We report the construction of a single-component optogenetic Rac1 (opto-Rac1) to control actin polymerization by dynamic membrane recruitment. Opto-Rac1 is a fusion of wildtype human Rac1 small GTPase to the C-terminal region of BcLOV4, a LOV (light-oxygen-voltage) photoreceptor that rapidly binds the plasma membrane upon blue-light activation via a direct electrostatic interaction with anionic membrane phospholipids. Translocation of the fused wildtype Rac1 effector permits its activation by GEFs (guanine nucleotide exchange factors) and consequent actin polymerization and lamellipodia formation, unlike in existing single-chain systems that operate by allosteric photo-switching of constitutively active Rac1 or the heterodimerization-based (i.e. two-component) membrane recruitment of a Rac1-activating GEF. Opto-Rac1 induction of lamellipodia formation was spatially restricted to the patterned illumination field and was efficient, requiring sparse stimulation duty ratios of ∼1-2% (at the sensitivity threshold for flavin photocycling) to cause significant changes in cell morphology. This work exemplifies how the discovery of LOV proteins of distinct signal transmission modes can beget new classes of optogenetic tools for controlling cellular function.

中文翻译：

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从膜脂质结合的RGS-LOV工程改造而来的光遗传Rac1，用于诱导型片状脂蛋白形成。

我们报告了通过动态膜募集来控制肌动蛋白聚合的单组分光遗传性Rac1（光-Rac1）的建设。Opto-Rac1是野生型人Rac1小GTP酶与BcLOV4的C端区域的融合体，BcLOV4是一种LOV（光氧电压）感光器，在蓝光激活后会通过与阴离子膜的直接静电相互作用迅速结合质膜。磷脂。融合的野生型Rac1效应子的易位使其可以被GEFs（鸟嘌呤核苷酸交换因子）激活，从而导致肌动蛋白聚合和片状脂蛋白形成，与现有的单链系统不同，该单链系统通过组成性活性Rac1的变构光开关或基于异源二聚化的（即Rac1激活的GEF的两组分膜募集。薄层脂蛋白形成的Opto-Rac1诱导在空间上受限于图案化的照明场，并且效率很高，要求稀疏的刺激占空比为1-2％（在黄素光循环的敏感度阈值下）以引起细胞形态的显着变化。这项工作例证了如何发现不同信号传输模式的LOV蛋白如何获得控制细胞功能的新型光遗传学工具。