Growth cone advance requires EB1 as revealed by genomic replacement with a light-sensitive variant.
Growth cone advance requires EB1 as revealed by genomic replacement with a light-sensitive variant.
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DOI：
10.7554/elife.84143
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发表时间：
2023-01-30
期刊：
影响因子：
7.7
通讯作者：
Wittmann T
中科院分区：
文献类型：
--
作者：
Dema A;Charafeddine R;Rahgozar S;van Haren J;Wittmann T
分析动态细胞内细胞生物过程的一个挑战是缺乏足够快速且特异性干扰细胞内蛋白质活性的方法。我们之前通过在功能域之间插入蓝光控制的蛋白质二聚化模块，开发了微管加末端追踪蛋白 EB1 的光敏变体。在这里，我们描述了一种先进的方法，在单个基因组编辑步骤中用这种光敏变体取代内源性 EB1，从而在人类诱导多能干细胞 (hiPSC) 和 hiPSC 衍生的神经元中实现这种方法。我们证明，发育中的皮层神经元中的急性和局部光遗传学 EB1 失活会诱导生长锥周围的微管解聚以及随后的神经突回缩。此外，前进的生长锥会受到蓝光照射区域的排斥。这些表型独立于神经元 EB1 同源物 EB3，揭示了 EB1 介导的微管和末端相互作用在神经元形态发生和神经突引导中的直接动态作用。
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 EB1-mediated microtubule plus end interactions in neuron morphogenesis and neurite guidance.