Optogenetics: optical control of a photoactivatable Rac in living cells.
Optogenetics: optical control of a photoactivatable Rac in living cells.
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DOI：
10.1007/978-1-4939-2080-8_15
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发表时间：
2015
影响因子：
--
通讯作者：
T. Yin;Yi I. Wu
中科院分区：
文献类型：
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作者：
T. Yin;Yi I. Wu
光遗传学的最新发展已经将信号传导的光学控制扩展到细胞内蛋白，包括Rac，Rho家族中的小G蛋白。将一个来自燕麦（Avena sativa）向光蛋白的蓝光敏感LOV（light，oxygen，or voltage）结构域通过一个在植物向光蛋白中保守的α螺旋（Jα）融合到一个组成型活性突变体Rac的N端。融合的LOV结构域在黑暗中封闭下游效应物与Rac的结合。蓝光照射引起LOV结构域的构象变化和Jα螺旋的解旋，从而缓解空间抑制。LOV结构域包含黄素作为光子吸收辅因子，并且可以通过光以可逆和可重复的方式激活。在培养的细胞中，蓝光的全局照明迅速激活Rac，并导致细胞铺展和膜皱褶。局部和脉冲照明产生梯度的Rac活动和诱导定向迁移。在这一章中，我们将详细描述的技术，并提出了一些应用程序的例子，使用光活化的Rac（PA-Rac）在活细胞。
Recent developments in optogenetics have extended optical control of signaling to intracellular proteins, including Rac, a small G protein in the Rho family. A blue light-sensing LOV (light, oxygen, or voltage) domain derived fromAvena sativa(oat) phototropin was fused to the N-terminus of a constitutively active mutant of Rac, via an α-helix (Jα) that is conserved among plant phototropins. The fused LOV domain occluded binding of downstream effectors to Rac in the dark. Exposure to blue light caused a conformational change of the LOV domain and unwinding of the Jα helix, relieving steric inhibition. The LOV domain incorporates a flavin as the photon-absorbing cofactor and can be activated by light in a reversible and repeatable fashion. In cultured cells, global illumination with blue light rapidly activated Rac and led to cell spreading and membrane ruffling. Localized and pulsed illumination generated a gradient of Rac activity and induced directional migration. In this chapter, we will describe the techniques in detail and present some examples of applications of using photoactivatable Rac (PA-Rac) in living cells.