Identification and characterization of proteins interacting with the Caenorhabditis elegans spindle component LIN-5

During cell division, the mitotic spindle segregates chromosomes and determines the position of the cleavage furrow. Thus, spindle function and position are critical for both genetic stability and the generation of cells with different sizes and fates. To better understand these processes, I have studied the Caenorhabditis elegans lin-5 gene, which encodes a novel coiled-coil component of the spindle apparatus. Previous work in our lab established a role for lin-5 in both chromosome segregation and spindle positioning.; To gain further insight into the mechanism of lin-5 function, we used a combination of approaches to identify proteins that interact with LIN-5. Using biochemical techniques, we identified an interaction between LIN-5 and the paralogs GPR-1 and GPR-2 (G&barbelow; p&barbelow;rotein r&barbelow;egulator). GPR-1/GPR-2 contain a GoLoco/GPR domain which mediates interaction with Gα·GDP and depend upon LIN-5 for their localization to the mitotic spindle and cell cortex. Loss of LIN-5, GPR-1/GPR-2 or the two Gα homologs GOA-1 and GPA-16 results in similar defects in chromosome segregation and spindle positioning, indicating a role for LIN-5 and GPR-1/GPR-2 in G protein signaling. LIN-5 and GPR-1/GPR-2 appear to act downstream of spindle positioning signals from the PAR polarity proteins and the tyrosine kinase-related proteins MES-1 and SRC-1. These results indicate that LIN-5 and GPR-1/GPR-2 together activate G protein signaling to affect spindle force and may be regulated by polarity determinants.; We also used genetic and two-hybrid approaches to characterize regions important for LIN-5 function and to reveal other LIN-5 partners. Genetic screens identified the coiled-coil domain as a region important for LIN-5 function, and two-hybrid assays suggested that LIN-5 homodimerizes through this domain. LIN-5 binding to interactors isolated in a two-hybrid screen required both the LIN-5 coiled-coil and C-terminal domains, while binding to GPR-1/GPR-2 occurred through the C-terminus. Two-hybrid screening also identified another potential LIN-5 partner, LFI-1, which is homologous to PUMA1 (Parascaris univalens Mitotic Antigen) and localizes to the mitotic spindle and cortex. Furthermore, two-hybrid assays revealed a correlation between intact LIN-5 function and its ability to bind LFI-1, and studies of a lin-5 temperature-sensitive strain indicated that LIN-5 must have functions in addition to localizing GPR-1/GPR-2.