Optogenetic dissection of mitotic spindle positioning in vivo

The position of the mitotic spindle determines the plane of cell cleavage, and thereby daughter cell location, size, and content. Spindle positioning is driven by dynein-mediated pulling forces exerted on astral microtubules, which requires an evolutionarily conserved complex of G alpha center dot GDP, GPR-1/2(Pins/LGN), and LIN-5(Mud/NuMA) proteins. To examine individual functions of the complex components, we developed a genetic strategy for light-controlled localization of endogenous proteins in C. elegans embryos. By replacing G alpha and GPR-1/2 with a light-inducible membrane anchor, we demonstrate that G alpha center dot GDP, G alpha center dot GTP, and GPR-1/2 are not required for pulling-force generation. In the absence of G a and GPR-1/2, cortical recruitment of LIN-5, but not dynein itself, induced high pulling forces. The light-controlled localization of LIN-5 overruled normal cell-cycle and polarity regulation and provided experimental control over the spindle and cellcleavage plane. Our results define G alpha center dot 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.