Data for: Growth cone advance requires EB1 as revealed by genomic replacement with a light-sensitive variant

A challenge in analyzing dynamic intracellular cell biological processes is the dearth of methodologies that are sufficiently fast and specific to perturb intracellular protein activities. We previously developed a light-sensitive variant of the microtubule plus end-tracking protein EB1 by inserting a blue light-controlled protein dimerization module between functional domains. Here, we describe an advanced method to replace endogenous EB1 with this light-sensitive variant in a single genome editing step, thereby enabling this approach in human induced pluripotent stem cells (hiPSCs) and hiPSC-derived neurons. We demonstrate that acute and local optogenetic EB1 inactivation in developing cortical neurons induces microtubule depolymerization in the growth cone periphery and subsequent neurite retraction. In addition, advancing growth cones are repelled from areas of blue light exposure. These phenotypes were independent of the neuronal EB1 homolog EB3, revealing a direct dynamic role of ..., This dataset contains raw data and analysis used to produce figures and conclusions associated with bioRxiv/eLife manuscript. Detailed description of the data collection and analysis methodology can be found in the associated manuscript., Raw microscopy data are provided as Nikon ND2 files that are native to Nikon Elements microscopy software. ND2 files can also be opened with ImageJ/Fiji with the Bioformats plugin (Note: metadata may not be imported correctly into ImageJ), or with a freely available Nikon Elements Viewer.
Data analysis on which manuscript figures are based are provided as Microsoft Excel spreadsheets.
Raw sequence data are provided in AB1 and SEQ file formats.

分析动态细胞内生物学过程的一大核心挑战，在于缺乏足够快速且特异性充足的细胞内蛋白质活性扰动方法。我们此前曾在功能结构域之间插入蓝光调控的蛋白质二聚化模块，开发出微管正端跟踪蛋白EB1（microtubule plus end-tracking protein EB1）的光敏感变体。本文介绍一种先进方法，可通过单步基因组编辑将内源性EB1替换为该光敏感变体，从而实现在人类诱导多能干细胞（human induced pluripotent stem cells, hiPSCs）及其分化获得的神经元中应用该技术。我们证实，在发育中的皮层神经元内实施急性局部光遗传EB1失活，可诱导生长锥周边区域的微管解聚，并引发后续的神经突回缩。此外，处于推进状态的生长锥会被蓝光照射区域排斥。这些表型与神经元EB1同源蛋白EB3无关，揭示了……本数据集包含用于生成bioRxiv/eLife关联手稿的图表与结论的原始数据及分析结果。数据收集与分析方法的详细描述可参阅该关联手稿。原始显微数据以尼康ND2文件格式提供，该格式为尼康Elements显微软件的原生文件格式。ND2文件也可通过搭载Bioformats插件的ImageJ/Fiji打开（注意：元数据可能无法正确导入ImageJ），或通过免费提供的尼康Elements Viewer打开。支撑手稿图表的数据分析结果以Microsoft Excel电子表格格式提供。原始测序数据以AB1和SEQ文件格式提供。