title: Kinetochores require oligomerization of Dam1 complex to maintain microtubule attachments against tension and promote biorientation.
abstract: Kinetochores assemble on centromeric DNA and present arrays of proteins that attach directly to the dynamic ends of microtubules. Kinetochore proteins coordinate at the microtubule interface through oligomerization, but how oligomerization contributes to kinetochore function has remained unclear. Here, using a combination of biophysical assays and live-cell imaging, we find that oligomerization of the Dam1 complex is required for its ability to form microtubule attachments that are robust against tension in vitro and in vivo. An oligomerization-deficient Dam1 complex that retains wild-type microtubule binding activity is primarily defective in coupling to disassembling microtubule ends under mechanical loads applied by a laser trap in vitro. In cells, the oligomerization-deficient Dam1 complex is unable to support stable bipolar alignment of sister chromatids, indicating failure of kinetochore-microtubule attachments under tension. We propose that oligomerization is an essential and conserved feature of kinetochore components that is required for accurate chromosome segregation during mitosis.
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Neil T
authors.initials: NT
authors.lastname: Umbreit
authors.affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Miller
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Jerry F
authors.initials: JF
authors.lastname: Tien
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Jérôme Cattin
authors.initials: JC
authors.lastname: Ortolá
authors.affiliation: Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Long
authors.initials: L
authors.lastname: Gui
authors.affiliation: Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Kelly K
authors.initials: KK
authors.lastname: Lee
authors.affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
authors.forename: Sue
authors.initials: S
authors.lastname: Biggins
authors.affiliation: Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Charles L
authors.initials: CL
authors.lastname: Asbury
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Trisha N
authors.initials: TN
authors.lastname: Davis
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D055592:Biophysical Phenomena; D018797:Cell Cycle Proteins / Q000737:chemistry*; D002503:Centromere / Q000648:ultrastructure; D002850:Chromatography, Gel; D004247:DNA / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry; D018386:Kinetochores / Q000737:chemistry*; D008164:Luminescent Proteins / Q000737:chemistry; D008869:Microtubule-Associated Proteins / Q000737:chemistry*; D008870:Microtubules / Q000737:chemistry*; D008938:Mitosis; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000166:cytology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D018797:Cell Cycle Proteins; C116003:DAM1 protein, S cerevisiae; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins; D029701:Saccharomyces cerevisiae Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D004247:DNA