of F-actin is stabilized in lamellipodia of XTC cells. (C) Alternatively, AIP1-associate filament severing/disruption may occur preferentially at the proximity of the barbed end within a single filament. (D) Cooperation of nucleation and filament severing/disruption to facilitate leading edge protrusion. Release of free barbed ends from AIP1-associated filament disruption processes may amplify the Arp2/3 complex-initiated actin nucleation increase at the leading edge by repeatedly acting along the retrograde flow. The severing and end-to-end annealing may also be relevant to actin remodeling activities that confer filament angle and length alternation along the aging of network seen in published electron micrographs [10], [48].

Mentions: The size of actin oligomers, if they exist, required to diffuse off the actin network is currently unknown. Detailed electron microscopy [48] demonstrates that the intercross between filaments occurs every 14–16 nm on average which corresponds to ∼5 actin subunits. The detergent-extracted dendritic nucleation network in the actin comet tail of Listeria monocytogenes in macrophages largely consists of short filaments less than 0.3 µm (typically ∼0.1 µm; Figure 8 in [49]). From these observations, the size of diffusible actin oligomers can be estimated to be within the range between 5 and 30 subunits. To achieve fragmentation of CP-bound F-actin to these sizes, a severing rate of 0.1∼0.02 s−1 is required. Hence the determined AIP1-mediated filament severing rate, 1.8×10−3 s−1, falls short of the severing rate predicted in our frequent filament severing-annealing hypothesis at least by one order.


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)

Models for mode and function of AIP1-associated filament disruption.(A) AIP1 may be associated with only a fraction of filament severing events within the network. (B) CP may preferentially associate with the dynamic filaments prone to AIP-mediated filament severing/disruption. We estimate that roughly a half of F-actin is stabilized in lamellipodia of XTC cells. (C) Alternatively, AIP1-associate filament severing/disruption may occur preferentially at the proximity of the barbed end within a single filament. (D) Cooperation of nucleation and filament severing/disruption to facilitate leading edge protrusion. Release of free barbed ends from AIP1-associated filament disruption processes may amplify the Arp2/3 complex-initiated actin nucleation increase at the leading edge by repeatedly acting along the retrograde flow. The severing and end-to-end annealing may also be relevant to actin remodeling activities that confer filament angle and length alternation along the aging of network seen in published electron micrographs [10], [48].
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004921-g008: Models for mode and function of AIP1-associated filament disruption.(A) AIP1 may be associated with only a fraction of filament severing events within the network. (B) CP may preferentially associate with the dynamic filaments prone to AIP-mediated filament severing/disruption. We estimate that roughly a half of F-actin is stabilized in lamellipodia of XTC cells. (C) Alternatively, AIP1-associate filament severing/disruption may occur preferentially at the proximity of the barbed end within a single filament. (D) Cooperation of nucleation and filament severing/disruption to facilitate leading edge protrusion. Release of free barbed ends from AIP1-associated filament disruption processes may amplify the Arp2/3 complex-initiated actin nucleation increase at the leading edge by repeatedly acting along the retrograde flow. The severing and end-to-end annealing may also be relevant to actin remodeling activities that confer filament angle and length alternation along the aging of network seen in published electron micrographs [10], [48].
Mentions: The size of actin oligomers, if they exist, required to diffuse off the actin network is currently unknown. Detailed electron microscopy [48] demonstrates that the intercross between filaments occurs every 14–16 nm on average which corresponds to ∼5 actin subunits. The detergent-extracted dendritic nucleation network in the actin comet tail of Listeria monocytogenes in macrophages largely consists of short filaments less than 0.3 µm (typically ∼0.1 µm; Figure 8 in [49]). From these observations, the size of diffusible actin oligomers can be estimated to be within the range between 5 and 30 subunits. To achieve fragmentation of CP-bound F-actin to these sizes, a severing rate of 0.1∼0.02 s−1 is required. Hence the determined AIP1-mediated filament severing rate, 1.8×10−3 s−1, falls short of the severing rate predicted in our frequent filament severing-annealing hypothesis at least by one order.

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