Dip1 Defines a Class of Arp2/3 Complex Activators that Function without Preformed Actin Filaments

Wagner, Andrew R.; Luan, Qing; Liu, Su-Ling; Nolen, Brad J.

doi:10.1016/j.cub.2013.08.029

Cited by 70 publications

(118 citation statements)

References 48 publications

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“…Therefore, whether direct actin monomer tethering is required for potent activation by any NPF likely depends on the identity and mechanism of the NPF and is not a fundamental requirement of the branching nucleation reaction. Consistent with this hypothesis, WISH/DIP/SPIN90 (WDS) family proteins can potently activate the Arp2/3 complex but lack known actin monomer-binding domains (5). Although little is known about this class of NPFs, we previously used the cross-linking assay to show that WDS protein Dip1 stimulates formation of the shortpitch conformation, and our data here suggest this stimulation is sufficient for Dip1-mediated activation of the complex (5).…”

Section: Discussionsupporting

confidence: 75%

“…Consistent with this hypothesis, WISH/DIP/SPIN90 (WDS) family proteins can potently activate the Arp2/3 complex but lack known actin monomer-binding domains (5). Although little is known about this class of NPFs, we previously used the cross-linking assay to show that WDS protein Dip1 stimulates formation of the shortpitch conformation, and our data here suggest this stimulation is sufficient for Dip1-mediated activation of the complex (5). Like the WDS family proteins, type II NPFs such as cortactin and fission yeast myosin I interact with the Arp2/3 complex and actin filaments but not with actin monomers (37,(42)(43)(44).…”

Section: Discussionmentioning

confidence: 83%

“…To do so, we first separated the cross-linked complex from the non-cross-linked complex using an affinity column charged with GST-tagged Dip1, a recently described Arp2/3 complex activator which binds preferentially to the cross-linked complex (SI Appendix, Fig. S2) (5). This protocol yielded 98% pure cross-linked complex (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Assembly of new actin filaments is limited by a slow nucleation step, and cells rely on three classes of actin filament nucleators-tandem WH2 domain proteins, formins, and the Arp2/3 (Actin-related proteins 2/3) complex-to control precisely when and where actin filament networks assemble (1,2). The Arp2/3 complex is unique among actin filament nucleators in that it typically nucleates branched (as opposed to linear) actin filaments (3)(4)(5). Branched actin filaments are important for assembling the dendritic networks required for lamellipodial protrusion and endocytosis (6,7).…”

mentioning

confidence: 99%

“…The Arp2/3 complex is tightly regulated, and multiple factors, including preformed actin filaments, actin monomers, ATP, and a class of proteins called "nucleation-promoting factors" (NPFs) are typically required for activation (5)(6)(7). In some species, phosphorylation of the Arp2/3 complex is also required for activation (8).…”

mentioning

confidence: 99%

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Role and structural mechanism of WASP-triggered conformational changes in branched actin filament nucleation by Arp2/3 complex

Rodnick-Smith

Luan

Liu

et al. 2016

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

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The Arp2/3 (Actin-related proteins 2/3) complex is activated by WASP (Wiskott-Aldrich syndrome protein) family proteins to nucleate branched actin filaments that are important for cellular motility. WASP recruits actin monomers to the complex and stimulates movement of Arp2 and Arp3 into a "short-pitch" conformation that mimics the arrangement of actin subunits within filaments. The relative contribution of these functions in Arp2/3 complex activation and the mechanism by which WASP stimulates the conformational change have been unknown. We purified budding yeast Arp2/3 complex held in or near the short-pitch conformation by an engineered covalent cross-link to determine if the WASP-induced conformational change is sufficient for activity. Remarkably, cross-linked Arp2/3 complex bypasses the need for WASP in activation and is more active than WASP-activated Arp2/3 complex. These data indicate that stimulation of the short-pitch conformation is the critical activating function of WASP and that monomer delivery is not a fundamental requirement for nucleation but is a specific requirement for WASP-mediated activation. During activation, WASP limits nucleation rates by releasing slowly from nascent branches. The cross-linked complex is inhibited by WASP's CA region, even though CA potently stimulates cross-linking, suggesting that slow WASP detachment masks the activating potential of the short-pitch conformational switch. We use structure-based mutations and WASP-Arp fusion chimeras to determine how WASP stimulates movement toward the short-pitch conformation. Our data indicate that WASP displaces the autoinhibitory Arp3 C-terminal tail from a hydrophobic groove at Arp3′s barbed end to destabilize the inactive state, providing a mechanism by which WASP stimulates the short-pitch conformation and activates Arp2/3 complex.actin | Arp2/3 | WASP

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Section: Discussionsupporting

confidence: 75%

Section: Discussionmentioning

confidence: 83%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Role and structural mechanism of WASP-triggered conformational changes in branched actin filament nucleation by Arp2/3 complex

Rodnick-Smith

Luan

Liu

et al. 2016

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

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Benefits and challenges of reconstituting the actin cortex

Waechtler,

Jayasankar,

Morin

et al. 2024

Cytoskeleton

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The cell's ability to change shape is a central feature in many cellular processes, including cytokinesis, motility, migration, and tissue formation. The cell constructs a network of contractile proteins underneath the cell membrane to form the cortex, and the reorganization of these components directly contributes to cellular shape changes. The desire to mimic these cell shape changes to aid in the creation of a synthetic cell has been increasing. Therefore, membrane‐based reconstitution experiments have flourished, furthering our understanding of the minimal components the cell uses throughout these processes. Although biochemical approaches increased our understanding of actin, myosin II, and actin‐associated proteins, using membrane‐based reconstituted systems has further expanded our understanding of actin structures and functions because membrane–cortex interactions can be analyzed. In this review, we highlight the recent developments in membrane‐based reconstitution techniques. We examine the current findings on the minimal components needed to recapitulate distinct actin structures and functions and how they relate to the cortex's impact on cellular mechanical properties. We also explore how co‐processing of computational models with wet‐lab experiments enhances our understanding of these properties. Finally, we emphasize the benefits and challenges inherent to membrane‐based, reconstitution assays, ranging from the advantage of precise control over the system to the difficulty of integrating these findings into the complex cellular environment.

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Control of polarized assembly of actin filaments in cell motility

Carlier¹,

Pernier²,

Montaville³

et al. 2015

Cell. Mol. Life Sci.

101

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Actin cytoskeleton remodeling, which drives changes in cell shape and motility, is orchestrated by a coordinated control of polarized assembly of actin filaments. Signal responsive, membrane-bound protein machineries initiate and regulate polarized growth of actin filaments by mediating transient links with their barbed ends, which elongate from polymerizable actin monomers. The barbed end of an actin filament thus stands out as a hotspot of regulation of filament assembly. It is the target of both soluble and membrane-bound agonists as well as antagonists of filament assembly. Here, we review the molecular mechanisms by which various regulators of actin dynamics bind, synergize or compete at filament barbed ends. Two proteins can compete for the barbed end via a mutually exclusive binding scheme. Alternatively, two regulators acting individually at barbed ends may be bound together transiently to terminal actin subunits at barbed ends, leading to the displacement of one by the other. The kinetics of these reactions is a key in understanding how filament length and membrane-filament linkage are controlled. It is also essential for understanding how force is produced to shape membranes by mechano-sensitive, processive barbed end tracking machineries like formins and by WASP-Arp2/3 branched filament arrays. A combination of biochemical and biophysical approaches, including bulk solution assembly measurements using pyrenyl-actin fluorescence, single filament dynamics, single molecule fluorescence imaging and reconstituted self-organized filament assemblies, have provided mechanistic insight into the role of actin polymerization in motile processes.

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Dip1 Defines a Class of Arp2/3 Complex Activators that Function without Preformed Actin Filaments

Cited by 70 publications

References 48 publications

Role and structural mechanism of WASP-triggered conformational changes in branched actin filament nucleation by Arp2/3 complex

Role and structural mechanism of WASP-triggered conformational changes in branched actin filament nucleation by Arp2/3 complex

Benefits and challenges of reconstituting the actin cortex

Control of polarized assembly of actin filaments in cell motility

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