Xu Yineng, Wang Bei, Bush Inle, Saunders Harriet Aj, Wildonger Jill, Han Chun
Weill Institute for Cell and Molecular Biology, Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.
Department of Biochemistry, University of Wisconsin-Madison, 440 Henry Mall, Madison, WI 53706, USA.
bioRxiv. 2023 Oct 2:2023.09.30.560303. doi: 10.1101/2023.09.30.560303.
Animal development involves numerous molecular events, whose spatiotemporal properties largely determine the biological outcomes. Conventional methods for studying gene function lack the necessary spatiotemporal resolution for precise dissection of developmental mechanisms. Optogenetic approaches are powerful alternatives, but most existing tools rely on exogenous designer proteins that produce narrow outputs and cannot be applied to diverse or endogenous proteins. To address this limitation, we developed OptoTrap, a light-inducible protein trapping system that allows manipulation of endogenous proteins tagged with GFP or split GFP. This system turns on fast and is reversible in minutes or hours. We generated OptoTrap variants optimized for neurons and epithelial cells and demonstrate effective trapping of endogenous proteins of diverse sizes, subcellular locations, and functions. Furthermore, OptoTrap allowed us to instantly disrupt microtubules and inhibit the kinesin-1 motor in specific dendritic branches of sensory neurons. Using OptoTrap, we obtained direct evidence that microtubules support the growth of highly dynamic dendrites. Similarly, targeted manipulation of Kinesin heavy chain revealed differential spatiotemporal requirements of kinesin-1 in the patterning of low- and high-order dendritic branches, suggesting that different cargos are needed for the growth of these branches. OptoTrap allows for precise manipulation of endogenous proteins in a spatiotemporal manner and thus holds great promise for studying developmental mechanisms in a wide range of cell types and developmental stages.
动物发育涉及众多分子事件，其时空特性在很大程度上决定了生物学结果。传统的基因功能研究方法缺乏精确剖析发育机制所需的时空分辨率。光遗传学方法是强大的替代方法，但大多数现有工具依赖于产生有限输出的外源设计蛋白，且无法应用于多种或内源性蛋白。为解决这一局限性，我们开发了OptoTrap，一种光诱导蛋白捕获系统，可用于操纵标记有绿色荧光蛋白（GFP）或分裂GFP的内源性蛋白。该系统开启迅速，在数分钟或数小时内即可逆转。我们生成了针对神经元和上皮细胞优化的OptoTrap变体，并证明其能有效捕获不同大小、亚细胞定位和功能的内源性蛋白。此外，OptoTrap使我们能够立即破坏微管，并在感觉神经元的特定树突分支中抑制驱动蛋白-1运动。利用OptoTrap，我们获得了直接证据，证明微管支持高度动态树突的生长。同样，对驱动蛋白重链的靶向操纵揭示了驱动蛋白-1在低阶和高阶树突分支模式形成中的不同时空需求，这表明这些分支的生长需要不同的货物。OptoTrap能够以时空方式精确操纵内源性蛋白，因此在研究广泛的细胞类型和发育阶段的发育机制方面具有巨大潜力。
