Identification of critical domains and putative partners for the Caenorhabditis elegans spindle component LIN-5
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Successful cell division requires proper assembly, placement and functioning of the spindle apparatus that segregates the chromosomes. The Caenorhabditis elegans gene lin-5 encodes a novel coiled-coil component of the spindle required for spindle positioning and chromosome segregation. To gain further insights into lin-5 function, we screened for dominant suppressors of the partial loss-of-function phenotype associated with the mutation lin-5(ev571ts), and isolated 68 suppressing mutations. Eight out of the ten suppressors sequenced contained intragenic missense mutations immediately upstream of the lesion in lin-5(ev571ts). These probably help to stabilize protein-protein interactions mediated by the coiled-coil domain. This domain was found to be required for binding to several putative LIN-5 interacting (LFI) proteins identified in yeast two-hybrid screens. Interestingly, interaction with the coiled-coil protein LFI-1 was specifically reduced by the lin-5(ev571ts) mutation and restored by a representative intragenic suppressor mutation. Immunostaining experiments showed that LIN-5 and LFI-1 may co-localize around the kinetochore microtubules during metaphase, indicating potential interaction in vivo. The coiled-coil domain of LIN-5 was also found to mediate homodimerization, while the C-terminal region of LIN-5 was sufficient for interaction with GPR-1, a recently identified component of a LIN-5 spindle-regulatory complex. A single amino-acid substitution in the N-terminal region of LIN-5, encoded by the e1457 allele, abolished all LIN-5 interactions. Taken together, our results indicate that the spindle functions of LIN-5 depend on interactions with multiple protein partners, and that these interactions are mediated through several different domains of LIN-5.
Keywordslin-5 Two-hybrid analysis Caenorhabditis elegans Mitotic spindle gpr-1/gpr-2
We are indebted to D. Merz and J. Culotti for the gift of the ev571 allele, and are grateful to Brenda Schulman and members of P. Kim’s lab for advice and discussions on protein structure. M. Esteban and C. Goday are acknowledged for the gift of mAB403 antibodies and Yuji Kohara for several plasmids. We thank Audrey Perreault and Dayalan Srinivasan for experimental assistance, members of the Hart and van den Heuvel labs for helpful discussions, and Mike Boxem, Mako Saito and Dayalan Srinivasan for critically reading the manuscript. The Caenorhabditis Genetics Center, supported by the National Institutes of Health National Center for Research Resources, provided several strains for this work. This work was supported by grants from the National Institutes of Health and March of Dimes Birth Defects Foundation to S.v.d.H. M.L. received a grant from the Medical Foundation. R.F.G. was supported by a Howard Hughes Medical Institute Predoctoral Fellowship. This work has been carried out in compliance with the current laws governing genetic experimentation in the United States