Tai Kiuchi

Cofilin promotes stimulus-induced lamellipodium formation by generating an abundant supply of actin monomers

Cofilin stimulates actin filament disassembly and accelerates actin filament turnover. Cofilin is also involved in stimulus-induced actin filament assembly during lamellipodium formation. However, it is not clear whether this occurs by replenishing the actin monomer pool, through filament disassembly, or by creating free barbed ends, through its severing activity. Using photoactivatable Dronpa-actin, we show that cofilin is involved in producing more than half of all cytoplasmic actin monomers and that the rate of actin monomer incorporation into the tip of the lamellipodium is dependent on the size of this actin monomer pool. Finally, in cofilin-depleted cells, stimulus-induced actin monomer incorporation at the cell periphery is attenuated, but the incorporation of microinjected actin monomers is not. We propose that cofilin contributes to stimulus-induced actin filament assembly and lamellipodium extension by supplying an abundant pool of cytoplasmic actin monomers.

F- and G-actin homeostasis regulates mechanosensitive actin nucleation by formins

Physical force evokes rearrangement of the actin cytoskeleton. Signalling pathways such as tyrosine kinases, stretch-activated Ca2+ channels and Rho GTPases are involved in force sensing. However, how signals are transduced to actin assembly remains obscure. Here we show mechanosensitive actin polymerization by formins (formin homology proteins). Cells overexpressing mDia1 increased the amount of F-actin on release of cell tension. Fluorescence single-molecule speckle microscopy revealed rapid induction of processive actin assembly by mDia1 on cell cortex deformation. mDia1 lacking the Rho-binding domain and other formins exhibited mechanosensitive actin nucleation, suggesting Rho-independent activation. Mechanosensitive actin nucleation by mDia1 required neither Ca2+ nor kinase signalling. Overexpressing LIM kinase abrogated the induction of processive mDia1. Furthermore, s-FDAPplus (sequential fluorescence decay after photoactivation) analysis revealed a rapid actin monomer increase on cell cortex deformation. Our direct visualization of the molecular behaviour reveals a mechanosensitive actin filament regeneration mechanism in which G-actin released by actin remodelling plays a pivotal role.

Measurements of spatiotemporal changes in G-actin concentration reveal its effect on stimulus-induced actin assembly and lamellipodium extension.

The cytoplasmic concentration of G-actin is a critical parameter for determining the extent of stimulus-induced G-actin assembly and cell extension.

Multitarget super-resolution microscopy with high-density labeling by exchangeable probes

We have developed a multitarget super-resolution microscopy technique called image reconstruction by integrating exchangeable single-molecule localization (IRIS). IRIS uses protein fragment–based probes that directly associate with and dissociate from their targets over durations on the order of tens of milliseconds. By integrating single-molecule localization and sequential labeling, IRIS enables unprecedented labeling density along multiple cellular structures. IRIS can be used to discern the area-specific proximity between cytoskeletal components and focal adhesions within a single cell.

Visualization of cofilin-actin and Ras-Raf interactions by bimolecular fluorescence complementation assays using a new pair of split Venus fragments

The bimolecular fluorescence complementation (BiFC) assay is a method for visualizing protein-protein interactions in living cells. To visualize the cofilin-actin interaction in living cells, a series of combinations of the N- and C-terminal fragments of Venus fused upstream or downstream of cofilin and actin were screened systematically. A new pair of split Venus fragments, Venus (1-210) fused upstream of cofilin and Venus (210-238) fused downstream of actin, was the most effective combination for visualizing the specific interaction between cofilin and actin in living cells. This pair of Venus fragments was also effective for detecting the active Ras-dependent interaction between H-Ras and Raf1 and the Ca(2+)-dependent interaction between calmodulin and its target M13 peptide. In vitro BiFC assays using the pair of purified BiFC probes provided the means to detect the specific interactions between cofilin and actin and between H-Ras and Raf1. In vivo and in vitro BiFC assays using the newly identified pair of Venus fragments will serve as a useful tool for measuring protein-protein interactions with high specificity and low background fluorescence and could be applied to the screening of inhibitors that block protein-protein interactions.

CaMKIIβ-mediated LIM-kinase activation plays a crucial role in BDNF-induced neuritogenesis.

LIM‐kinase 1 (LIMK1) regulates actin cytoskeletal reorganization by phosphorylating and inactivating actin‐depolymerizing factor and cofilin. We examined the role of LIMK1 in brain‐derived neurotrophic factor (BDNF)‐induced neuritogenesis in primary‐cultured rat cortical neurons. Knockdown of LIMK1 or expression of a kinase‐dead LIMK1 mutant suppressed BDNF‐induced enhancement of primary neurite formation. By contrast, expression of an active form of LIMK1 promoted primary neuritogenesis in the absence of BDNF. BDNF‐induced neuritogenesis was inhibited by KN‐93, an inhibitor of Ca2+/calmodulin‐dependent protein kinases (CaMKs), but not by STO‐609, an inhibitor of CaMK‐kinase (CaMKK). CaMKK activity is required for the activation of CaMKI and CaMKIV, but not CaMKII, which suggests that CaMKII is principally involved in BDNF‐induced enhancement of neuritogenesis. Knockdown of CaMKIIβ, but not CaMKIIα, suppressed BDNF‐induced neuritogenesis. Active CaMKIIβ promoted neuritogenesis, and this promotion was inhibited by knockdown of LIMK1, indicating that CaMKIIβ is involved in BDNF‐induced neuritogenesis via activation of LIMK1. Furthermore, in vitro kinase assays revealed that CaMKIIβ phosphorylates LIMK1 at Thr‐508 in the kinase domain and activates the cofilin‐phosphorylating activity of LIMK1. In summary, these results suggest that CaMKIIβ‐mediated activation of LIMK1 plays a crucial role in BDNF‐induced enhancement of primary neurite formation.

Distributed Actin Turnover in the Lamellipodium and FRAP Kinetics

Studies of actin dynamics at the leading edge of motile cells with single-molecule speckle (SiMS) microscopy have shown a broad distribution of EGFP-actin speckle lifetimes and indicated actin polymerization and depolymerization over an extended region. Other experiments using FRAP with the same EGFP-actin as a probe have suggested, by contrast, that polymerization occurs exclusively at the leading edge. We performed FRAP experiments on XTC cells to compare SiMS to FRAP on the same cell type. We used speckle statistics obtained by SiMS to model the steady-state distribution and kinetics of actin in the lamellipodium. We demonstrate that a model with a single diffuse actin species is in good agreement with FRAP experiments. A model including two species of diffuse actin provides an even better agreement. The second species consists of slowly diffusing oligomers that associate to the F-actin network throughout the lamellipodium or break up into monomers after a characteristic time. Our work motivates studies to test the presence and composition of slowly diffusing actin species that may contribute to local remodeling of the actin network and increase the amount of soluble actin.

New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales

This paper introduces a new, easy-to-use method of fluorescence single-molecule speckle microscopy for actin with nanometer-scale accuracy. This new method reveals that actin flows in front of mature focal adhesions (FAs) are fast and biased toward FAs, suggesting that mature FAs are actively engaged in pulling and remodeling the local actin network.

LIM Kinase Has a Dual Role in Regulating Lamellipodium Extension by Decelerating the Rate of Actin Retrograde Flow and the Rate of Actin Polymerization

Background: LIMK1 regulates actin dynamics by inactivating cofilin. Results: LIMK1 knockdown accelerated actin polymerization and retrograde flow, but the effect on retrograde flow was more efficient. Conclusion: LIMK1 has a dual role in regulating lamellipodium extension by decelerating actin retrograde flow and polymerization. LIMK1 contributes to lamellipodium extension by decelerating actin retrograde flow. Significance: The dual role of LIMK1 in lamellipodium extension was clarified. Lamellipodium extension is crucial for cell migration and spreading. The rate of lamellipodium extension is determined by the balance between the rate of actin polymerization and the rate of actin retrograde flow. LIM kinase 1 (LIMK1) regulates actin dynamics by phosphorylating and inactivating cofilin, an actin-depolymerizing protein. We examined the role of LIMK1 in lamellipodium extension by measuring the rates of actin polymerization, actin retrograde flow, and lamellipodium extension using time-lapse imaging of fluorescence recovery after photobleaching. In the non-extending lamellipodia of active Rac-expressing N1E-115 cells, LIMK1 expression decelerated and LIMK1 knockdown accelerated actin retrograde flow. In the extending lamellipodia of neuregulin-stimulated MCF-7 cells, LIMK1 knockdown accelerated both the rate of actin polymerization and the rate of actin retrograde flow, but the accelerating effect on retrograde flow was greater than the effect on polymerization, thus resulting in a decreased rate of lamellipodium extension. These results indicate that LIMK1 has a dual role in regulating lamellipodium extension by decelerating actin retrograde flow and polymerization, and in MCF-7 cells endogenous LIMK1 contributes to lamellipodium extension by decelerating actin retrograde flow more effectively than decelerating actin polymerization.

Live-cell imaging of G-actin dynamics using sequential FDAP

Various microscopic techniques have been developed to understand the mechanisms that spatiotemporally control actin filament dynamics in live cells. Kinetic data on the processes of actin assembly and disassembly on F-actin have been accumulated. However, the kinetics of cytoplasmic G-actin, a key determinant for actin polymerization, has remained unclear because of a lack of appropriate methods to measure the G-actin concentration quantitatively. We have developed two new microscopic techniques based on the fluorescence decay after photoactivation (FDAP) time-lapse imaging of photoswitchable Dronpa-labeled actin. These techniques, sequential FDAP (s-FDAP) and multipoint FDAP, were used to measure the time-dependent changes in and spatial distribution of the G-actin concentration in live cells. Use of s-FDAP provided data on changes in the G-actin concentration with high temporal resolution; these data were useful for the model analysis of actin assembly processes in live cells. The s-FDAP analysis also provided evidence that the cytoplasmic G-actin concentration substantially decreases after cell stimulation and that the extent of stimulus-induced actin assembly and cell size extension are linearly correlated with the G-actin concentration before cell stimulation. The advantages of using s-FDAP and multipoint FDAP to measure spatiotemporal G-actin dynamics and the roles of G-actin concentration and ADF/cofilin in stimulus-induced actin assembly and lamellipodium extension in live cells are discussed.