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Formins promote processive elongation of actin filaments for cytokinetic contractile rings and other cellular structures. In vivo, these structures are exposed to tension, but the effect of tension on these processes was unknown. Here we used single-molecule imaging to investigate the effects of tension on actin polymerization mediated by yeast formin Bni1p. Small forces on the filaments dramatically slowed formin-mediated polymerization in the absence of profilin, but resulted in faster polymerization in the presence of profilin. We propose that force shifts the conformational equilibrium of the end of a filament associated with formin homology 2 domains toward the closed state that precludes polymerization, but that profilin-actin associated with formin homology 1 domains reverses this effect. Thus, physical forces strongly influence actin assembly by formin Bni1p.A host of proteins regulate the actin cytoskeleton by controlling filament nucleation, elongation, capping, branching, and bundling. Members of the formin family of proteins nucleate new filaments and remain processively attached to barbed ends while promoting the elongation of unbranched filaments (1, 2). Formin homology (FH)2 domains form a donut-shaped head-to-tail homodimer that encircles the fast-growing barbed end of actin filaments and promotes nucleation and polymerization (1, 3). When an actin monomer binds to the barbed end of a filament, one FH2 domain steps onto the new subunit, allowing the formin to remain attached to the filament through thousands of cycles of subunit addition ( Fig. 1A) (2, 4). The FH2-bound end of the filament binds incoming actin monomers when in an "open" conformation but not in the "closed" conformation. As a consequence, FH2 domains slow barbed-end elongation compared with free barbed ends, a phenomenon termed "gating" (1, 5). Despite gating, FH2 domains can promote rapid filament elongation when coupled to FH1 domains (6), which are located N-terminal to the FH2 domain (7). Multiple polyproline tracks in FH1 domains bind the small actin-binding protein profilin, which mediates association of several profilin-actin complexes in close proximity to the end of a filament. Diffusive motions of the FH1 domain transfer actin rapidly to the filament barbed end (5), allowing elongation at rates faster than addition of subunits from the bulk phase.Actin filaments are subject to tension in cells, yet the influence of tension on formin-mediated polymerization was unknown, and theories predicted different outcomes in the absence and presence of profilin. Kozlov and colleagues proposed that the elasticity of the FH2 domain and the formin-barbed end binding energy govern the polymerization rate (8, 9). Their simulations suggested that tension increases the rate of polymerization by lowering the activation barrier for subunit addition and energetically favoring FH2 domain stepping onto the incoming subunit (8). Vavylonis et al. (10) postulated that force-induced stretching of FH1 domains slows the transfer of profilin-actin t...

Cellular viability requires tight regulation of actin cytoskeletal dynamics. Distinct families of nucleation-promoting factors enable the rapid assembly of filament nuclei that elongate and are incorporated into diverse and specialized actin-based structures. In addition to promoting filament nucleation, the formin family of proteins directs the elongation of unbranched actin filaments. Processive association of formins with growing filament ends is achieved through continuous barbed end binding of the highly conserved, dimeric formin homology (FH) 2 domain. In cooperation with the FH1 domain and C-terminal tail region, FH2 dimers mediate actin subunit addition at speeds that can dramatically exceed the rate of spontaneous assembly. Here, I review recent biophysical, structural, and computational studies that have provided insight into the mechanisms of formin-mediated actin assembly and dynamics.

Although our understanding of globular protein folding continues to advance, the irregular tertiary structures and high cooperativity of globular proteins complicates energetic dissection. Recently, proteins with regular, repetitive tertiary structures have been identified that sidestep limitations imposed by globular protein architecture. Here we review recent studies of repeat-protein folding. These studies uniquely advance our understanding of both the energetics and kinetics of protein folding. Equilibrium studies provide detailed maps of local stabilities, access to energy landscapes, insights into cooperativity, determination of nearest-neighbor interaction parameters using statistical thermodynamics, relationships between consensus sequences and repeat-protein stability. Kinetic studies provide insight into the influence of short-range topology on folding rates, the degree to which folding proceeds by parallel (versus localized) pathways, and the factors that select among multiple potential pathways. The recent application of force spectroscopy to repeat-protein unfolding is providing a unique route to test and extend many of these findings. KeywordsRepeat-protein; Ankyrin repeat; protein folding; energy landscape; atomic force microscopy In the last twenty-five years, advances in experimental studies and in computation and theory have greatly improved our understanding of protein folding. On the experimental side, advances have come from new and improved techniques, including site-directed mutagenesis, hydrogen exchange (HX) methods, improvements to rapid mixing devices, and development of single-molecule fluorescence and force spectroscopy. Experimental advances have also come in the form of generalizations and insights from expanding databases of thermodynamic and kinetic constants for protein folding [1][2][3][4][5].On the computational side, advances in our understanding of protein folding have come from huge increases in computer speed and storage capacity, in innovative methods to study energetics of folding such as ensemble-based approaches and replica exchange methods [6,7], and distributed computing methods [8]. As with experiment, computational studies of folding, and in particular, fold prediction, have greatly benefited from expanding databases of protein structure [9,10]. On the theoretical side, the application of ideas from condensed-matter *Correspondence: barrick@jhu.edu, (410) 516-0409. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptArch Biochem Biophys. Author manuscript; available in PMC 2009 January ...

SUMMARY Latrunculin A (LatA), a toxin from the red sea sponge Latrunculia magnifica, is the most widely used reagent to depolymerize actin filaments in experiments on live cells. LatA binds actin monomers and sequesters them from polymerization [1, 2]. Low concentrations of LatA result in rapid (tens of seconds) disassembly of actin filaments in animal [3] and yeast cells [2]. Depolymerization is usually assumed to result from sequestration of actin monomers. Our observations of single muscle actin filaments by TIRF microscopy showed that LatA bound ATP-actin monomers with a higher affinity (Kd = 0.1 μM) than ADP-Pi-actin (Kd = 0.4 μM) or ADP-actin (Kd = 4.7 μM). LatA also slowly severed filaments and increased the depolymerization rate at both ends of filaments freshly assembled from ATP-actin to the rates of ADP-actin. This rate plateaued at LatA concentrations >60 μM. LatA did not change the depolymerization rates of ADP- actin filaments or ADP-Pi-actin filaments generated with 160 mM phosphate in the buffer. LatA did not increase the rate of phosphate release from bulk samples of filaments assembled from ATP-actin. Thermodynamic analysis showed that LatA binds weakly to actin filaments with a Kd >100 μM. We propose that concentrations of LatA much lower than this Kd promote phosphate dissociation only from both ends of filaments, resulting in depolymerization limited by the rate of ADP-actin dissociation. Thus, one must consider both rapid actin depolymerization and severing in addition to sequestering actin monomers when interpreting the effects of LatA on cells.

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Profilin binds not only to actin monomers but also to the barbed end of the actin filament, where it inhibits association of subunits. To address open questions about the interactions of profilin with barbed ends, we measured the effects of a wide range of concentrations of Homo sapiens profilin 1 on the rate of elongation of individual skeletal muscle actin filaments by total internal reflection fluorescence microscopy. Much higher concentrations of profilin were required to stop elongation by AMP-PNP-actin monomers than ADP-actin monomers. High concentrations of profilin depolymerized barbed ends much faster than the spontaneous dissociation rates of Mg-ATP-, Mg-AMP-PNP-, Mg-ADP-Pi- or Mg-ADP-actin subunits. Fitting a thermodynamic model to these data allowed us to determine the affinities of profilin and profilin-actin for barbed ends and the influence of the nucleotide bound to actin on these interactions. Profilin has a much higher affinity for ADP-actin filament barbed ends (Kd = 1 μM) than AMP-PNP-actin filament barbed ends (Kd = 226 μM). ADP-actin monomers associated with profilin bind to ADP-actin filament barbed ends 10% as fast as free ADP-actin monomers, but bound profilin does not affect the rate of association of AMP-PNP-actin monomers with barbed ends. The differences in the affinities of AMP-PNP- and ADP-bound barbed ends for profilin and profilin-actin suggest that conformations of barbed end subunits differ from those of monomers and change upon nucleotide hydrolysis and phosphate release. A structural model revealed minor steric clashes between profilin and actin subunits at the barbed end that explain the biochemical results.

Background: Formin FH1 domains deliver subunits to actin filament barbed ends. Results: The transfer rate of formin Bni1p depends on FH1 polyproline track sequences and positions. Conclusion:The two FH1 domains of formin dimers deliver actin independently of one another. Significance: Sequence features of other FH1 domains are similar to Bni1p, suggesting a common strategy to maximize actin polymerization rates.