Actin filament curvature biases branching direction

Abstract

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

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

“…S1 A and B; Movie S1), monomeric actin (sparsely labeled with the green-emitting dye Alexa Fluor 488) and the VCA portion of S. cerevisiae Wiskott-Aldrich syndrom protein (WASp/Las17; unlabeled), and observed filament assembly by total internal reflection fluorescence (TIRF) microscopy. As previously seen using labeled actin and unlabeled Arp2/3 complex, we observed formation of branched filament networks (4,5,7,(19)(20)(21)(22)(23)(24)(25)(26)(27)(28), and branches rarely if ever dissociated over tens of minutes (26). We saw that 90% (143/160) of branch junctions displayed well-defined spots of orange Arp2/3 complex fluorescence that typically persisted for tens or hundreds of seconds (Fig.…”

“…4B; open [A 1 ] vs. closed [A 2 ]) (7-10) and that only mother filament binding of closed Arp2/3 complex can ultimately lead to branch formation (B). The formation of the initial closed Arp2/3-filament complex (A 2 F) is reversible, but the Arp-filament interaction is stabilized by subsequent activation processes (13,17), which may include binding of actin monomers (13,15), changes in the nucleotide state of Arp2 and/or Arp3 (16,25), additional Arp2/3 complex conformational changes (6,10,17), or alteration of the mother filament structure or curvature (6,28,31). Because the experiments were conducted at either zero or maximally activating concentrations A B actin-AF488 Arp2/3 binding heat map merge of VCA, the scheme does not explicitly show VCA binding steps or define the occupancies of the multiple VCA-binding sites (10,14,15).…”

Actin Mechanics and Fragmentation

“…The filament binding affinity of the Arp2/3 complex depends on the filament curvature (79), raising the possibility that a branched network exhibiting a compressive load could respond both by modulating the branch density and possibly by cofilinmediated severing and filament subunit exchange. Together, this will increase the branch density and decrease average filament length, resulting in increased force generation network stiffness as a result of the applied load.…”

The other side of the coin: Functional and structural versatility of ADF/cofilins

“…These observations suggest that Abp1 may enhance the activity of the Arp2/3 complex by promoting its interaction with the actin filament. On the other hand, the activity of the Arp2/3 complex is sensitive to the conformation of the actin filaments (Risca et al, 2012) and ADF-H domain proteins can affect the structure of the filament. Based on these connections, it is hypothesized that, besides the direct interaction with the Arp2/3 complex, Abp1 can also alter the conformation of actin filament structure that would favor the nucleation by the Arp2/3 complex machinery.…”