Abstract
During cell division, the mitotic spindle segregates replicated chromosomes to opposite poles of the cell, while the position of the spindle determines the plane of cleavage. Spindle positioning and chromosome segregation depend on pulling forces on microtubules extending from the centrosomes to the cell cortex. Critical in pulling force generation is the cortical anchoring of cytoplasmic dynein by a conserved ternary complex of Gα, GPR-1/2, and LIN-5 proteins in C. elegans (Gα–LGN–NuMA in mammals). Previously, we showed that the polarity kinase PKC-3 phosphorylates LIN-5 to control spindle positioning in early C. elegans embryos. Here, we investigate whether additional LIN-5 phosphorylations regulate cortical pulling forces, making use of targeted alteration of in vivo phosphorylated residues by CRISPR/Cas9-mediated genetic engineering. Four distinct in vivo phosphorylated LIN-5 residues were found to have critical functions in spindle positioning. Two of these residues form part of a 30 amino acid binding site for GPR-1, which we identified by reverse two-hybrid screening. We provide evidence for a dual-kinase mechanism, involving GSK3 phosphorylation of S659 followed by phosphorylation of S662 by casein kinase 1. These LIN-5 phosphorylations promote LIN-5–GPR-1/2 interaction and contribute to cortical pulling forces. The other two critical residues, T168 and T181, form part of a cyclin-dependent kinase consensus site and are phosphorylated by CDK1-cyclin B in vitro. We applied a novel strategy to characterize early embryonic defects in lethal T168,T181 knockin substitution mutants, and provide evidence for sequential LIN-5 N-terminal phosphorylation and dephosphorylation in dynein recruitment. Our data support that phosphorylation of multiple LIN-5 domains by different kinases contributes to a mechanism for spatiotemporal control of spindle positioning and chromosome segregation.
Highlights
Animal development and tissue homeostasis depend critically on cell divisions that create cells with specific shapes and functions, in the right numbers and at the proper positions
C. elegans LIN-5 participates in chromosome segregation and is essential for positioning the spindle and cell cleavage plane during asymmetric cell division
Our current analysis reveals four additional phosphorylated residues that are critical for LIN-5 function. Two of these residues contribute to the interaction of LIN-5 with its binding partner GPR-1/2, whereas the other two residues are critical in dynein motor recruitment by LIN-5
Summary
Animal development and tissue homeostasis depend critically on cell divisions that create cells with specific shapes and functions, in the right numbers and at the proper positions. The spindle apparatus plays a central role in the cell division process, as it segregates the chromosomes in mitosis and determines the plane of cell cleavage during cytokinesis [1,2,3]. Asymmetric cell divisions create cell diversity and allow maintenance of tissue-specific stem cells, by combining self-renewal with the generation of differentiating daughter cells (Reviews: [4,5]). Tight control of the spindle function and position is needed to coordinate chromosome segregation with cleavage plane determination, which is essential for genetic stability, tissue integrity and stem cell maintenance in a wide variety of evolutionary contexts
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
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
