Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA.
Mol Biol Cell. 2012 Oct;23(19):3814-26. doi: 10.1091/mbc.E12-05-0400. Epub 2012 Aug 23.
Actin filaments are dynamically reorganized to accommodate ever-changing cellular needs for intracellular transport, morphogenesis, and migration. Formins, a major family of actin nucleators, are believed to function as direct effectors of Rho GTPases, such as the polarity regulator Cdc42p. However, the presence of extensive redundancy has made it difficult to assess the in vivo significance of the low-affinity Rho GTPase-formin interaction and specifically whether Cdc42p polarizes the actin cytoskeleton via direct formin binding. Here we exploit a synthetically rewired budding yeast strain to eliminate the redundancy, making regulation of the formin Bni1p by Cdc42p essential for viability. Surprisingly, we find that direct Cdc42p-Bni1p interaction is dispensable for Bni1p regulation. Alternative paths linking Cdc42p and Bni1p via "polarisome" components Spa2p and Bud6p are also collectively dispensable. We identify a novel regulatory input to Bni1p acting through the Cdc42p effector, Gic2p. This pathway is sufficient to localize Bni1p to the sites of Cdc42p action and promotes a polarized actin organization in both rewired and wild-type contexts. We suggest that an indirect mechanism linking Rho GTPases and formins via Rho effectors may provide finer spatiotemporal control for the formin-nucleated actin cytoskeleton.
肌动蛋白丝动态重组，以适应细胞内运输、形态发生和迁移等不断变化的细胞需求。成核因子（formin）是肌动蛋白成核的主要家族之一，被认为是 Rho GTPases（如极性调节剂 Cdc42p）的直接效应物。然而，广泛的冗余性使得评估低亲和力 Rho GTPase-成核因子相互作用的体内意义变得困难，特别是 Cdc42p 是否通过直接成核因子结合使肌动蛋白细胞骨架极化。在这里，我们利用一种人工重新布线的出芽酵母菌株来消除冗余性，使 Cdc42p 对成核因子 Bni1p 的调控成为生存所必需的。令人惊讶的是，我们发现 Cdc42p-Bni1p 相互作用对于 Bni1p 的调控是可有可无的。通过“极性体”成分 Spa2p 和 Bud6p 将 Cdc42p 和 Bni1p 连接起来的替代途径也是可有可无的。我们确定了一种作用于 Bni1p 的新型调节输入，通过 Cdc42p 效应物 Gic2p 发挥作用。该途径足以将 Bni1p 定位到 Cdc42p 作用的部位，并在重新布线和野生型环境中促进极化的肌动蛋白组织。我们认为，通过 Rho 效应物将 Rho GTPases 和成核因子间接连接起来的机制可能为成核因子引发的肌动蛋白细胞骨架提供更精细的时空控制。
