Arp2/3 Complex from<i>Acanthamoeba</i>Binds Profilin and Cross-links Actin Filaments

Mullins, R. Dyche; Kelleher, Joseph F.; Xu, James; Pollard, Thomas D.

doi:10.1091/mbc.9.4.841

Cited by 87 publications

(71 citation statements)

References 44 publications

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“…Relatively high concentrations of the Arp2/3 complex were required to raise the elastic modulus of actin filaments to levels achieved with much lower FLNa concentrations (53), implying that the Arp2/3 complex caused actin filaments to form bundles, and electron micrographs showed actin filament bundle formation (54). Fig.…”

Section: Effect Of the Arp2/3 Complex And Flna On The Diffusion Ofmentioning

confidence: 89%

Comparison of Filamin A-induced Cross-linking and Arp2/3 Complex-mediated Branching on the Mechanics of Actin Filaments

Nakamura

Osborn

Janmey

et al. 2002

Journal of Biological Chemistry

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We compared the effects of human filamin A (FLNa) and the activated human Arp2/3 complex on mechanical properties of actin filaments. As little as 1 FLNa to 800 polymerizing actin monomers induces a sharp concentration-dependent increase in the apparent viscosity of 24 M actin, a parameter classically defined as a gel point. The activated Arp2/3 complex, at concentrations up to 1:25 actins had no detectable actin gelation activity, even in the presence of phalloidin, to stabilize actin filaments against debranching. Increasing the activated Arp2/3 complex to actin ratio raises the FLNa concentration required to induce actin gelation, an effect ascribable to Arp2/3-mediated actin nucleation resulting in actin filament length diminution. Time lapse video microscopy of microparticles attached to actin filaments or photoactivation of fluorescence revealed actin filament immobilization by FLNa in contrast to diffusion of Arp2/3-branched actin filaments. The experimental results support theories predicting that polymer branching absent cross-linking does not lead to polymer gelation and are consistent with the observation that cells deficient in actin filament cross-linking activity have unstable surfaces. They suggest complementary roles for actin branching and cross-linking in cellular actin mechanics in vivo.The major ingredient of living cells is water, but unlike water, cells exhibit elastic as well as viscous properties. The elasticity of cells, historically designated as gel-like, results from the interactions of intracellular solute, principally cytoskeletal polymers. One of these polymers, actin, dominates the periphery of motile non-muscle cells and accounts for their ability to protrude against resistance as they spread and crawl and to resist surface deformations. A basic prerequisite for this material property is solute cohesion (1, 2).Pure actin filaments in the absence of chemical cross-linking form a relatively soft material (3, 4), a finding predicted by recent theories for the rheological behavior of semi-flexible rods (5-7). Moreover, estimates of the actin polymer concentration (ϳ1 mM) (8) and of the average actin filament length (less than 1 m in actively protruding cells (9, 10)) predict that the actin filaments would disperse unless tethered and that, therefore, cross-linking of these filaments is necessary to provide sufficient coherence to account for the protrusive activity and deformation resistance of cells. Cells contain many actin filament-cross-linking proteins to accomplish this task (11).A phase transition that marks the cross-linking of actin filaments into a cohesive structure is an easily discernable abrupt consistency increase inducible by imposing a critical number of actin filament-cross-linking molecules (12). At this transition point, the system converts from predominantly overlapping linear chains into a giant continuously linked molecular structure, operationally defined as a gel (13). As with other polymers (1), the length of actin filaments is directly proportional to their...

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Section: Effect Of the Arp2/3 Complex And Flna On The Diffusion Ofmentioning

confidence: 89%

Comparison of Filamin A-induced Cross-linking and Arp2/3 Complex-mediated Branching on the Mechanics of Actin Filaments

Nakamura

Osborn

Janmey

et al. 2002

Journal of Biological Chemistry

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“…Experiments with L27 monomers alone were checked using WinNonLin; data fit in all cases to a single-species model with monomeric molecular weight, and no higher aggregation states could be fit. Analysis of heterodimers with one labeled species was performed as described (34,35). Proteins used for analytical ultracentrifugation experiments were purified as described above or specifically labeled as follows: purified H.s.…”

Section: Methodsmentioning

confidence: 99%

Coordinated Folding and Association of the LIN-2, -7 (L27) Domain

Harris¹,

Venkatasubrahmanyam²,

Lim

2002

Journal of Biological Chemistry

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LIN-2, -7 (L27) homology domains are putative protein-protein interaction modules found in several scaffold proteins involved in the assembly of polarized cellsignaling structures. These specific interaction pairs are well conserved across metazoan species, from worms to man. We have expressed and purified L27 domains from multiple species and find that certain domains from proteins such as Caenorhabditis elegans LIN-2 and LIN-7 can specifically heterodimerize. Biophysical analysis of interacting L27 domains demonstrates that the domains interact with a 1:1 stoichiometry. Circular dichroism studies reveal that the domains appear to function as an obligate heterodimer; individually the domains are largely unfolded, but when associated they show a significant increase in helicity, as well as a cooperative unfolding transition. These novel obligate interacting pairs are likely to play a key role in regulating the organization of signaling proteins at polarized cell structures.

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“…The Arp2/3 actin-organizing complex is activated, in other cell types, by Cdc42 and Rac, albeit by distinct mechanisms (Mullins et al, 1998). To determine whether Arp proteins participate in Rac-mediated organization of their cytoskeleton, we exposed bone-residing cytokine-starved WT and LysMRacDKO osteoclasts to M-CSF, which we established activates Rac.…”

Section: Deletion Of Rac Impairs Arp3 Localizationmentioning

confidence: 99%

Rac deletion in osteoclasts causes severe osteopetrosis

Croke

Ross

Korhonen

et al. 2011

Journal of Cell Science

106

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SummaryCdc42 mediates bone resorption principally by stimulating osteoclastogenesis. Whether its sister GTPase, Rac, meaningfully impacts upon the osteoclast and, if so, by what means, is unclear. We find that whereas deletion of Rac1 or Rac2 alone has no effect, variable reduction of Rac1 in osteoclastic cells of Rac2 2/2 mice causes severe osteopetrosis. Osteoclasts lacking Rac1 and Rac2 in combination (Rac double-knockout, RacDKO), fail to effectively resorb bone. By contrast, osteoclasts are abundant in RacDKO osteopetrotic mice and, unlike those deficient in Cdc42, express the maturation markers of the cells normally. Hence, the osteopetrotic lesion of RacDKO mice largely reflects impaired function, and not arrested differentiation, of the resorptive polykaryon. The dysfunction of RacDKO osteoclasts represents failed cytoskeleton organization as evidenced by reduced motility of the cells and their inability to spread or generate the key resorptive organelles (i.e. actin rings and ruffled borders), which is accompanied by abnormal Arp3 distribution. The cytoskeleton-organizing capacity of Rac1 is mediated through its 20-amino-acid effector domain. Thus, Rac1 and Rac2 are mutually compensatory. Unlike Cdc42 deficiency, their combined absence does not impact upon differentiation but promotes severe osteopetrosis by dysregulating the osteoclast cytoskeleton.

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Arp2/3 Complex fromAcanthamoebaBinds Profilin and Cross-links Actin Filaments

Cited by 87 publications

References 44 publications

Comparison of Filamin A-induced Cross-linking and Arp2/3 Complex-mediated Branching on the Mechanics of Actin Filaments

Comparison of Filamin A-induced Cross-linking and Arp2/3 Complex-mediated Branching on the Mechanics of Actin Filaments

Coordinated Folding and Association of the LIN-2, -7 (L27) Domain

Rac deletion in osteoclasts causes severe osteopetrosis

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