title: Microtubule-dependent changes in assembly of microtubule motor proteins and mitotic spindle checkpoint proteins at PtK1 kinetochores.
abstract: The ability of kinetochores to recruit microtubules, generate force, and activate the mitotic spindle checkpoint may all depend on microtubule- and/or tension-dependent changes in kinetochore assembly. With the use of quantitative digital imaging and immunofluorescence microscopy of PtK1 tissue cells, we find that the outer domain of the kinetochore, but not the CREST-stained inner core, exhibits three microtubule-dependent assembly states, not directly dependent on tension. First, prometaphase kinetochores with few or no kinetochore microtubules have abundant punctate or oblate fluorescence morphology when stained for outer domain motor proteins CENP-E and cytoplasmic dynein and checkpoint proteins BubR1 and Mad2. Second, microtubule depolymerization induces expansion of the kinetochore outer domain into crescent and ring morphologies around the centromere. This expansion may enhance recruitment of kinetochore microtubules, and occurs with more than a 20- to 100-fold increase in dynein and relatively little change in CENP-E, BubR1, and Mad2 in comparison to prometaphase kinetochores. Crescents disappear and dynein decreases substantially upon microtubule reassembly. Third, when kinetochores acquire their full metaphase complement of kinetochore microtubules, levels of CENP-E, dynein, and BubR1 decrease by three- to sixfold in comparison to unattached prometaphase kinetochores, but remain detectable. In contrast, Mad2 decreases by 100-fold and becomes undetectable, consistent with Mad2 being a key factor for the "wait-anaphase" signal produced by unattached kinetochores. Like previously found for Mad2, the average amounts of CENP-E, dynein, or BubR1 at metaphase kinetochores did not change with the loss of tension induced by taxol stabilization of microtubules.
authors.affiliation: Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599-3280, USA.
authors.forename: D B
authors.initials: DB
authors.lastname: Hoffman
authors.forename: C G
authors.initials: CG
authors.lastname: Pearson
authors.forename: T J
authors.initials: TJ
authors.lastname: Yen
authors.forename: B J
authors.initials: BJ
authors.lastname: Howell
authors.forename: E D
authors.initials: ED
authors.lastname: Salmon
mesh_terms: D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004398:Dyneins / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D018386:Kinetochores / Q000378:metabolism*; D064246:Mad2 Proteins; D008677:Metaphase; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D015739:Nocodazole / Q000494:pharmacology; D017239:Paclitaxel / Q000494:pharmacology; D000075223:Poly-ADP-Ribose Binding Proteins; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011506:Proteins / Q000378:metabolism; D012097:Repressor Proteins; D015398:Signal Transduction; D008941:Spindle Apparatus / Q000378:metabolism*
chemical_list: C489444:BUB3 protein, human; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D011506:Proteins; D012097:Repressor Proteins; C068688:centromere protein E; D011494:Protein Kinases; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases; D004398:Dyneins; D017239:Paclitaxel; D015739:Nocodazole