Chromosome segregation requires proper assembly and positioning of the mitotic spindle, established through a balance between microtubule dynamics and forces generated by microtubule-associated proteins. To further dissect the complicated process of chromosome segregation, we have focused on the
lin-5 gene in C. elegans. Animals mutant for
lin-5 fail chromosome alignment and segregation, and show mispositioning of the spindle in early embryonic divisions. These processes are all mediated by microtubules and microtubule-associated proteins. We thus hypothesize that
lin-5, which encodes a novel coiled-coil component of the spindle apparatus, affects spindle dynamics or force generation. However, the mechanisms by which
lin-5 functions remain unknown. To gain further insight into the function of
lin-5 , we decided to identify proteins that interact with LIN-5. In a two-hybrid screen, LIN-5 was found to interact with itself and five other proteins. RNAi of these interactors did not produce a Lin-5-like phenotype, nor did it enhance or suppress the defects in
lin-5(
ev571 ts ) mutants . As an alternative approach, we examined the two-hybrid interactions in the context of
lin-5 mutations, since such mutations may prevent binding to key partners. We introduced two mutations previously identified in vivo ,
ev571 ts and
e1457 , to determine whether they disrupted any two-hybrid interactions. The
e1457 product, though expressed in yeast, failed to bind any of the interactors. The
ev571 ts fusion protein showed decreased binding to three of the interactors. The loss of one or more of these interactions might be the cause of the
ev571 ts phenotype. Therefore, we tested a previously isolated intragenic
lin-5(
ev571 ts ) suppressor mutations for restoration of LIN-5 interactions. We found that predominantly binding to the interactor ZC8.4 was restored. ZC8.4 is predicted to encode a homolog of PUMA1, a component of the mitotic apparatus in the nematode Parascaris univalens. The correlation between intact
lin-5 function and its ability to bind ZC8.4 suggests in vivo relevance of this interaction. To further explore this possibility, we compared the localization of ZC8.4 and LIN-5. ZC8.4 staining coincided with that of LIN-5 at the centrosomes, kinetocore microtubules, and cell cortex. In contrast to the overlapping localization in mitosis, ZC8.4 also localized to the nucleus of interphase cells whereas LIN-5 remained cytoplasmic. Together, the two-hybrid data and the co-localization of ZC8.4 and LIN-5 suggest that an interaction between these two coiled-coil proteins plays a role in one or more of the mitotic functions of LIN-5.