Cofilin Increases the Bending Flexibility of Actin Filaments: Implications for Severing and Cell Mechanics

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147

Mechanical cues affect many important biological processes in metazoan cells, such as migration, proliferation, and differentiation. Such cues are thought to be detected by specialized mechanosensing molecules linked to the cytoskeleton, an intracellular network of protein filaments that provide mechanical rigidity to the cell and drive cellular shape change. The most abundant such filament, actin, forms branched networks nucleated by the actin-related protein (Arp) 2/3 complex that support or induce membrane protrusions and display adaptive behavior in response to compressive forces. Here we show that filamentous actin serves in a mechanosensitive capacity itself, by biasing the location of actin branch nucleation in response to filament bending. Using an in vitro assay to measure branching from curved sections of immobilized actin filaments, we observed preferential branch formation by the Arp2/3 complex on the convex face of the curved filament. To explain this behavior, we propose a fluctuation gating model in which filament binding or branch nucleation by Arp2/3 occur only when a sufficiently large, transient, local curvature fluctuation causes a favorable conformational change in the filament, and we show with Monte Carlo simulations that this model can quantitatively account for our experimental data. We also show how the branching bias can reinforce actin networks in response to compressive forces. These results demonstrate how filament curvature can alter the interaction of cytoskeletal filaments with regulatory proteins, suggesting that direct mechanotransduction by actin may serve as a general mechanism for organizing the cytoskeleton in response to force.actin-based motility | autocatalytic branching | bending fluctuations | worm-like chain | force sensing

Section: Discussionsupporting

confidence: 76%

Actin filament curvature biases branching direction

Risca

Wang²,

Chaudhuri

et al. 2012

163

147

“…We evaluated the salt dependence of actin filament flexural rigidity by directly visualizing filaments undergoing thermally driven fluctuations in shape and calculating their bending persistence lengths (L p ) from the average angular correlation along their contour length (34,35). Because filament contour lengths are comparable to L p , small changes in filament rigidity yield readily detectable changes in filament shape (Fig.…”

Section: Resultsmentioning

confidence: 99%

Identification of cation-binding sites on actin that drive polymerization and modulate bending stiffness

Kang

Bradley

McCullough

et al. 2012

Self Cite

148

The assembly of actin monomers into filaments and networks plays vital roles throughout eukaryotic biology, including intracellular transport, cell motility, cell division, determining cellular shape, and providing cells with mechanical strength. The regulation of actin assembly and modulation of filament mechanical properties are critical for proper actin function. It is well established that physiological salt concentrations promote actin assembly and alter the overall bending mechanics of assembled filaments and networks. However, the molecular origins of these salt-dependent effects, particularly if they involve nonspecific ionic strength effects or specific ion-binding interactions, are unknown. Here, we demonstrate that specific cation binding at two discrete sites situated between adjacent subunits along the long-pitch helix drive actin polymerization and determine the filament bending rigidity. We classify the two sites as "polymerization" and "stiffness" sites based on the effects that mutations at the sites have on salt-dependent filament assembly and bending mechanics, respectively. These results establish the existence and location of the cation-binding sites that confer salt dependence to the assembly and mechanics of actin filaments.ion-linkage | structural bioinformatics | persistence length | polyelectrolyte T he polymerization of the protein actin into double-stranded helical filaments powers many eukaryotic cell movements and provides cells with mechanical strength and integrity (1-4). Filament formation is favored when the total actin concentration exceeds the critical concentration (C c ) for assembly-defined as the monomer concentration at steady state for ATP-actin, or the dissociation constant for the reversible-equilibrium binding reaction of monomer binding to ADP-actin filament ends. Accordingly, the C c of ADP-actin is linked to the filament subunit interaction free energy such that lower C c values reflect greater thermodynamic stability (5).The effects of solution ionic conditions on the assembly and stability of actin filaments have been investigated for several decades (6-12). The actin C c and (monomer and filament) conformation depend on the nucleotide-associated divalent cation (Ca 2þ or Mg 2þ ) as well as the type and concentration of ions in solution (6, 7, 13-15), a behavior shared among characterized actins and their bacterial homologs (16). However, it is not firmly established if these salt effects on actin filament assembly and mechanics originate from nonspecific ion effects (e.g., electrostatic screening, counterion condensation, etc.) and/or specific ion binding interactions, potentially at discrete sites. Identification of saturable cation binding sites with different affinities favors specific and discrete binding sites on monomers (8-10, 17), but the location of these sites and their contributions to filament assembly and stiffness are unknown.Here we identify distinct cation-binding sites at subunit interfaces that regulate actin filament assembly and rigidity. Sit...

“…Microscope slides were cleaned with absolute ethanol then 0.1 M KOH, followed by extensive rinsing with MilliQ water. The actin filament L p values were determined from an angular correlation analysis of digitized filament images (>20 images, n = 200-500 individual filaments) using a custom MATLAB script as described in detail (11,12,19,27).…”

Section: Methodsmentioning

confidence: 99%

“…Alteration of WT yeast actin filament flexibility and severing by yeast cofilin must originate from E167-and stiffness cation-independent perturbations. However, the severing activities of both yeast and vertebrate cofilins correlate with the ability to alter filament mechanical properties (12), favoring a shared mechanical mechanism for severing (5,11,12,15,19,41). These differences among cofilin orthologs are presumably explained by their relatively low sequence identity (∼40%), which may afford yeast cofilin with additional or distinct (yeast) actin interactions from vertebrate cofilin (42).…”

Section: Significancementioning

confidence: 99%

“…Filaments with bound cofilin have altered twist (8,9) and are more compliant in both bending and twisting than bare filaments (10)(11)(12)(13). It has been suggested that deformations in filament shape promote fragmentation at or near regions of topological and mechanical discontinuities, such as boundaries between bare and cofilin-decorated segments along partially decorated filaments (5,12,(14)(15)(16)(17)(18).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Site-specific cation release drives actin filament severing by vertebrate cofilin

Kang

Bradley

Cao

et al. 2014

Self Cite

Actin polymerization powers the directed motility of eukaryotic cells. Sustained motility requires rapid filament turnover and subunit recycling. The essential regulatory protein cofilin accelerates network remodeling by severing actin filaments and increasing the concentration of ends available for elongation and subunit exchange. Although cofilin effects on actin filament assembly dynamics have been extensively studied, the molecular mechanism of cofilin-induced filament severing is not understood. Here we demonstrate that actin filament severing by vertebrate cofilin is driven by the linked dissociation of a single cation that controls filament structure and mechanical properties. Vertebrate cofilin only weakly severs Saccharomyces cerevisiae actin filaments lacking this "stiffness cation" unless a stiffness cation-binding site is engineered into the actin molecule. Moreover, vertebrate cofilin rescues the viability of a S. cerevisiae cofilin deletion mutant only when the stiffness cation site is simultaneously introduced into actin, demonstrating that filament severing is the essential function of cofilin in cells. This work reveals that site-specific interactions with cations serve a key regulatory function in actin filament fragmentation and dynamics.cytoskeleton | persistence length | mechanics | electron cryomicroscopy A ctin polymerization powers the directed motility of eukaryotic cells and some pathogenic bacteria (1-3). Actin assembly also plays critical roles in endocytosis, cytokinesis, and establishment of cell polarity. Sustained motility requires filament disassembly and subunit recycling. The essential regulatory protein cofilin severs actin filaments (4-6), which accelerates actin network reorganization by increasing the concentration of filament ends available for subunit exchange (7).Cofilin binding alters the structure and mechanical properties of filaments, which effectively introduces local "defects" that compromise filament integrity and promote severing (5). Filaments with bound cofilin have altered twist (8, 9) and are more compliant in both bending and twisting than bare filaments (10-13). It has been suggested that deformations in filament shape promote fragmentation at or near regions of topological and mechanical discontinuities, such as boundaries between bare and cofilin-decorated segments along partially decorated filaments (5,12,(14)(15)(16)(17)(18).Cations modulate actin filament structure and mechanical properties (19) and cofilin dissociates filament-associated cations (20), leading us to hypothesize that cation-binding interactions regulate filament severing by cofilin. Cations bind filaments at two discrete and specific sites positioned between adjacent subunits along the long-pitch helix of the filament (19, 21). These cation binding sites are referred to as "polymerization" and "stiffness" sites based on their roles in filament assembly and mechanics, respectively. These discrete sites bind both monovalent and divalent cations with a range of affinities (low millimolar f...