Optogenetics reveals Cdc42 local activation by scaffold-mediated positive feedback and Ras GTPase
Optogenetics reveals Cdc42 local activation by scaffold-mediated positive feedback and Ras GTPase
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DOI：
10.1371/journal.pbio.3000600
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发表时间：
2020-01-01
期刊：
影响因子：
9.8
通讯作者：
Martin, Sophie G.
中科院分区：
文献类型：
--
作者：
Lamas, Iker;Merlini, Laura;Martin, Sophie G.
小 GTPase Cdc42 的局部活性对于细胞极化至关重要。虽然支架介导的正反馈被认为可以打破芽殖酵母细胞的对称性并产生单个 Cdc42 活性区，但尚未在其他生物体中探究类似调节的存在。在这里，我们使用裂殖酵母裂殖酵母的杆状细胞来解决这个问题，该细胞在两个细胞极都表现出活性 Cdc42-GTP 区域。我们实施了 CRY2-CIB1 光遗传学系统，用于将急性光依赖性蛋白招募到质膜上，从而可以直接证明正反馈。事实上，组成型活性 Cdc42 的光遗传学募集导致鸟嘌呤核苷酸交换因子 (GEF) Scd1 和内源性 Cdc42 的共同募集，其方式依赖于支架蛋白 Scd2。我们证明，当正反馈通过 GEF 和 Cdc42 效应器 p21 激活激酶 1 (Pak1) 之间的工程相互作用发挥作用时，Scd2 功能是可有可无的。值得注意的是，这种重新连接的正反馈赋予细胞活力，并允许细胞形成 2 个活性 Cdc42 区域，即使缺乏其他必需的 Cdc42 激活剂也是如此。这些细胞进一步揭示，小 GTPase Ras1 在定位 GEF Scd1 和促进其活性方面发挥着作用，从而增强了正反馈。我们得出的结论是，由 Ras 活性门控的支架介导的正反馈为杆状形成提供了强大的极化。
Local activity of the small GTPase Cdc42 is critical for cell polarization. Whereas scaffold-mediated positive feedback was proposed to break symmetry of budding yeast cells and produce a single zone of Cdc42 activity, the existence of similar regulation has not been probed in other organisms. Here, we address this problem using rod-shaped cells of fission yeast Schizosaccharomyces pombe, which exhibit zones of active Cdc42-GTP at both cell poles. We implemented the CRY2-CIB1 optogenetic system for acute light-dependent protein recruitment to the plasma membrane, which allowed to directly demonstrate positive feedback. Indeed, optogenetic recruitment of constitutively active Cdc42 leads to co-recruitment of the guanine nucleotide exchange factor (GEF) Scd1 and endogenous Cdc42, in a manner dependent on the scaffold protein Scd2. We show that Scd2 function is dispensable when the positive feedback operates through an engineered interaction between the GEF and a Cdc42 effector, the p21-activated kinase 1 (Pak1). Remarkably, this rewired positive feedback confers viability and allows cells to form 2 zones of active Cdc42 even when otherwise essential Cdc42 activators are lacking. These cells further revealed that the small GTPase Ras1 plays a role in both localizing the GEF Scd1 and promoting its activity, which potentiates the positive feedback. We conclude that scaffold-mediated positive feedback, gated by Ras activity, confers robust polarization for rod-shape formation.