Abstract
INF2 (inverted formin 2) is a formin protein with unique biochemical effects on actin. In addition to the common formin ability to accelerate actin nucleation and elongation, INF2 can also sever filaments and accelerate their depolymerization. Although we understand key attributes of INF2-mediated severing, we do not understand the mechanism by which INF2 accelerates depolymerization subsequent to severing. Here, we show that INF2 can create short filaments (<60 nm) that continuously turn over actin subunits through a combination of barbed end elongation, severing, and WH2 motif-mediated depolymerization. This pseudo-steady state condition occurs whether starting from actin filaments or monomers. The rate-limiting step of the cycle is nucleotide exchange of ADP for ATP on actin monomers after release from the INF2/actin complex. Profilin addition has two effects: 1) to accelerate filament turnover 6-fold by accelerating nucleotide exchange and 2) to shift the equilibrium toward polymerization, resulting in longer filaments. In sum, our findings show that the combination of multiple interactions of INF2 with actin can work in concert to increase the ATP turnover rate of actin. Depending on the ratio of INF2:actin, this increased flux can result in rapid filament depolymerization or maintenance of short filaments. We also show that high concentrations of cytochalasin D accelerate ATP turnover by actin but through a different mechanism from that of INF2.
Highlights
The formin INF2 can accelerate both actin polymerization and depolymerization
The abundant actin monomer binding protein, profilin, has multiple effects on dynamics: 1) it inhibits pointed end addition of ATP-actin monomers [13,14,15], focusing elongation to the barbed end; 2) it inhibits spontaneous nucleation of actin monomers [13], thereby forcing de novo filament assembly to be triggered by nucleation factors such as Arp2/3 complex, formins, or COWs [16, 17]; and 3) it accelerates nucleotide exchange on actin monomers (18 –20), allowing recently depolymerized ADP-actin monomers to recharge with ATP for subsequent rounds of polymerization
INF2 Causes Continuous ATP Turnover on Actin after Apparent Depolymerization—Previously, we showed that an INF2 construct containing the formin homology 1 (FH1), formin homology 2 (FH2), and C-terminal domains (FFC; Fig. 1) potently severed and depolymerized filaments [23, 25]
Summary
The formin INF2 can accelerate both actin polymerization and depolymerization. Results: ATP hydrolysis continues even after apparent complete actin depolymerization by INF2, and profilin accelerates the process. The abundant actin monomer binding protein, profilin, has multiple effects on dynamics: 1) it inhibits pointed end addition of ATP-actin monomers [13,14,15], focusing elongation to the barbed end; 2) it inhibits spontaneous nucleation of actin monomers [13], thereby forcing de novo filament assembly to be triggered by nucleation factors such as Arp2/3 complex, formins, or COWs (compound WH2-containing proteins) [16, 17]; and 3) it accelerates nucleotide exchange on actin monomers (18 –20), allowing recently depolymerized ADP-actin monomers to recharge with ATP for subsequent rounds of polymerization. In the presence of profilin, this process results in the assembly of highly dynamic filaments of relatively uniform filaments length, providing a potential mechanism for assembly of short, transient actin filaments
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