Actin assembly factors regulate the gelation kinetics and architecture of F-actin networks

Abstract

Dynamic regulation of the actin cytoskeleton is required for diverse cellular processes. Proteins regulating the assembly kinetics of the cytoskeletal biopolymer F-actin are known to impact the architecture of actin cytoskeletal networks in vivo, but the underlying mechanisms are not well understood. Here, we demonstrate that changes to actin assembly kinetics with physiologically relevant proteins profilin and formin (mDia1 and Cdc12) have dramatic consequences on the architecture and gelation kinetics of otherwise biochemically identical cross-linked F-actin networks. Reduced F-actin nucleation rates promote the formation of a sparse network of thick bundles, whereas increased nucleation rates result in a denser network of thinner bundles. Changes to F-actin elongation rates also have marked consequences. At low elongation rates, gelation ceases and a solution of rigid bundles is formed. By contrast, rapid filament elongation accelerates dynamic arrest and promotes gelation with minimal F-actin density. These results are consistent with a recently developed model of how kinetic constraints regulate network architecture and underscore how molecular control of polymer assembly is exploited to modulate cytoskeletal architecture and material properties.

Figure 1

Quantification of assembly kinetics of cross-linked profilin-actin networks. (A) Images of bundles formed by 5 μM actin, 1.2 μM α-actinin, and 15 μM SpPrf. Scale bar = 30 μm. (B) S(q,t) calculated for sample described in (A) for times from 45 to 1245 s. (C) S(q,t) at q = 0.0316 μm⁻¹ and linear bundle density over time. (D) Vector map of bundle displacements obtained in regions identified in (A) at 245 s (top) and 845 s (bottom); scale bar = 5 μm. (E) The mean speed of bundle mobility is plotted as a function of time. Dynamic arrest is defined when mean speed is reduced to 30 nm/s, near our resolution limit. (F) Time course of the spontaneous assembly of 5 μM Mg-ATP-actin monomers (10% pyrene labeled) in the presence of 15 μM SpPrf.

Figure 2

Profilin slows network assembly and promotes the formation of a network of sparse, thick bundles. (A) Time course of the spontaneous assembly of 5 μM Mg-ATP-actin monomers (10% pyrene labeled) in the presence of the indicated concentrations of SpPrf. (B) The 95% F-actin polymerization time from (A) as a function of SpPrf concentration. (C) Representative images of fluorescent (Alexa 488) phalloidin labeling of F-actin in networks forming via a spontaneous assembly of 5 μM Mg-ATP-actin, and 1.2 μM α-actinin, in the presence of varying concentrations of SpPrf. Polymerization is initiated at 0 s; scale bar = 30 μm. (D) The time to steady-state bundle density (open squares), S(q,t) steady state (solid circles), and dynamic arrest (solid diamonds) as a function of SpPrf concentration. (E) Steady-state linear bundle density as a function of SpPrf concentration. (F) Average steady-state bundle intensity as a function of SpPrf concentration.

Figure 3

Cdc12 formin accelerates dynamics and increases the bundle density in profilin-actin networks. (A) Time course of the spontaneous assembly of 5 μM Mg-ATP-actin monomers (10% pyrene labeled) in the presence of 15 μM SpPrf and the indicated concentrations of Cdc12. (B) The 95% F-actin polymerization time from (A) as a function of Cdc12 concentration. (C) Representative images of fluorescent (Alexa 488) phalloidin labeling of F-actin in networks forming via a spontaneous assembling of 5 μM Mg-ATP-actin, 1.2 μM α-actinin, and 15 μM SpPrf in the presence of varying concentrations of Cdc12. Polymerization is initiated at t = 0 s; scale bar = 30 μm. (D) The time to steady-state bundle density (open squares), steady-state S(q,t) (solid circles), and dynamic arrest (solid diamonds) as a function of Cdc12 concentration. (E) Steady-state linear bundle density as a function of Cdc12 concentration. (F) Average bundle intensity as a function of Cdc12 concentration.

Figure 4

Network gelation is inhibited at low filament elongation rates. (A) Time course of the spontaneous assembly of 5 μM Mg-ATP-actin monomers (10% pyrene labeled) in the presence of 500 nM Cdc12 and the indicated concentrations of SpPrf. (B) Calculated barbed end concentration from (A) and elongation rate per formin-bound filament as a function of SpPrf concentration. (C) Representative images of fluorescent (Alexa 488) phalloidin labeling of F-actin in networks forming via a spontaneous assembling of 5 μM Mg-ATP-actin, 1.2 μM α-actinin, and 500 nM Cdc12 in the presence of varying concentrations of SpPrf. Polymerization is initiated at 0 s; scale bar = 30 μm. (D) The time to steady-state bundle density (open squares), structure factor S(q,t) (solid circles), and dynamic arrest (solid diamonds) for the samples described above as a function of SpPrf concentration. (E) The mean instantaneous speed as a function of time for bundles formed with 0.5 μM Cdc12 (black square), 3.3 μM Cdc12 (open circles, 5 μM Cdc12 (solid triangles), and 15 μM Cdc12 (solid circles). Only samples with 5 and 15 μM Cdc12 reached the threshold for dynamic arrest (horizontal dashed line). (F) Steady-state linear bundle density for samples with 500 nM Cdc12 as a function of SpPrf concentration.

Figure 5

Rapid filament elongation by mDia1 formin promotes fast network assembly at low F-actin concentrations. (A) Time course of the spontaneous assembly of 5 μM Mg-ATP-actin monomers (10% pyrene labeled) in the presence of 15 μM pfn1 and the indicated concentrations of mDia1. (B) Calculated barbed end concentration (left, red circles) and the 95% F-actin polymerization time (right, blue squares) from (A) as a function of mDia1 concentration. (C) Representative images of fluorescent (Alexa 488) phalloidin-labeling of F-actin in networks forming via a spontaneous assembly of 5 μM Mg-ATP-actin, 1.2 μM α-actinin, and 15 μM pfn1 in the presence of varying concentrations of mDia1. Polymerization is initiated at t = 0 s; scale bar = 30 μm. (D) The time to steady-state bundle density (solid circles), S(q,t) steady state (open squares), and dynamic arrest (solid diamonds) as a function of mDia1 concentration. (E) Steady-state linear bundle density as a function of mDia1 concentration. (F) Concentration of actin polymer at gelation time as a function of mDia1 concentration.

Figure 6

Network architecture is impacted by actin assembly kinetics. (A) Diagram of the effects of increasing nucleation rates on structure as a function of time. At low nucleation rates, networks form over a long period of time and at steady state take on well-spaced bundled structures. At accelerated nucleation rates, more filaments are present at early times, and rapidly form a network composed of lower intensity, but higher density, bundles. (B) Diagram of the effects of increasing elongation rates on network architecture as a function of time. At high elongation rates, actin is able to rapidly form an integrated network with very few actin filaments by reducing the time at which filaments overlap each other. At low elongation rates, a network is unable to form before actin is fully polymerized and forms thick and rigid bundles unable to form an interconnected gel. (C) Plot of the percent of actin monomers polymerized at gelation time across samples used in Figs. 3 and 5. The arrow indicates increasing concentrations of formin. Blue squares, correspond to 0, 5, 10, 25, 50, and 100 nM concentrations of mDia1. Red triangles correspond to concentrations 0, 50, 100, 250, and 500 nM of Cdc12.