Instantaneous inactivation of cofilin reveals its function of F-actin disassembly in lamellipodia

Vitriol, Eric A.; Wise, Ariel L.; Berginski, Mathew E.; Bamburg, James R.; Zheng, James

doi:10.1091/mbc.e13-03-0156

Cited by 52 publications

(41 citation statements)

References 48 publications

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“…8). A similar increase in lamellipodial F-actin has been recently described after instantaneous inactivation of cofilin (5). These data suggest that CIN depletion enhanced P-cofilin levels that led to a decrease in filament disassembly.…”

Section: Discussionsupporting

confidence: 86%

“…C ofilin is one crucial mediator of actin cytoskeletal dynamics during cell motility (1)(2)(3)(4)(5). At the cell edge, cofilin severs F-actin filaments, generating substrates for Arp2/3-mediated branching activity and contributing to F-actin depolymerization by creating a new pointed end and F-actin assembly by increasing the pool of polymerization-competent actin monomers (G-actin) (6,7).…”

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confidence: 99%

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Chronophin coordinates cell leading edge dynamics by controlling active cofilin levels

Delorme-Walker

Seo

Gohla

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Cofilin, a critical player of actin dynamics, is spatially and temporally regulated to control the direction and force of membrane extension required for cell locomotion. In carcinoma cells, although the signaling pathways regulating cofilin activity to control cell direction have been established, the molecular machinery required to generate the force of the protrusion remains unclear. We show that the cofilin phosphatase chronophin (CIN) spatiotemporally regulates cofilin activity at the cell edge to generate persistent membrane extension. We show that CIN translocates to the leading edge in a PI3-kinase-, Rac1-, and cofilindependent manner after EGF stimulation to activate cofilin, promotes actin free barbed end formation, accelerates actin turnover, and enhances membrane protrusion. In addition, we establish that CIN is crucial for the balance of protrusion/retraction events during cell migration. Thus, CIN coordinates the leading edge dynamics by controlling active cofilin levels to promote MTLn3 cell protrusion.ofilin is one crucial mediator of actin cytoskeletal dynamics during cell motility (1-5). At the cell edge, cofilin severs F-actin filaments, generating substrates for Arp2/3-mediated branching activity and contributing to F-actin depolymerization by creating a new pointed end and F-actin assembly by increasing the pool of polymerization-competent actin monomers (G-actin) (6, 7). Because of its ability to sever actin filaments and thus, modulate actin dynamics, the precise spatial and temporal regulation of cofilin activity at the cell leading edge is crucial to cell protrusion, chemotaxis, and motility both in vitro and in vivo (2,(8)(9)(10)(11)(12)(13). Misregulation of cofilin activity and/or expression is directly related to diseases, including tumor metastasis (14-18) and Alzheimer's disease (19).Several mechanisms regulate tightly the activation of cofilin in response to upstream stimuli, including interaction with phosphatidylinositol (4,5)-bisphosphate (20-22), local pH changes (23,24), and phosphorylation at a single regulatory serine (Ser3) (8,25). The phosphorylation of cofilin, leading to its inactivation, is catalyzed by two kinase families: the LIM-kinases [LIMKs(Lin11, Isl-1, and Mec-3 domain)] and the testicular kinases (25-27). Two primary families of ser/thr phosphatases dephosphorylate and reactivate the actin-depolymerizing and -severing functions of cofilin: slingshot (SSH) (28) and chronophin (CIN) (29).SSH was identified as a cofilin phosphatase through genetic studies in Drosophila (28). The most active and abundant SSH isoform, SSH-1L, has been implicated in such biological processes as cell division, growth cone motility/morphology, neurite extension, and actin dynamics during membrane protrusion (30). SSH dephosphorylates a number of actin regulatory proteins in addition to cofilin, including LIMK1 (31) and Coronin 1B (32). CIN is a haloacid dehydrogenase-type phosphatase, a family of enzymes with activity in mammalian cells that has been poorly characterized. CIN dephosph...

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Section: Discussionsupporting

confidence: 86%

mentioning

confidence: 99%

Chronophin coordinates cell leading edge dynamics by controlling active cofilin levels

Delorme-Walker

Seo

Gohla

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Two possible solutions to this paradox have been proposed: (1) cofilin-mediated severing might create free barbed ends and polymerizing actin filaments that can activate the Arp2/3 complex , and (2) SPIN90 (also known as NCKIPSD)-mediated activation of the Arp2/3 complex without pre-existing actin filaments (Wagner et al, 2013). However, neither mechanism seems to be satisfactory as knockdown and CALI of cofilin results in enlargement of lamellipodia and ruffles and increased F-actin levels in cells employing the mesenchymal mode of migration (Hotulainen et al, 2005;Rogers et al, 2003;Sidani et al, 2007;Vitriol et al, 2013), and full-length SPIN90 does not activate the Arp2/3 complex (Fukuoka et al, 2001 and data not shown). In this regard, the involvement of another actin nucleator in the mechanism regulating initial activation of the Arp2/3 complex would resolve this paradox.…”

Section: Discussionmentioning

confidence: 99%

Initiation of lamellipodia and ruffles involves cooperation between mDia1 and the Arp2/3 complex

Isogai

Kammen

Leyton-Puig

et al. 2015

Journal of Cell Science

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Protrusion of lamellipodia and ruffles requires polymerization of branched actin filaments by Arp2/3 complex. Although regulation of Arp2/3-complex activity has been extensively investigated, the mechanism of initiation of lamellipodia and ruffles remains poorly understood. Here we show that mDia1 acts in concert with the Arp2/3 complex to promote initiation of lamellipodia and ruffles. We find that mDia1 is an EGF-regulated actin nucleator involved in membrane ruffling using a combination of knockdown and rescue experiments. At the molecular level, mDia1 polymerizes linear actin filaments activating the Arp2/3 complex and localizes within nascent and mature membrane ruffles. We employ functional complementation experiments and optogenetics to show that mDia1 cooperates with the Arp2/3 complex in initiating ruffles. Finally, we show that genetic and pharmacological interference with this cooperation hampers ruffling and cell migration. Thus, we propose that the lamellipodium/ruffle-initiating machinery consists of two actin nucleators that act sequentially to regulate membrane protrusion and cell migration.

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“…Without changing the localization of the actin regulator cofilin, CALI was performed separately in cofilin-depleted cells by using KillerRed coupled to either a constitutively active cofilin mutant (cofilin S3A ) or a dominantnegative cofilin mutant (cofilin S3E ) (Vitriol et al, 2013). Cells that express either cofilin S3A or cofilin S3E fused to KillerRed show no obvious change in the quantity or distribution of lamellipodial F-actin.…”

Section: Cali With Killerredmentioning

confidence: 99%

Chromophore-assisted laser inactivation – towards a spatiotemporal–functional analysis of proteins, and the ablation of chromatin, organelle and cell function

Sano

Watanabe

Matsunaga

2014

Journal of Cell Science

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Chromophore-assisted laser or light inactivation (CALI) has been employed as a promising technique to achieve spatiotemporal knockdown or loss-of-function of target molecules in situ. CALI is performed using photosensitizers as generators of reactive oxygen species (ROS). There are two CALI approaches that use either transgenic tags with chemical photosensitizers, or genetically encoded fluorescent protein fusions. Using spatially restricted microscopy illumination, CALI can address questions regarding, for example, protein isoforms, subcellular localization or phase-specific analyses of multifunctional proteins that other knockdown approaches, such as RNA interference or treatment with chemicals, cannot. Furthermore, rescue experiments can clarify the phenotypic capabilities of CALI after the depletion of endogenous targets. CALI can also provide information about individual events that are involved in the function of a target protein and highlight them in multifactorial events. Beyond functional analysis of proteins, CALI of nuclear proteins can be performed to induce cell cycle arrest, chromatin-or locus-specific DNA damage. Even at organelle levelsuch as in mitochondria, the plasma membrane or lysosomes -CALI can trigger cell death. Moreover, CALI has emerged as an optogenetic tool to switch off signaling pathways, including the optical depletion of individual neurons. In this Commentary, we review recent applications of CALI and discuss the utility and effective use of CALI to address open questions in cell biology.

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Instantaneous inactivation of cofilin reveals its function of F-actin disassembly in lamellipodia

Cited by 52 publications

References 48 publications

Chronophin coordinates cell leading edge dynamics by controlling active cofilin levels

Chronophin coordinates cell leading edge dynamics by controlling active cofilin levels

Initiation of lamellipodia and ruffles involves cooperation between mDia1 and the Arp2/3 complex

Chromophore-assisted laser inactivation – towards a spatiotemporal–functional analysis of proteins, and the ablation of chromatin, organelle and cell function

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