Abstract
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 Gα∙GDP, GPR-1/2Pins/LGN, and LIN-5Mud/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 Gα and GPR-1/2 with a light-inducible membrane anchor, we demonstrate that Gα∙GDP, Gα∙GTP, and GPR-1/2 are not required for pulling-force generation. In the absence of Gα 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 cell-cleavage plane. Our results define Gα∙GDP-GPR-1/2Pins/LGN as a regulatable membrane anchor, and LIN-5Mud/NuMA as a potent activator of dynein-dependent spindle-positioning forces.
Highlights
Animal cells control the position of the spindle to determine the plane of cell cleavage
Our previous studies and CRISPR/Cas9-assisted endogenous tagging demonstrated that cytoplasmic dynein and the G –GPR-1/2–LIN-5 complex overlap and function together a in pulling force generation at the cell cortex of C. elegans early blastomeres (Figure 1b)
Recent advances in CRISPR/Cas9-mediated genome engineering and optogenetics hold far-reaching potential for cell and developmental biology (Johnson and Toettcher, 2018; Waaijers and Boxem, 2014). We combined these strategies to systematically control the localization of endogenous proteins in the C. elegans early embryo by light-induced ePDZ–LOV heterodimerization, to determine their individual contributions in spindle positioning
Summary
Animal cells control the position of the spindle to determine the plane of cell cleavage. Work in C. elegans demonstrated that cortical pulling forces position the spindle through a protein complex that consists of a heterotrimeric G protein alpha subunit, GOA-1Gao or GPA-16Gai (together referred to as G ), a TPR-GoLoco domain protein GPR-1/2, and the coiled-coil a protein LIN-5 (Colombo et al, 2003; Gotta and Ahringer, 2001; Gotta et al, 2003; Grill et al, 2001; Lorson et al, 2000; Miller and Rand, 2000; Srinivasan et al, 2003) This complex, and the closely related Drosophila Gai/o–Pins–Mud and mammalian Gai/o–LGN–NuMA protein complexes, recruit the microtubule motor dynein to the cell cortex (Bellaıche et al, 2001; Bowman et al, 2006; Du and Macara, 2004; Du et al, 2001; Izumi et al, 2004; Nguyen-Ngoc et al, 2007; Schaefer et al, 2001; Zheng et al, 2010; Zhu et al, 2011) (Figure 1a).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
