title: Mps1 phosphorylation of Dam1 couples kinetochores to microtubule plus ends at metaphase.
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.
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Shimogawa
authors.forename: Beth
authors.initials: B
authors.lastname: Graczyk
authors.forename: Melissa K
authors.initials: MK
authors.lastname: Gardner
authors.forename: Susan E
authors.initials: SE
authors.lastname: Francis
authors.forename: Erin A
authors.initials: EA
authors.lastname: White
authors.forename: Michael
authors.initials: M
authors.lastname: Ess
authors.forename: Jeffrey N
authors.initials: JN
authors.lastname: Molk
authors.forename: Cristian
authors.initials: C
authors.lastname: Ruse
authors.forename: Sherry
authors.initials: S
authors.lastname: Niessen
authors.forename: John R
authors.initials: JR
authors.lastname: Yates
authors.forename: Eric G D
authors.initials: EG
authors.lastname: Muller
authors.forename: Kerry
authors.initials: K
authors.lastname: Bloom
authors.forename: David J
authors.initials: DJ
authors.lastname: Odde
authors.forename: Trisha N
authors.initials: TN
authors.lastname: Davis
mesh_terms: D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D020090:Chromosome Segregation / Q000502:physiology*; D036681:Fluorescence Recovery After Photobleaching; D018386:Kinetochores / Q000378:metabolism*; D008677:Metaphase / Q000502:physiology*; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D008941:Spindle Apparatus / Q000502:physiology*; D014057:Tomography, X-Ray Computed
chemical_list: D018797:Cell Cycle Proteins; C116003:DAM1 protein, S cerevisiae; D008869:Microtubule-Associated Proteins; D029701:Saccharomyces cerevisiae Proteins; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; C093416:MPS1 protein, S cerevisiae