Department of Neuroscience and Cell Biology, Yale University School of Medicine, New Haven, United States.
Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, United States.
Elife. 2020 Nov 11;9:e63230. doi: 10.7554/eLife.63230.
Light-inducible dimerization protein modules enable precise temporal and spatial control of biological processes in non-invasive fashion. Among them, Magnets are small modules engineered from the photoreceptor Vivid by orthogonalizing the homodimerization interface into complementary heterodimers. Both Magnets components, which are well-tolerated as protein fusion partners, are photoreceptors requiring simultaneous photoactivation to interact, enabling high spatiotemporal confinement of dimerization with a single excitation wavelength. However, Magnets require concatemerization for efficient responses and cell preincubation at 28°C to be functional. Here we overcome these limitations by engineering an optimized Magnets pair requiring neither concatemerization nor low temperature preincubation. We validated these 'enhanced' Magnets (eMags) by using them to rapidly and reversibly recruit proteins to subcellular organelles, to induce organelle contacts, and to reconstitute OSBP-VAP ER-Golgi tethering implicated in phosphatidylinositol-4-phosphate transport and metabolism. eMags represent a very effective tool to optogenetically manipulate physiological processes over whole cells or in small subcellular volumes.
光诱导二聚体蛋白模块能够以非侵入性的方式精确控制生物过程的时间和空间。其中，Magnets 是从小型光受体 Vivid 中设计出来的模块，通过将同源二聚化界面正交化为互补异二聚体，实现了对生物过程的精确控制。这两个 Magnets 组件都可以作为蛋白质融合伴侣很好地耐受，并且都是需要同时光激活才能相互作用的光受体，从而能够以单一激发波长实现高时空限制的二聚化。然而，Magnets 需要串联化才能产生有效的响应，并且需要在 28°C 下进行细胞预孵育才能发挥作用。在这里，我们通过设计一对不需要串联化和低温预孵育的优化 Magnets 对来克服这些限制。我们通过使用这些优化后的 Magnets (eMags) 来快速可逆地将蛋白质募集到亚细胞细胞器，诱导细胞器接触，以及重建涉及磷脂酰肌醇-4-磷酸转运和代谢的 OSBP-VAP ER-Golgi 连接，验证了这些 eMags 的有效性。eMags 是一种非常有效的工具，可以在整个细胞或小亚细胞体积中对生理过程进行光遗传学操作。
