Actin turnover–dependent fast dissociation of capping protein in the dendritic nucleation actin network: evidence of frequent filament severing

Miyoshi, Takushi; Tsuji, Takahiro; Higashida, Chiharu; Hertzog, Maud; Fujita, Akiko; Narumiya, Shuh; Scita, Giorgio; Watanabe, Naoki

doi:10.1083/jcb.200604176

Cited by 123 publications

(275 citation statements)

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“…Consistent with CP regulation in vivo, estimates of the half-life of CP on the barbed end near the plasma membrane in living cells are approximately three orders of magnitude shorter than CP's half-life on the barbed in vitro (i.e., ∼2-15 s in cells vs. ∼30 min for pure proteins) (8,18). To date, two direct regulators of CP activity have been identified.…”

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confidence: 74%

“…Importantly, two predictions of this model have already been confirmed. First, the half-life of CP on barbed ends near the plasma membrane in living cells is much closer to the half-life of the CP:CARMIL complex on the barbed end in vitro (∼8 s) (22) than to the half-life of CP on the barbed end in vitro (∼1,800 s) (8,18). Second, cells engineered to express a version of CP that cannot see the CPI motif in CARMIL and related proteins (e.g., CD2AP or CKIP) phenocopy Arp2/3-inhibited cells (29).…”

Section: Discussionmentioning

confidence: 99%

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V-1 regulates capping protein activity in vivo

Jung

Alexander

et al. 2016

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

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Capping Protein (CP) plays a central role in the creation of the Arp2/ 3-generated branched actin networks comprising lamellipodia and pseudopodia by virtue of its ability to cap the actin filament barbed end, which promotes Arp2/3-dependent filament nucleation and optimal branching. The highly conserved protein V-1/Myotrophin binds CP tightly in vitro to render it incapable of binding the barbed end. Here we addressed the physiological significance of this CP antagonist in Dictyostelium, which expresses a V-1 homolog that we show is very similar biochemically to mouse V-1. Consistent with previous studies of CP knockdown, overexpression of V-1 in Dictyostelium reduced the size of pseudopodia and the cortical content of Arp2/3 and induced the formation of filopodia. Importantly, these effects scaled positively with the degree of V-1 overexpression and were not seen with a V-1 mutant that cannot bind CP. V-1 is present in molar excess over CP, suggesting that it suppresses CP activity in the cytoplasm at steady state. Consistently, cells devoid of V-1, like cells overexpressing CP described previously, exhibited a significant decrease in cellular F-actin content. Moreover, V-1-null cells exhibited pronounced defects in macropinocytosis and chemotactic aggregation that were rescued by V-1, but not by the V-1 mutant. Together, these observations demonstrate that V-1 exerts significant influence in vivo on major actin-based processes via its ability to sequester CP. Finally, we present evidence that V-1's ability to sequester CP is regulated by phosphorylation, suggesting that cells may manipulate the level of active CP to tune their "actin phenotype."T he addition of Capping Protein (CP) to seed-initiated actin polymerization assays results in the rapid cessation of polymerization because CP binds with very high affinity to the fastgrowing barbed end of the actin filament to block further monomer addition (1). Direct extrapolation of this simple, potent biochemical property would suggest that the cell's content of F-actin should rise and fall as its content of CP is artificially forced to fall and rise, respectively. Indeed, this finding was reported many years ago in Dictyostelium amoeba (2). This simple view of CP's role in regulating actin assembly in vivo falls short of the whole story, however. The additional complexity arises from the critical relationship between CP and the Arp2/3 complex, the major actin nucleating machine that generates the branched actin networks comprising lamellipodia and pseudopodia (3). At the heart of this relationship is the fact that CP increases the rate of Arp2/3-dependent filament nucleation and promotes optimal branching by rapidly capping filaments (4). As a result, CP promotes actin-related proteins 2 and 3 (Arp2/3)-driven actin assembly and motility (4, 5). This effect was evident from early solution experiments focused on defining the function of the Arp2/3 complex (6), verified by in vitro reconstitution of the Arp2/3-dependent motility of Listeria (5), and explained mecha...

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confidence: 74%

Section: Discussionmentioning

confidence: 99%

V-1 regulates capping protein activity in vivo

Jung

Alexander

et al. 2016

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

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“…Because data from these techniques can hide local differences in actin assembly, researchers have recently used additional methods such as single-molecule and speckle microscopy to analyze the fine-grained spatial and temporal dynamics of actin rearrangements (Watanabe and Mitchison, 2002;Ponti et al, 2004). Single-molecule imaging has also been used to study actinassociated proteins such as the Arp2/3 complex and capping protein, which have different lifetimes in the actin network (Ponti et al, 2005;Miyoshi et al, 2006;Iwasa and Mullins, 2007). However, single-molecule imaging has not been applied to nucleation-promoting factors such as the SCAR/WAVE complex, and it is unknown whether the SCAR/WAVE complex incorporates into the actin network.…”

Section: Introductionmentioning

confidence: 99%

Diffusion, capture and recycling of SCAR/WAVE and Arp2/3 complexes observed in cells by single-molecule imaging

Millius

Watanabe

Weiner

2012

Journal of Cell Science

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SummaryThe SCAR/WAVE complex drives lamellipodium formation by enhancing actin nucleation by the Arp2/3 complex. Phosphoinositides and Rac activate the SCAR/WAVE complex, but how SCAR/WAVE and Arp2/3 complexes converge at sites of nucleation is unknown. We analyzed the single-molecule dynamics of WAVE2 and p40 (subunits of the SCAR/WAVE and Arp2/3 complexes, respectively) in XTC cells. We observed lateral diffusion of both proteins and captured the transition of p40 from diffusion to network incorporation. These results suggest that a diffusive 2D search facilitates binding of the Arp2/3 complex to actin filaments necessary for nucleation. After nucleation, the Arp2/3 complex integrates into the actin network and undergoes retrograde flow, which results in its broad distribution throughout the lamellipodium. By contrast, the SCAR/WAVE complex is more restricted to the cell periphery. However, with single-molecule imaging, we also observed WAVE2 molecules undergoing retrograde motion. WAVE2 and p40 have nearly identical speeds, lifetimes and sites of network incorporation. Inhibition of actin retrograde flow does not prevent WAVE2 association and disassociation with the membrane but does inhibit WAVE2 removal from the actin cortex. Our results suggest that membrane binding and diffusion expedites the recruitment of nucleation factors to a nucleation site independent of actin assembly, but after network incorporation, ongoing actin polymerization facilitates recycling of SCAR/WAVE and Arp2/3 complexes.

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“…Because the primary barbed end capping protein in cells, CP is probably a major focal point for regulation. Indeed, the huge discrepancy between the half-life of CP bound to the barbed end in vitro (ϳ30 min) (18,19) and in vivo (ϳ1 s) (20) suggests that CP activity is significantly controlled by regulatory molecules in vivo. In fact, several proteins have been found to bind CP to alter its activity (1,2,(21)(22)(23)(24).…”

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confidence: 99%

Molecular Basis for Barbed End Uncapping by CARMIL Homology Domain 3 of Mouse CARMIL-1

Zwolak

Uruno

Piszczek

et al. 2010

Journal of Biological Chemistry

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Capping protein (CP) is a ubiquitously expressed, 62-kDa heterodimer that binds the barbed end of the actin filament with ϳ0.1 nM affinity to prevent further monomer addition. CARMIL is a multidomain protein, present from protozoa to mammals, that binds CP and is important for normal actin dynamics in vivo. The CARMIL CP binding site resides in its CAH3 domain (CARMIL homology domain 3) located at or near the protein's C terminus. CAH3 binds CP with ϳ1 nM affinity, resulting in a complex with weak capping activity (30 -200 nM). Solution assays and single-molecule imaging show that CAH3 binds CP already present on the barbed end, causing a 300-fold increase in the dissociation rate of CP from the end (i.e. uncapping). Here we used nuclear magnetic resonance (NMR) to define the molecular interaction between the minimal CAH3 domain (CAH3a/b) of mouse CARMIL-1 and CP. Specifically, we show that the highly basic CAH3a subdomain is required for the high affinity interaction of CAH3 with a complementary "acidic groove" on CP opposite its actin-binding surface. This CAH3a-CP interaction orients the CAH3b subdomain, which we show is also required for potent anti-CP activity, directly adjacent to the basic patch of CP, shown previously to be required for CP association to and high affinity interaction with the barbed end. The importance of specific residue interactions between CP and CAH3a/b was confirmed by site-directed mutagenesis of both proteins. Together, these results offer a mechanistic explanation for the barbed end uncapping activity of CARMIL, and they identify the basic patch on CP as a crucial regulatory site. Capping protein (CP)4 is a ubiquitously expressed, 62-kDa ␣/␤ heterodimer that binds the barbed end of the actin filament with high affinity (K d ϭ 0.1 nM) to prevent further actin monomer association and dissociation, thereby limiting the extent of filament elongation in vivo (1, 2). Consistent with such a central role in actin filament assembly, CP is one of only five proteins required for the reconstitution of actin-based motility in vitro (3-5), and cells lacking CP have profound deficiencies in actin cytoskeleton assembly (6 -10).Determination of the CP crystal structure led to the "tentacles" model of barbed end capping by CP (11). The two structurally homologous CP subunits form a central ␤-sheet, which comprises the bulk of the protein core, above which there are two antiparallel ␣-helices, one belonging to each subunit (11). At the end of these helices, each subunit contains a C-terminal "tentacle," which, on CP␣, is composed of an unstructured region punctuated in the middle by a short, 4-residue helix and, on CP␤, is composed of a longer amphipathic helix that protrudes from the protein core. Based on crystallographic evidence, it was proposed that these C-terminal tentacles are flexible in solution, allowing them to bind and cap the barbed end. Extensive mutational studies in yeast (12) and vertebrate (13) CP that focused on the tentacles provided strong support for the tentacles model of ca...

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Actin turnover–dependent fast dissociation of capping protein in the dendritic nucleation actin network: evidence of frequent filament severing

Cited by 123 publications

References 53 publications

V-1 regulates capping protein activity in vivo

V-1 regulates capping protein activity in vivo

Diffusion, capture and recycling of SCAR/WAVE and Arp2/3 complexes observed in cells by single-molecule imaging

Molecular Basis for Barbed End Uncapping by CARMIL Homology Domain 3 of Mouse CARMIL-1

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