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


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.


lin-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

Supplementary material

supp.pdf (415 kb)
(PDF 415 KB)


  1. Albertson DG, Sulston JE, White JG (1978) Cell cycling and DNA replication in a mutant blocked in cell division in the nematode Caenorhabditis elegans. Dev Biol 63:165–178PubMedGoogle Scholar
  2. Berger B, Wilson DB, Wolf E, Tonchev T, Milla M, Kim PS (1995) Predicting coiled coils by use of pairwise residue correlations. Proc Natl Acad Sci USA 92:8259–8263PubMedGoogle Scholar
  3. Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77:71–94PubMedGoogle Scholar
  4. Bulgheresi S, Kleiner E, Knoblich JA (2001) Inscuteable-dependent apical localization of the microtubule-binding protein Cornetto suggests a role in asymmetric cell division. J Cell Sci 114:3655–62PubMedGoogle Scholar
  5. Compton DA, Cleveland DW (1994) NuMA, a nuclear protein involved in mitosis and nuclear reformation. Curr Opin Cell Biol 6:343–346CrossRefPubMedGoogle Scholar
  6. Compton DA, Szilak I, Cleveland DW (1992) Primary structure of NuMA, an intranuclear protein that defines a novel pathway for segregation of proteins at mitosis. J Cell Biol 116:1395–408PubMedGoogle Scholar
  7. Doe CQ, Bowerman B (2001) Asymmetric cell division: fly neuroblast meets worm zygote. Curr Opin Cell Biol 13:68–75Google Scholar
  8. Esteban M, Giovinazzo G, Hera A, Goday C (1998) PUMA1: a novel protein that associates with the centrosomes, spindle and centromeres in the nematode Parascaris. J Cell Sci 111:723–735PubMedGoogle Scholar
  9. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811PubMedGoogle Scholar
  10. Gonczy P (2002) Mechanisms of spindle positioning: focus on flies and worms. Trends Cell Biol 12:332–339CrossRefPubMedGoogle Scholar
  11. Gotta M, Dong Y, Peterson YK, Lanier SM, Ahringer J (2003) Asymmetrically distributed C. elegans homologs of AGS3/PINS control spindle position in the early embryo. Curr Biol 13:1029–1037CrossRefPubMedGoogle Scholar
  12. Haren L, Merdes A (2002) Direct binding of NuMA to tubulin is mediated by a novel sequence motif in the tail domain that bundles and stabilizes microtubules. J Cell Sci 115:1815–1824PubMedGoogle Scholar
  13. Horvitz HR, Sulston JE (1980) Isolation and genetic characterization of cell-lineage mutants of the nematode Caenorhabditis elegans. Genetics 96:435–454PubMedGoogle Scholar
  14. Liao H, Winkfein RJ, Mack G, Rattner JB, Yen TJ (1995) CENP-F is a protein of the nuclear matrix that assembles onto kinetochores at late G2 and is rapidly degraded after mitosis. J Cell Biol 130:507–518PubMedGoogle Scholar
  15. Lorson MA, Horvitz HR, van den Heuvel S (2000) LIN-5 is a novel component of the spindle apparatus required for chromosome segregation and cleavage plane specification in Caenorhabditis elegans. J Cell Biol 148:73–86CrossRefPubMedGoogle Scholar
  16. Lupas A (1996) Coiled coils: new structures and new functions. Trends Biochem Sci 21:375–382PubMedGoogle Scholar
  17. Lupas A, Van Dyke M, Stock J (1991) Predicting coiled coils from protein sequences. Science 252:1162–1164PubMedGoogle Scholar
  18. Merdes A, Ramyar K, Vechio J, Cleveland D (1996) A complex of NuMA and cytoplasmic dynein is essential for mitotic spindle assembly. Cell 87:447–458PubMedGoogle Scholar
  19. O’Neil KT, DeGrado WF (1990) A thermodynamic scale for the helix-forming tendencies of the commonly occurring amino acids. Science 250:646–651PubMedGoogle Scholar
  20. Oegema K, Marshall WF, Sedat JW, Alberts BM (1997) Two proteins that cycle asynchronously between centrosomes and nuclear structures: Drosophila CP60 and CP190. J Cell Sci 110:1573–1583PubMedGoogle Scholar
  21. Reboul J, et al (2001) Open-reading-frame sequence tags (OSTs) support the existence of at least 17,300 genes in C. elegans. Nat Genet 27:332–336CrossRefPubMedGoogle Scholar
  22. Riddle DL, Blumenthal T, Meyer BJ, Priess JR (1997) C. elegans II. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  23. Schaefer M, Shevchenko A, Knoblich JA (2000) A protein complex containing Inscuteable and the Galpha-binding protein Pins orients asymmetric cell divisions in Drosophila. Curr Biol 10:353–362CrossRefPubMedGoogle Scholar
  24. Simmer F, Tijsterman M, Parrish S, Koushika SP, Nonet ML, Fire A, Ahringer J, Plasterk RH (2002) Loss of the putative RNA-directed RNA polymerase RRF-3 makes C. elegans hypersensitive to RNAi. Curr Biol 12:1317–9CrossRefPubMedGoogle Scholar
  25. Simmer F, Moorman C, van der Linden AM, Kuijk E, van den Berghe PV, Kamath R, Fraser AG, Ahringer J, Plasterk RH (2003) Genome-wide RNAi of C. elegans using the hypersensitive rrf-3 strain reveals novel gene functions. PLoS Biol 1:E12CrossRefPubMedGoogle Scholar
  26. Srinivasan DG, Fisk RM, Xu H, van den Heuvel S (2003) A complex of LIN-5 and GPR proteins regulates G protein signaling and spindle function in C. elegans. Genes Dev 17:1225–1239CrossRefPubMedGoogle Scholar
  27. Sulston JE, Horvitz HR (1977) Post-embryonic cell lineages of nematode, Caenorhabditis elegans. Dev Biol 56:110–156PubMedGoogle Scholar
  28. Timmons L, Court DL, Fire A (2001) Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene 263:103–112CrossRefPubMedGoogle Scholar
  29. Triteeraprapab S, Richie TL, Tuan RS, Shepley KJ, Dinman JD, Neubert TA, Scott AL (1995) Molecular cloning of a gene expressed during early embryonic development in Onchocerca volvulus. Mol Biochem Parasitol 69:161–171CrossRefPubMedGoogle Scholar
  30. Vidal M, Brachmann RK, Fattaey A, Harlow E, Boeke JD (1996) Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions. Proc Natl Acad Sci USA 93:10315–10320CrossRefPubMedGoogle Scholar
  31. Walhout AJ, Vidal M (1999) A genetic strategy to eliminate self-activator baits prior to high-throughput yeast two-hybrid screens. Genome Res 9:1128–1134CrossRefPubMedGoogle Scholar
  32. Walhout AJ, Vidal M (2001) High-throughput yeast two-hybrid assays for large-scale protein interaction mapping. Methods 24:297–306CrossRefPubMedGoogle Scholar
  33. Walhout AJ, Sordella R, Lu X, Hartley JL, Temple GF, Brasch MA, Thierry-Mieg N, Vidal M (2000a) Protein interaction mapping in C. elegans using proteins involved in vulval development. Science 287:116–122CrossRefPubMedGoogle Scholar
  34. Walhout AJ, Temple GF, Brasch MA, Hartley JL, Lorson MA, van den Heuvel S, Vidal M (2000b) GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol 328:575–592PubMedGoogle Scholar
  35. Yang CH, Lambie EJ, Snyder M (1992) NuMA: an unusually long coiled-coil related protein in the mammalian nucleus. J Cell Biol 116:1303–17PubMedGoogle Scholar
  36. Yu F, Morin X, Cai Y, Yang X, Chia W (2000) Analysis of partner of inscuteable, a novel player of Drosophila asymmetric divisions, reveals two distinct steps in Inscuteable apical localization. Cell 100:399–409PubMedGoogle Scholar
  37. Yu F, Ong CT, Chia W, Yang X (2002) Membrane targeting and asymmetric localization of Drosophila Partner of Inscuteable are discrete steps controlled by distinct regions of the protein. Mol Cell Biol 22:4230–4240CrossRefPubMedGoogle Scholar

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

Personalised recommendations