Spatiotemporal control of small GTPases with light using the LOV domain.
Spatiotemporal control of small GTPases with light using the LOV domain.
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DOI：
10.1016/b978-0-12-385075-1.00016-0
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发表时间：
2011
影响因子：
--
通讯作者：
Hahn, Klaus M.
中科院分区：
文献类型：
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作者：
Wu, Yi I.;Wang, Xiaobo;He, Li;Montell, Denise;Hahn, Klaus M.
生命系统中的信号网络通过亚细胞区室化和精确的激活时间来协调。这些时空方面确保了信号的保真度，同时有助于下游事件的多样性。这是通过开发分子工具来研究的，这些工具在生命系统中产生局部和精确定时的蛋白质活性。为了研究负责真实的时间细胞骨架变化的分子事件，我们产生了Rho家族GTP酶的版本，其与下游效应子的相互作用受光控制。将GTP酶接枝到向光素LOV（光、氧或电压）结构域。拟南芥NPH 1：一种具有推定氧化还原敏感结构域的蛋白激酶。Science 278，2120-2123.）通过LOV C末端的α螺旋。基因编码的光活化Rac控制活细胞的运动性。Nature 461，104-108.）。LOV结构域在空间上阻断GTdR活性位点，直到其被照射。暴露于400-500 nm的光引起连接LOV结构域与GT酶的螺旋解旋，从而缓解空间抑制。这种变化是可逆的，可重复的，只要关掉灯，蛋白质就可以恢复到它的非活性状态。LOV结构域包含黄素作为活性发色团。这种天然存在的分子在细胞或动物中表达LOV融合体时简单地掺入，允许在不同系统中容易地控制GTdR功能。在培养的单细胞中，光激活的Rac导致膜褶皱、突起和迁移。在果蝇卵巢中的集体迁移边缘细胞中，单个细胞中的光活化Rac（PA-Rac）的局灶性激活足以重定向整个组。单个细胞中的PA-Rac还挽救了整个组中内源性指导受体信号传导丧失引起的表型。这些发现表明，边界细胞簇内的细胞进行通信，并集体指导。在这里，我们使用详细的例子描述了PA-Rac的优化和应用，我们希望这些例子能够帮助其他人将该方法应用于不同的蛋白质和各种不同的细胞，组织和生物体。
Signaling networks in living systems are coordinated through subcellular compartmentalization and precise timing of activation. These spatiotemporal aspects ensure the fidelity of signaling while contributing to the diversity and of downstream events. This is studied through development of molecular tools that generate localized and precisely timed protein activity in living systems. To study the molecular events responsible for cytoskeletal changes in real time, we generated versions of Rho family GTPases whose interactions with downstream effectors is controlled by light. GTPases were grafted to the phototropin LOV (light, oxygen, or voltage) domain. Arabidopsis NPH1: A protein kinase with a putative redox-sensing domain. Science 278, 2120–2123.) via an alpha helix on the LOV C-terminus. A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461, 104–108.). The LOV domain sterically blocked the GTPase active site until it was irradiated. Exposure to 400–500 nm light caused unwinding of the helix linking the LOV domain to the GTPase, relieving steric inhibition. The change was reversible and repeatable, and the protein could be returned to its inactive state simply by turning off the light. The LOV domain incorporates a flavin as the active chromophore. This naturally occurring molecule is incorporated simply upon expression of the LOV fusion in cells or animals, permitting ready control of GTPase function in different systems. In cultured single cells, light-activated Rac leads to membrane ruffling, protrusion, and migration. In collectively migrating border cells in the Drosophila ovary, focal activation of photoactivatable Rac (PA-Rac) in a single cell is sufficient to redirect the entire group. PA-Rac in a single cell also rescues the phenotype caused by loss of endogenous guidance receptor signaling in the whole group. These findings demonstrate that cells within the border cell cluster communicate and are guided collectively. Here, we describe optimization and application of PA-Rac using detailed examples that we hope will help others apply the approach to different proteins and in a variety of different cells, tissues, and organisms.