title: Assembling the protein architecture of the budding yeast kinetochore-microtubule attachment using FRET.
abstract: BACKGROUND
  The kinetochore is a multiprotein machine that couples chromosome movement to microtubule (MT) polymerization and depolymerization. It uses numerous copies of at least three MT-binding proteins to generate bidirectional movement. The nanoscale organization of these proteins within the kinetochore plays an important role in shaping the mechanisms that drive persistent, bidirectional movement of the kinetochore.
  
  
  RESULTS
  We used fluorescence resonance energy transfer (FRET) between genetically encoded fluorescent proteins fused to kinetochore subunits to reconstruct the nanoscale organization of the budding yeast kinetochore. We performed >60 FRET and high-resolution colocalization measurements involving the essential MT-binding kinetochore components: Ndc80, Dam1, Spc105, and Stu2. These measurements reveal that neighboring Ndc80 complexes within the kinetochore are narrowly distributed along the length of the MT. Dam1 complex molecules are concentrated near the MT-binding domains of Ndc80. Stu2 localizes in high abundance within a narrowly defined territory within the kinetochore centered ∼20 nm on the centromeric side of the Dam1 complex.
  
  
  CONCLUSIONS
  Our data show that the MT attachment site of the budding yeast kinetochore is well organized. Ndc80, Dam1, and Stu2 are all narrowly distributed about their average positions along the kinetochore-MT axis. The relative organization of these components, their narrow distributions, and their known MT-binding properties together elucidate how their combined actions generate persistent, bidirectional kinetochore movement coupled to MT polymerization and depolymerization.
authors.affiliation: Biophysics, University of Michigan, 930 North University Avenue, Ann Arbor, MI 48109-1055, USA.
authors.forename: Pavithra
authors.initials: P
authors.lastname: Aravamudhan
authors.affiliation: Cell and Developmental Biology, University of Michigan, 109 Zina Pitcher Place, Ann Arbor, MI 48109, USA.
authors.forename: Isabella
authors.initials: I
authors.lastname: Felzer-Kim
authors.affiliation: Sastra University, Tirumalaisamudram, Thanjavur, Tamil Nadu 613402, India.
authors.forename: Kaushik
authors.initials: K
authors.lastname: Gurunathan
authors.affiliation: Biophysics, University of Michigan, 930 North University Avenue, Ann Arbor, MI 48109-1055, USA; Cell and Developmental Biology, University of Michigan, 109 Zina Pitcher Place, Ann Arbor, MI 48109, USA. Electronic address: ajitj@umich.edu.
authors.forename: Ajit P
authors.initials: AP
authors.lastname: Joglekar
mesh_terms: D018797:Cell Cycle Proteins / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D018386:Kinetochores / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D004718:Saccharomycetales / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; C116003:DAM1 protein, S cerevisiae; D008869:Microtubule-Associated Proteins; D046912:Multiprotein Complexes; C112472:NDC80 protein, S cerevisiae; D009687:Nuclear Proteins; C109414:STU2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins