Acceleration of Yeast Actin Polymerization by Yeast Arp2/3 Complex Does Not Require an Arp2/3-activating Protein

101

137

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.

Section: Individual Arp2/3 Complexes Bind the Sides Of Preexisting Actinsupporting

confidence: 51%

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

Smith

Daugherty-Clarke

Goode

et al. 2013

101

137

“…Our cross-linking experiments showed that, even without WASP, a small fraction of the complex adopts the short-pitch conformation (Fig. 2 A and C), consistent with the weak NPF-independent activity of the budding yeast complex (13,38). Therefore, we reasoned that cross-linking could be used to trap a fraction of the complex in the short-pitch conformation to test its influence on nucleation activity directly.…”

Section: Resultsmentioning

confidence: 99%

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

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

“…If such a "direct-coupling" mechanism is valid one might intuitively expect mutations that modify the rates of F-actin polymerization to exhibit proportional changes in mitochondrial mobility. The act1-v159n mutant is known to stabilize F-actin and thereby increase the forward rate of F-actin assembly while decreasing the disassembly rate (21). Our experiments on this mutant show an enhancement of the long-time diffusion coefficient for length scales ≥0.78 μm (see Fig.…”

Section: Discussionmentioning

confidence: 64%

“…The act1-v159n protein is thought to stabilize F-actin by enhancing the forward polymerization rate. For example, Wen et al observed in vitro enhanced polymerization rates of yeast act1-v159n protein in the presence of the actin filament nucleation complex Arp2∕3 (21). At the elevated temperature of 36°C we observe an enhancement of the dynamics for act1-V159N in comparison to wild-type mitochondria for d G ≥ 0.79 μm.…”

mentioning

confidence: 82%

Actin polymerization driven mitochondrial transport in mating S. cerevisiae

Senning

Marcus²

2009

The dynamic microenvironment of cells depends on macromolecular architecture, equilibrium fluctuations, and nonequilibrium forces generated by cytoskeletal proteins. We studied the influence of these factors on the motions of mitochondria in mating S. cerevisiae using Fourier imaging correlation spectroscopy (FICS). Our measurements provide detailed length-scale dependent information about the dynamic behavior of mitochondria. We investigate the influence of the actin cytoskeleton on mitochondrial motion and make comparisons between conditions in which actin network assembly and disassembly is varied either by using disruptive pharmacological agents or mutations that alter the rates of actin polymerization. Under physiological conditions, nonequilibrium dynamics of the actin cytoskeleton leads to 1.5-fold enhancement of the long-time mitochondrial diffusion coefficient and a transient subdiffusive temporal scaling of the mean-square displacement (MSD ∝ τ α , with α ¼ 2∕3). We find that nonequilibrium forces associated with actin polymerization are a predominant factor in driving mitochondrial transport. Moreover, our results lend support to an existing model in which these forces are directly coupled to mitochondrial membrane surfaces.anomalous diffusion | Arp2/3-mediated actin polymerization | Fourier imaging correlation spectroscopy | intracellular transport | mitochondrial dynamics T he intracellular environment is a dynamic multicomponent fluid with relaxations spanning a broad range of length and time scales. As in ordinary fluids, in cells thermally generated inertial forces give rise to stochastic particle motions, which may be characterized by the particle mean-square displacement (MSD). Under many circumstances, thermal diffusion is a primary mechanism of intracellular transport. However, nonequilibrium forces that are generated by protein polymerization, motor proteins, and gradients in thermodynamic potentials can additionally influence particle motion. A central question in modeling cell transport is whether the cytoplasm may be viewed as a simple extension of a complex fluid at equilibrium or if nonequilibrium effects dominate the motions of intracellular species.For a spatially homogeneous fluid at equilibrium, the MSD scales linearly in time (i.e. MSD ∝ τ α with α ¼ 1). However, microscopic heterogeneity and viscoelasticity of multicomponent fluids results in "anomalous" particle motion (1-5). In the context of cell dynamics the motion of a macromolecule or larger particle may become hindered by obstacles in its immediate environment, leading to subdiffusive scaling of the MSD (i.e. α < 1), over an appreciable time range (4). For example, the motions of small proteins in bacteria appear to be diffusive (6-8), whereas those of larger particles, such as mRNA-protein clusters in E. coli and yolk granules in yeast, appear to be subdiffusive (2, 3). These differences in the dynamics of intracellular species and host organisms may be reconciled by accounting for the relative size of the mobile particles...