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Optogenetic dissection of mitotic spindle positioning in vivo
Daniel J. Dickinson,
Anna A. Akhmanova,
Sander J.L. van den Heuvel
Posted 14 May 2018
bioRxiv DOI: 10.1101/319772 (published DOI: 10.7554/elife.38198)
Posted 14 May 2018
The position of the mitotic spindle determines the plane of cell cleavage, and thereby the location, size, and content of daughter cells. Spindle positioning is driven by dynein-mediated pulling forces exerted on astral microtubules. This process requires an evolutionarily conserved complex of α-GDP, GPR-1,2/Pins/LGN, and LIN-5/Mud/NuMA proteins. It remains unknown whether this complex merely forms a membrane anchor for dynein, or whether the individual components have additional functions, for instance through Gα-GTP or dynein activation. To functionally dissect this system, we developed a genetic strategy for light-controlled localization of endogenous proteins in C. elegans embryos. Controlled germline expression and membrane recruitment of the Gα; regulators RIC-8/Ric-8A and RGS-7/Loco/RGS3, and replacement of Gα with a light-inducible membrane anchor demonstrated that Gα-GTP signaling is dispensable for pulling force generation. In the absence of Gα, cortical recruitment of GPR-1,2 or LIN-5, but not dynein itself, induced high pulling forces. Local recruitment of LIN-5 overruled normal cell-cycle and polarity regulation, and provided experimental control over the spindle and cell cleavage plane. Our results define Gα-GDP-GPR-1,2/Pins/LGN as a regulatable membrane anchor, and LIN-5/Mud/NuMA as a potent activator of dynein-dependent spindle positioning forces. This study also highlights the possibilities for optogenetic control of endogenous proteins within an animal system.
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