Molecular Genetics and Genomics

, Volume 271, Issue 5, pp 532–544 | Cite as

Identification of critical domains and putative partners for the Caenorhabditis elegans spindle component LIN-5

  • R. Fisk Green
  • M. Lorson
  • A. J. M. Walhout
  • M. Vidal
  • S. van den Heuvel
Original Paper

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.

Keywords

lin-5 Two-hybrid analysis  Caenorhabditis elegans Mitotic spindle  gpr-1/gpr-2 

Supplementary material

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Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • R. Fisk Green
    • 1
  • M. Lorson
    • 1
    • 3
  • A. J. M. Walhout
    • 2
    • 4
  • M. Vidal
    • 2
  • S. van den Heuvel
    • 1
  1. 1.Massachusetts General Hospital Cancer Center (Bldg. 149)Harvard Medical SchoolCharlestownUSA
  2. 2.Department of Cancer BiologyDana-Farber Cancer InstituteBostonUSA
  3. 3.Department of Veterinary Pathobiology, College of Veterinary MedicineUniversity of MissouriColumbiaUSA
  4. 4.Program in Molecular MedicineUniversity of Massachusetts Medical SchoolWorcesterUSA

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