Competition for delivery of profilin–actin to barbed ends limits the rate of formin-mediated actin filament elongation

Abstract

Formins direct the elongation of unbranched actin filaments by binding their barbed ends and processively stepping onto incoming actin monomers to incorporate them into the filament. Binding of profilin to actin monomers creates profilin–actin complexes, which then bind polyproline tracts located in formin homology 1 (FH1) domains. Diffusion of these natively disordered domains enables direct delivery of profilin–actin to the barbed end, speeding the rate of filament elongation. In this study, we investigated the mechanism of coordinated actin delivery from the multiple polyproline tracts in formin FH1 domains. We found that each polyproline tract can efficiently mediate polymerization, but that all tracts do not generate the same rate of elongation. In WT FH1 domains, the multiple polyproline tracts compete to deliver profilin–actin to the barbed end. This competition ultimately limits the rate of formin-mediated elongation. We propose that intrinsic properties of the filament-binding FH2 domain tune the efficiency of FH1-mediated elongation by directly regulating the rate of monomer incorporation at the barbed end. A strong correlation between competitive FH1-mediated profilin–actin delivery and FH2-regulated gating of the barbed end effectively limits the elongation rate, thereby obviating the need for evolutionary optimization of FH1 domain sequences.

Formin’s nucleation activity influences actin filament length

“…This mechanism also explains the assembly of very long formin-bound filaments (>20 µm) in elongation experiments monitored by TIRF microscopy [10, 17, 25]. These reactions utilize a low concentration of formin (typically <5 nM) and a concentration of actin monomers that is optimized to ensure a low rate of spontaneous nucleation.…”

“…Most eukaryotes express at least two formin isoforms that assemble unbranched actin filaments that are incorporated into cytoskeletal structures including cytokinetic rings, filopodia and stress fibers [23, 24]. Each formin isoform mediates a specific rate of filament elongation that depends on both the extent to which its FH2 domain gates the barbed end and the efficiency with which its FH1 domain delivers profilin-actin to the barbed end [17, 25]. Formin isoforms have also been shown to possess specific nucleation activities, though the mechanism underlying these differences is not well understood [26, 27].…”

Visualizing Molecules of FunctionalHumanProfilin

“…Profilin has strikingly different affinities for actin monomers (k D = 100 nM) and for the growing ends of actin filaments (k D = 225 µM) (Courtemanche and Pollard, 2013;Pernier et al, 2016;Funk et al, 2019;Pimm et al, 2020;Zweifel and Courtemanche, 2020). This property permits the effective disassociation of profilin from the ends of growing actin filament during polymerization (Courtemanche and Pollard, 2013;Pernier et al, 2016;Zweifel and Courtemanche, 2020). Many of the actin-assembly roles of profilin revolve around its ability to bind actin monomers.…”

“…Many of the actin-assembly roles of profilin revolve around its ability to bind actin monomers. However at high stochiometric concentrations, profilin binds actin filaments and may interfere with actin elongation (Jégou et al, 2011;Courtemanche and Pollard, 2013;Funk et al, 2019;Zweifel and Courtemanche, 2020). We performed two-color TIRF microscopy to visualize the localization of mApple-profilin with polymerizing actin filaments (Figure S3A).…”

“…Combined, these activities effectively regulate many properties of the actin monomer pool and bias actin polymerization in favor of straight filament architectures (Henty-Ridilla and Goode, 2015; Rotty et al, 2015;Suarez et al, 2015;Suarez and Kovar, 2016;Skruber et al, 2018). Despite similar concentrations, not all cellular profilin is bound to actin monomers (Goldschmidt-Clermont et al, 1992; Kaiser et al, 1999;Skruber et al, 2018Skruber et al, , 2020Zweifel and Courtemanche, 2020). Some profilin outcompetes actin bound to intracellular lipids present on membranes and vesicles to transmit distinct cellular signals (Lass-ing and Lindberg, 1985; Davey and Moens, 2020).…”