Mps1 Phosphorylation of Dam1 Couples Kinetochores to Microtubule Plus Ends at Metaphase

Michelle M. Shimogawa, Beth Graczyk, Melissa K. Gardner, Susan E. Francis, Erin A. White, Michael Ess, Jeffrey N. Molk, Cristian Ruse, Sherry Niessen, John R. Yates, Eric G.D. Muller, Kerry Bloom, David J. Odde, Trisha N. Davis

Research output: Contribution to journalArticlepeer-review

74 Scopus citations

Abstract

Background: Duplicated chromosomes are equally segregated to daughter cells by a bipolar mitotic spindle during cell division. By metaphase, sister chromatids are coupled to microtubule (MT) plus ends from opposite poles of the bipolar spindle via kinetochores. Here we describe a phosphorylation event that promotes the coupling of kinetochores to microtubule plus ends. Results: Dam1 is a kinetochore component that directly binds to microtubules. We identified DAM1-765, a dominant allele of DAM1, in a genetic screen for mutations that increase stress on the spindle pole body (SPB) in Saccharomyces cerevisiae. DAM1-765 contains the single mutation S221F. We show that S221 is one of six Dam1 serines (S13, S49, S217, S218, S221, and S232) phosphorylated by Mps1 in vitro. In cells with single mutations S221F, S218A, or S221A, kinetochores in the metaphase spindle form tight clusters that are closer to the SPBs than in a wild-type cell. Five lines of experimental evidence, including localization of spindle components by fluorescence microscopy, measurement of microtubule dynamics by fluorescence redistribution after photobleaching, and reconstructions of three-dimensional structure by electron tomography, combined with computational modeling of microtubule behavior strongly indicate that, unlike wild-type kinetochores, Dam1-765 kinetochores do not colocalize with an equal number of plus ends. Despite the uncoupling of the kinetochores from the plus ends of MTs, the DAM1-765 cells are viable, complete the cell cycle with the same kinetics as wild-type cells, and biorient their chromosomes as efficiently as wild-type cells. Conclusions: We conclude that phosphorylation of Dam1 residues S218 and S221 by Mps1 is required for efficient coupling of kinetochores to MT plus ends. We find that efficient plus-end coupling is not required for (1) maintenance of chromosome biorientation, (2) maintenance of tension between sister kinetochores, or (3) chromosome segregation.

Original languageEnglish (US)
Pages (from-to)1489-1501
Number of pages13
JournalCurrent Biology
Volume16
Issue number15
DOIs
StatePublished - Aug 8 2006

Bibliographical note

Funding Information:
We thank Sue Biggins, Michelle Jones, and Mark Winey for plasmids and strains and for helpful discussions. We thank Chad Pearson for helpful discussions. We thank Joy Bagley for initial analyses of the Dam1 complex and strains. This work was supported by the National Institute of General Medical Sciences (NIGMS) grant R01GM40506 to T.N.D. and National Center for Research Resources (NCRR) grant P41RR11823 to T.N.D. (for J.R.Y. and E.M.), National Science Foundation Career Award BES9984955 and NIGMS grant R01GM071522 to D.J.O., and NIGMS grant R01GM32238 to K.S.B. NIGMS grant T32GM07270 supported M.M.S. Electron microscopy and E.A.W. were supported by NCRR grant P41RR00592.

Keywords

  • CELLCYCLE

Fingerprint

Dive into the research topics of 'Mps1 Phosphorylation of Dam1 Couples Kinetochores to Microtubule Plus Ends at Metaphase'. Together they form a unique fingerprint.

Cite this