Direct Observation of the Uncapping of Capping Protein-capped Actin Filaments by CARMIL Homology Domain 3

Fujiwara, Ikuko; Remmert, Kirsten; Hammer, John A.

doi:10.1074/jbc.m109.031203

Cited by 52 publications

(74 citation statements)

References 39 publications

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“…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%

“…CP was purified from Escherichia coli strain BL21-RILP harboring the expression plasmid pET-28B containing both the α and β subunits of D.d. CP, essentially as described for mouse CP (22), but by using the CAH3 domain of D.d. CARMIL fused to GST as the affinity matrix.…”

Section: Fcsmentioning

confidence: 99%

“…CP bound to CPI binds the barbed end with an affinity of ∼50 nM, which, although still quite significant, is ∼100-fold weaker than free CP (20,21). CPI also dramatically accelerates the dissociation of CP already present on the barbed end (i.e., it robustly uncaps CP-capped filaments) (22). Structural studies indicate that CPI reduces CP's affinity for the barbed end in an allosteric fashion by restricting fluctuations within CP to a Significance F-actin is assembled downstream of major actin nucleators like the actin-related proteins 2/3 (Arp2/3) complex and formins.…”

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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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Section: Discussionmentioning

confidence: 99%

Section: Fcsmentioning

confidence: 99%

mentioning

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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“…The single-molecule fluorescence colocalization techniques used here are powerful tools for directly observing binding and deciphering complex reaction mechanisms (32)(33)(34)(35)(36). These techniques allowed us to directly measure key features of Arp2/3 complexmediated branch formation, including Arp2/3 binding and dissociation rates, branch formation efficiency, and activation time, and to determine how these parameters are affected by VCA.…”

Section: Discussionmentioning

confidence: 99%

Pathway of actin filament branch formation by Arp2/3 complex revealed by single-molecule imaging

Smith

Daugherty-Clarke

Goode

et al. 2013

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

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137

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Actin filament nucleation by actin-related protein (Arp) 2/3 complex is a critical process in cell motility and endocytosis, yet key aspects of its mechanism are unknown due to a lack of real-time observations of Arp2/3 complex through the nucleation process. Triggered by the verprolin homology, central, and acidic (VCA) region of proteins in the Wiskott-Aldrich syndrome protein (WASp) family, Arp2/3 complex produces new (daughter) filaments as branches from the sides of preexisting (mother) filaments. We visualized individual fluorescently labeled Arp2/3 complexes dynamically interacting with and producing branches on growing actin filaments in vitro. Branch formation was strikingly inefficient, even in the presence of VCA: only ∼1% of filament-bound Arp2/3 complexes yielded a daughter filament. VCA acted at multiple steps, increasing both the association rate of Arp2/3 complexes with mother filament and the fraction of filament-bound complexes that nucleated a daughter. The results lead to a quantitative kinetic mechanism for branched actin assembly, revealing the steps that can be stimulated by additional cellular factors.

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“…This type of uncapping mechanism is completely consistent with the fact that the preformed complex of CP and CAH3 still has weak barbed end capping activity. Indeed, just as sequestering and the inability to uncap CP-capped barbed ends are probably mechanistically coupled in V-1, weak capping and barbed end uncapping are probably mechanistically coupled in CARMIL (34). Importantly, recent single-molecule imaging of CP-capped actin filaments following the addition of the CAH3 domain-containing fragment of mCARMIL-1 used here (CAH3a/b) has confirmed both the uncapping activity of the CAH3 domain and the ability of the CAH3-CP complex to cap the barbed end weakly (34).…”

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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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Direct Observation of the Uncapping of Capping Protein-capped Actin Filaments by CARMIL Homology Domain 3

Cited by 52 publications

References 39 publications

V-1 regulates capping protein activity in vivo

V-1 regulates capping protein activity in vivo

Pathway of actin filament branch formation by Arp2/3 complex revealed by single-molecule imaging

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

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