Mahanta Biswaprakash, Courtemanche Naomi
Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, USA.
Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, USA.
J Biol Chem. 2025 Jan;301(1):108071. doi: 10.1016/j.jbc.2024.108071. Epub 2024 Dec 10.
Formins play crucial roles in actin polymerization by nucleating filaments and regulating their elongation. Formins bind the barbed ends of filaments via their dimeric FH2 domains, which step processively onto incoming actin subunits during elongation. Actin monomers can bind formin-bound barbed ends directly or undergo diffusion-mediated delivery through interactions with formin FH1 domains and profilin. Despite its fundamental importance, a clear mechanism governing processive FH2 stepping has remained elusive. In this study, we systematically characterized the polymerization behavior of the Saccharomyces cerevisiae formin Bni1p using in vitro reconstitution assays and stochastic simulations. We found that Bni1p assembles populations of filaments with lengths that depend nonlinearly on the rate of elongation. This processive behavior is dictated by a variable probability of dissociation that depends on the reaction conditions. Bni1p dissociates from barbed ends with a basal off-rate, which enables prolonged filament assembly over the course of a long lifetime at the barbed end. A bias toward FH1-mediated delivery as the dominant mechanism for polymerization curtails elongation by shortening the lifetime of the formin at the filament end. This facilitates the assembly of populations of filaments with similar average lengths, even when polymerization proceeds at different rates. Our results suggest a central role for formin FH1 domains in regulating processivity. The specific effects of FH1 domains on processivity are variable and likely tailored to the physiological function of each formin.
formin蛋白通过使肌动蛋白丝成核并调节其延伸，在肌动蛋白聚合过程中发挥关键作用。formin蛋白通过其二聚体FH2结构域结合肌动蛋白丝的带刺末端，在延伸过程中，该结构域逐步作用于进入的肌动蛋白亚基。肌动蛋白单体可以直接结合与formin蛋白结合的带刺末端，或者通过与formin蛋白的FH1结构域和脯氨酰肌动蛋白结合蛋白相互作用，以扩散介导的方式传递。尽管其具有根本重要性，但控制FH2结构域逐步移动的明确机制仍然难以捉摸。在本研究中，我们使用体外重组实验和随机模拟系统地表征了酿酒酵母formin蛋白Bni1p的聚合行为。我们发现，Bni1p组装的肌动蛋白丝群体的长度非线性地依赖于延伸速率。这种持续行为由解离的可变概率决定，该概率取决于反应条件。Bni1p以基础解离速率从带刺末端解离，这使得在带刺末端的长寿命过程中能够长时间组装肌动蛋白丝。作为聚合的主要机制，偏向于FH1介导的传递会通过缩短formin蛋白在丝末端的寿命来减少延伸。即使聚合以不同速率进行，这也有助于组装平均长度相似的肌动蛋白丝群体。我们的结果表明formin蛋白的FH1结构域在调节持续性方面发挥核心作用。FH1结构域对持续性的具体影响是可变的，并且可能根据每种formin蛋白的生理功能进行调整。
