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Optogenetic dissection of mitotic spindle positioning in vivo

By Lars-Eric Fielmich, Ruben Schmidt, Daniel J. Dickinson, Bob Goldstein, Anna A. Akhmanova, Sander J.L. van den Heuvel

Posted 14 May 2018
bioRxiv DOI: 10.1101/319772 (published DOI: 10.7554/elife.38198)

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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