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.

中文翻译：

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光遗传学揭示了通过支架介导的正反馈和Ras GTPase对Cdc42的局部激活。

小GTPase Cdc42的局部活性对于细胞极化至关重要。尽管提出了支架介导的正反馈以破坏发芽酵母细胞的对称性并产生Cdc42活性的单个区域，但尚未在其他生物中探究类似调控的存在。在这里，我们使用裂殖酵母粟酒裂殖酵母的杆状细胞解决了这个问题，所述杆状细胞在两个细胞极均显示出活性Cdc42-GTP区域。我们实施了CRY2-CIB1光遗传系统，将急性光依赖性蛋白募集到质膜上，从而可以直接证明阳性反馈。确实，组成性活性Cdc42的光遗传募集导致鸟嘌呤核苷酸交换因子（GEF）Scd1和内源性Cdc42的共募集，其方式取决于支架蛋白Scd2。我们显示，当正反馈通过GEF和Cdc42效应子，p21激活的激酶1（Pak1）之间的工程相互作用而起作用时，Scd2功能是可有可无的。值得注意的是，这种重新接线的正反馈赋予了活力，即使缺少其他必需的Cdc42激活剂，细胞也可以形成2个活性Cdc42区域。这些细胞进一步揭示了小GTPase Ras1在定位GEF Scd1和促进其活性中都发挥了作用，从而增强了正反馈。我们得出的结论是，由Ras活性控制的脚手架介导的正反馈赋予杆状形成强大的极化作用。这种重新接线的正反馈赋予了活力，即使缺少其他必需的Cdc42激活剂，细胞也可以形成2个活性Cdc42区域。这些细胞进一步揭示了小GTPase Ras1在定位GEF Scd1和促进其活性中都发挥了作用，从而增强了正反馈。我们得出的结论是，由Ras活性控制的支架介导的正反馈赋予杆形形成鲁棒的极化作用。这种重新接线的正反馈赋予了活力，即使缺少其他必需的Cdc42激活剂，细胞也可以形成2个活性Cdc42区域。这些细胞进一步揭示了小GTPase Ras1在定位GEF Scd1和促进其活性中都发挥了作用，从而增强了正反馈。我们得出的结论是，由Ras活性控制的支架介导的正反馈赋予杆形形成鲁棒的极化作用。