European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Heidelberg 69117, Germany.
European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Heidelberg 69117, Germany.
Dev Cell. 2021 Aug 23;56(16):2348-2363.e8. doi: 10.1016/j.devcel.2021.07.011. Epub 2021 Aug 6.
Many developmental regulators have complex and context-specific roles in different tissues and stages, making the dissection of their function extremely challenging. As regulatory processes often occur within minutes, perturbation methods that match these dynamics are needed. Here, we present the improved light-inducible nuclear export system (iLEXY), an optogenetic loss-of-function approach that triggers translocation of proteins from the nucleus to the cytoplasm. By introducing a series of mutations, we substantially increased LEXY's efficiency and generated variants with different recovery times. iLEXY enables rapid (t < 30 s), efficient, and reversible nuclear protein depletion in embryos, and is generalizable to proteins of diverse sizes and functions. Applying iLEXY to the Drosophila master regulator Twist, we phenocopy loss-of-function mutants, precisely map the Twist-sensitive embryonic stages, and investigate the effects of timed Twist depletions. Our results demonstrate the power of iLEXY to dissect the function of pleiotropic factors during embryogenesis with unprecedented temporal precision.
许多发育调控因子在不同组织和阶段具有复杂且特定于背景的作用，使得解析它们的功能极具挑战性。由于调控过程通常在数分钟内发生，因此需要与这些动态相匹配的扰动方法。在这里，我们提出了改进的光诱导核输出系统（iLEXY），这是一种光遗传学的功能丧失方法，可触发蛋白质从细胞核到细胞质的易位。通过引入一系列突变，我们大大提高了 LEXY 的效率，并产生了具有不同恢复时间的变体。iLEXY 可在胚胎中快速（t < 30 s）、高效且可逆地耗尽核蛋白，并且可推广到各种大小和功能的蛋白质。将 iLEXY 应用于果蝇主要调控因子 Twist，我们可以模拟功能丧失突变体，精确映射 Twist 敏感的胚胎阶段，并研究定时 Twist 耗竭的影响。我们的结果证明了 iLEXY 具有前所未有的时间精度解析胚胎发生中多效因子功能的强大功能。
