Interactions of Isolated C-terminal Fragments of Neural Wiskott-Aldrich Syndrome Protein (N-WASP) with Actin and Arp2/3 Complex

Gaucher, Jonathan; Mauge, Chloé; Didry, Dominique; Guichard, Bérengère; Renault, Louis; Carlier, Marie-France

doi:10.1074/jbc.m112.394361

Cited by 41 publications

(55 citation statements)

References 73 publications

(61 reference statements)

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“…However, testing this hypothesis has proven difficult, due to spontaneous polymerization of actin, which interferes with biochemical and structural studies of Arp2/3 complex requiring high sample concentrations under physiological salt conditions. While latrunculin (A or B) has been used in some studies to prevent actin polymerization 25, 32 , this toxin binds in the nucleotide-binding cleft at the pointed end of the actin monomer and is thus expected to interfere with proper interaction with the Arps, as suggested by a recent study 28 and analogous to its effect on actin-actin interactions 33 . To circumvent these problems, we devised two different strategies, namely crosslinking of actin to WCA and substitution of the N-terminal region of the W domain in WCA by gelsolin segment 1 (GS1) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In contrast, the other study found that the stoichiometry of the mammalian complex was 2:1 (WCA-Arp2/3 complex), both in the presence or the absence of actin-latrunculin B 25 . And both studies were challenged by a work that found that Arp2/3 complex binds a single WCA with or without actin 28 . Here, we tested the two competing models of activation, addressing specifically the role of actin in the interaction of mammalian Arp2/3 complex and NPFs.…”

Section: Resultsmentioning

confidence: 99%

“…Despite these considerations, the two-NPF model was immediately challenged by a study that found that Arp2/3 complex interacts with a single NPF, both in the absence or the presence of actin 28 . The ongoing debate, and the fact that our own SAXS analysis of the 1:1:1 complex 24 was used as supporting evidence in the latter study, motivated us to analyze the two competing models of Arp2/3 complex activation using different approaches.…”

Section: Discussionmentioning

confidence: 99%

“…Two groups have now reported that Arp2/3 complex binds two NPFs 25, 26 , which leads to more efficient activation of the complex and is also consistent with the observation that in cells NPFs are frequently clustered on membranes or bound to dimeric partners 27 . However, these reports were recently disputed by another study that found that Arp2/3 complex bound a single NPF in the presence or the absence of actin 28 .…”

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Structural analysis of the transitional state of Arp2/3 complex activation by two actin-bound WCAs

et al. 2014

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Actin filament nucleation and branching by Arp2/3 complex is activated by nucleation-promoting factors (NPFs), whose C-terminal WCA region contains binding sites for actin (W) and Arp2/3 complex (CA). It is debated whether one or two NPFs are required for activation. Here, we present conclusive evidence in support of the two-NPF model and show that actin plays a crucial role in the interactions of two mammalian NPFs, N-WASP and WAVE2, with Arp2/3 complex. Competition between actin-WCA and glia maturation factor (GMF) for binding to Arp2/3 complex suggests that during activation the first actin monomer binds at the barbed end of Arp2. Based on distance constrains obtained by time-resolved fluorescence resonance energy transfer, we define the relative position of the two actin-WCAs on Arp2/3 complex and propose an atomic model of the 11-subunit transitional complex.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Structural analysis of the transitional state of Arp2/3 complex activation by two actin-bound WCAs

et al. 2014

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“…25), full-length human gelsolin, VCA domain of human neural Wiskott-Aldrich syndrome protein (N-WASP), the Arp2/3 complex from bovine brain or Spectrin-actin seeds from human erythrocytes were purified as described previously25405152.…”

Section: Methodsmentioning

confidence: 99%

Actin activates Pseudomonas aeruginosa ExoY nucleotidyl cyclase toxin and ExoY-like effector domains from MARTX toxins

et al. 2016

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The nucleotidyl cyclase toxin ExoY is one of the virulence factors injected by the Pseudomonas aeruginosa type III secretion system into host cells. Inside cells, it is activated by an unknown eukaryotic cofactor to synthesize various cyclic nucleotide monophosphates. ExoY-like adenylate cyclases are also found in Multifunctional-Autoprocessing Repeats-in-ToXin (MARTX) toxins produced by various Gram-negative pathogens. Here we demonstrate that filamentous actin (F-actin) is the hitherto unknown cofactor of ExoY. Association with F-actin stimulates ExoY activity more than 10,000 fold in vitro and results in stabilization of actin filaments. ExoY is recruited to actin filaments in transfected cells and alters F-actin turnover. Actin also activates an ExoY-like adenylate cyclase MARTX effector domain from Vibrio nigripulchritudo. Finally, using a yeast genetic screen, we identify actin mutants that no longer activate ExoY. Our results thus reveal a new sub-group within the class II adenylyl cyclase family, namely actin-activated nucleotidyl cyclase (AA-NC) toxins.

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Mutations in actin used for structural studies partially disrupt β‐thymosin/WH2 domains interaction

et al. 2016

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Understanding the structural basis of actin cytoskeleton remodeling requires stabilization of actin monomers, oligomers, and filaments in complex with partner proteins, using various biochemical strategies. Here, we report a dramatic destabilization of the dynamic interaction with a model β-thymosin/WH2 domain induced by mutations in actin. This result underlines that mutant actins should be used with prudence to characterize interactions with intrinsically disordered partners as destabilization of dynamic interactions, although identifiable by NMR, may be invisible to other structural techniques. It also highlights how both β-thymosin/WH2 domains and actin tune local structure and dynamics in regulatory processes involving intrinsically disordered domains.

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Interactions of Isolated C-terminal Fragments of Neural Wiskott-Aldrich Syndrome Protein (N-WASP) with Actin and Arp2/3 Complex

Cited by 41 publications

References 73 publications

Structural analysis of the transitional state of Arp2/3 complex activation by two actin-bound WCAs

Structural analysis of the transitional state of Arp2/3 complex activation by two actin-bound WCAs

Actin activates Pseudomonas aeruginosa ExoY nucleotidyl cyclase toxin and ExoY-like effector domains from MARTX toxins

Mutations in actin used for structural studies partially disrupt β‐thymosin/WH2 domains interaction

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