Identification of critical domains and putative partners for the<Emphasis Type="Italic"> Caenorhabditis elegans</Emphasis> spindle component LIN-5 |
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| Authors: | R?Fisk Green M?Lorson A?J?M?Walhout M?Vidal Email author" target="_blank">S?van den?HeuvelEmail author |
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| Institution: | (1) Massachusetts General Hospital Cancer Center (Bldg. 149), Harvard Medical School, 13th Street, Charlestown, MA 02129, USA;(2) Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA;(3) Present address: Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211, USA;(4) Present address: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA |
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| Abstract: | 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.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by C. P. Hollenberg |
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| Keywords: | lin-5 Two-hybrid analysis Caenorhabditis elegans Mitotic spindle
gpr-1/gpr-2 |
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