title: Control of the spindle checkpoint by lateral kinetochore attachment and limited Mad1 recruitment.
abstract: We observed the dynamic recruitment of spindle checkpoint proteins Mad1 and Bub1 to detached kinetochores in budding yeast using real-time live-cell imaging and quantified recruitment in fixed cells. After induced de novo kinetochore assembly at one pair of sister centromeres, Mad1 appeared after the kinetochore protein Mtw1. Detached kinetochores were not associated with the nuclear envelope, so Mad1 does not anchor them to nuclear pore complexes (NPCs). Disrupting Mad1's NPC localization increased Mad1 recruitment to detached sister kinetochores. Conversely, increasing the number of detached kinetochores reduced the amount of Mad1 per detached kinetochore. Bub1 also relocalized completely from the spindle to detached sister centromeres after kinetochore assembly. After their capture by microtubules, Mad1 and Bub1 progressively disappeared from kinetochores. Sister chromatids that arrested with a lateral attachment to one microtubule exhibited half the Mad1 of fully detached sisters. We propose that detached kinetochores compete with alternate binding sites in the nucleus to recruit Mad1 and Bub1 from available pools that are small enough to be fully depleted by just one pair of detached kinetochores and that lateral attachment licenses Mad1 removal from kinetochores after a kinetic delay.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720.
authors.forename: Nathaniel I
authors.initials: NI
authors.lastname: Krefman
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720.
authors.forename: David G
authors.initials: DG
authors.lastname: Drubin
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720 gbarnes@berkeley.edu.
authors.forename: Georjana
authors.initials: G
authors.lastname: Barnes
mesh_terms: D059447:Cell Cycle Checkpoints; D018797:Cell Cycle Proteins / Q000378:metabolism*; D018386:Kinetochores / Q000378:metabolism*; D008870:Microtubules; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D008941:Spindle Apparatus / Q000378:metabolism*
chemical_list: C584280:Bub1 protein, S cerevisiae; D018797:Cell Cycle Proteins; C501382:MAD1 protein, S cerevisiae; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D017346:Protein Serine-Threonine Kinases