Pulling apart mitotic spindle positioning in the C. elegans early embryo

Abstract

Development and homeostasis of tissues and organisms depend on correct execution of cell division. To control the size, content, and position of daughter cells, the mother cell’s cleavage plane positiong is tightly controlled. The cell cleavage plane is instructed by the mitotic spindle, a microtubule-based structure that segregates the DNA over the daughter cells at the end of mitosis. The spindle (and thereby the cleavage plane) is positioned by cortical pulling forces exerted from the cell cortex on astral microtubules. The generation of pulling forces depends on dynamic astral microtubules, anchored to the cortex by a complex known as the cortical force generator (FG). The FG consists of a molecular switch Gα as a membrane anchor, which in its GDP-bound state can bind to GoLoco protein GPR-1,2 (Pins/LGN). GPR-1,2 can bind to the coiled-coil protein LIN-5 (Mud/NuMA), which interacts with the dynein motor protein complex that captures microtubule ends. For our studies we used the C. elegans early embryo, which undergoes highly reproducible asymmetric cell divisions. By using advanced fluorescent microscopy and genetic tools, we have studied this process in detail, focusing on the individual roles of the FG components. We identified the minimal binding region of LIN-5 in GPR-1,2. Using CRISPR/Cas9 technology, we studied the in vivo relevance of this region and the phosphorylation of 25 LIN-5 residues. We also identified the responsible kinase for some of these residues. These include cell cycle-, polarity-, and intracellular signalling kinases. We found that the protein APR-1 (tumour suppressor APC) is asymmetrically localized in the C. elegans early embryo. This asymmetry is regulated by PAR cell-polarity and results in an asymmetry in cortical microtubule dynamics. Using 3D modelling and numerical simulations, we were able to show compelling evidence that this contributes to asymmetric spindle positioning. The arrival of genome editing tools like CRISPR/Cas9 facilitates the detailed study of endogenous proteins in vivo. By tagging LIN-5 and dynein with fluorescent proteins, we could study their dynamics during asymmetric cell division. We uncovered two distinct populations of dynein at the cell cortex that contribute to cortical pulling forces and asymmetric cell division. One population depends on the microtubule end-binding proteins and serves as a back-up mechanism if dynein’s function is impaired. The other population depends on LIN-5 and is asymmetrically regulated downstream of PAR polarity. In combination with optogenetic heterodimerzation tools, CRISPR/Cas9 enables the controlled subcellular localization of endogenous proteins. To promote transgene expression, we developed a novel sequence optimization algorithm. We used these tools to reconstruct cortical force generators in vivo. We found that cortical dynein itself is insufficient for force generation, meaning that the LIN-5 complex is essential for activation of dynein-dependent spindle positioning. We also showed that the regulated Gα GDP/GTP hydrolysis cycle is essential for force generation, using controlled double knock-out of endogenous genes. These tools allowed us to optogenetically control the position of the spindle and cell cleavage plane in vivo. In summary, our work provides promising new techniques for the community and presents detailed novel insights in the mechanisms of mitotic spindle positioning.