Optogenetic Rac1 engineered from membrane lipid-binding RGS-LOV for inducible lamellipodia formation
Optogenetic Rac1 engineered from membrane lipid-binding RGS-LOV for inducible lamellipodia formation
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DOI：
10.1039/c9pp00434c
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发表时间：
2020-03-01
影响因子：
3.1
通讯作者：
Chow, Brian Y.
中科院分区：
文献类型：
--
作者：
Berlew, Erin E.;Kuznetsov, Ivan A.;Chow, Brian Y.
我们报道了单组分光遗传Rac1 （optorac1）的构建，通过动态膜募集来控制肌动蛋白聚合。Opto-Rac1是野生型人类Rac1小GTPase与BcLOV4 c端区域的融合，BcLOV4是一种光氧电压（LOV）光感受器，通过与阴离子膜磷脂的直接静电相互作用，在蓝光激活时迅速结合质膜。融合野生型Rac1效应物的易位允许其被GEF（鸟嘌呤核苷酸交换因子）激活，随后的肌动蛋白聚合和板脚形成，而不像现有的单链系统，通过组成活性Rac1的变构光开关或基于异源二聚化（即双组分）的膜募集Rac1激活GEF。Opto-Rac1对板足形成的诱导在空间上仅限于有图案的光照场，并且是有效的，需要类似于1-2%的稀疏刺激占空比（在黄素光循环的敏感阈值）才能引起细胞形态的显著变化。这项工作举例说明了不同信号传输模式的LOV蛋白的发现如何能够产生新的光遗传学工具来控制细胞功能。
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 similar to 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.