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Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation.

Howell BJ, McEwen BF, Canman JC, Hoffman DB, Farrar EM, Rieder CL, Salmon ED - J. Cell Biol. (2001)

Bottom Line: These proteins include the microtubule motors CENP-E and cytoplasmic dynein, and proteins involved with the mitotic spindle checkpoint, Mad2, Bub1R, and the 3F3/2 phosphoantigen.Depletion of these components did not disrupt kinetochore outer domain structure or alter metaphase kinetochore microtubule number.Thus, a major function of dynein/dynactin in mitosis is in a kinetochore disassembly pathway that contributes to inactivation of the spindle checkpoint.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina, Chapel Hill, NC 27599, USA. Bhowell@email.unc.edu

ABSTRACT
We discovered that many proteins located in the kinetochore outer domain, but not the inner core, are depleted from kinetochores and accumulate at spindle poles when ATP production is suppressed in PtK1 cells, and that microtubule depolymerization inhibits this process. These proteins include the microtubule motors CENP-E and cytoplasmic dynein, and proteins involved with the mitotic spindle checkpoint, Mad2, Bub1R, and the 3F3/2 phosphoantigen. Depletion of these components did not disrupt kinetochore outer domain structure or alter metaphase kinetochore microtubule number. Inhibition of dynein/dynactin activity by microinjection in prometaphase with purified p50 "dynamitin" protein or concentrated 70.1 anti-dynein antibody blocked outer domain protein transport to the spindle poles, prevented Mad2 depletion from kinetochores despite normal kinetochore microtubule numbers, reduced metaphase kinetochore tension by 40%, and induced a mitotic block at metaphase. Dynein/dynactin inhibition did not block chromosome congression to the spindle equator in prometaphase, or segregation to the poles in anaphase when the spindle checkpoint was inactivated by microinjection with Mad2 antibodies. Thus, a major function of dynein/dynactin in mitosis is in a kinetochore disassembly pathway that contributes to inactivation of the spindle checkpoint.

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ATP reduction does not disrupt kinetochore fibers, kinetochore outer plate structure, or microtubule attachment. (A) Prometaphase PtK1 cells were processed for tubulin immunofluorescence after treatment with saline alone, saline plus ATP inhibitors, or saline + ATP inhibitors followed by a 10-min rinse. Single plane images were taken by confocal microscopy. (B) Electron micrographs of kinetochores from metaphase-aligned chromosomes from an untreated PtK1 cell and a cell treated with Az/DOG for 30 min. Bars: (A) 10 μm; (B) 0.2 μm.
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fig2: ATP reduction does not disrupt kinetochore fibers, kinetochore outer plate structure, or microtubule attachment. (A) Prometaphase PtK1 cells were processed for tubulin immunofluorescence after treatment with saline alone, saline plus ATP inhibitors, or saline + ATP inhibitors followed by a 10-min rinse. Single plane images were taken by confocal microscopy. (B) Electron micrographs of kinetochores from metaphase-aligned chromosomes from an untreated PtK1 cell and a cell treated with Az/DOG for 30 min. Bars: (A) 10 μm; (B) 0.2 μm.

Mentions: ATP inhibitor treatment at similar concentrations has previously been shown to reduce spindle microtubule dynamics and induce astral microtubule growth, but have little effect on spindle morphology (Wadsworth and Salmon, 1988). To examine overall spindle structure during our ATP reduction assay, we obtained confocal images of immuofluorescently stained spindles in prometaphase PtK1 cells incubated with or without sodium azide (Az)/2-deoxyglucose (DOG) for 30 min. As seen in Fig. 2 A, prometaphase spindles remained bipolar and robust kinetochore fibers persisted after inhibitor treatment. Astral microtubule assembly was enhanced after inhibitor treatment as reported previously for BSC1 cells (Fig. 2 A; Wadsworth and Salmon, 1988). Cells progressed normally through mitosis after inhibitor washout (DeBrabender et al., 1981; unpublished data).


Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation.

Howell BJ, McEwen BF, Canman JC, Hoffman DB, Farrar EM, Rieder CL, Salmon ED - J. Cell Biol. (2001)

ATP reduction does not disrupt kinetochore fibers, kinetochore outer plate structure, or microtubule attachment. (A) Prometaphase PtK1 cells were processed for tubulin immunofluorescence after treatment with saline alone, saline plus ATP inhibitors, or saline + ATP inhibitors followed by a 10-min rinse. Single plane images were taken by confocal microscopy. (B) Electron micrographs of kinetochores from metaphase-aligned chromosomes from an untreated PtK1 cell and a cell treated with Az/DOG for 30 min. Bars: (A) 10 μm; (B) 0.2 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199338&req=5

fig2: ATP reduction does not disrupt kinetochore fibers, kinetochore outer plate structure, or microtubule attachment. (A) Prometaphase PtK1 cells were processed for tubulin immunofluorescence after treatment with saline alone, saline plus ATP inhibitors, or saline + ATP inhibitors followed by a 10-min rinse. Single plane images were taken by confocal microscopy. (B) Electron micrographs of kinetochores from metaphase-aligned chromosomes from an untreated PtK1 cell and a cell treated with Az/DOG for 30 min. Bars: (A) 10 μm; (B) 0.2 μm.
Mentions: ATP inhibitor treatment at similar concentrations has previously been shown to reduce spindle microtubule dynamics and induce astral microtubule growth, but have little effect on spindle morphology (Wadsworth and Salmon, 1988). To examine overall spindle structure during our ATP reduction assay, we obtained confocal images of immuofluorescently stained spindles in prometaphase PtK1 cells incubated with or without sodium azide (Az)/2-deoxyglucose (DOG) for 30 min. As seen in Fig. 2 A, prometaphase spindles remained bipolar and robust kinetochore fibers persisted after inhibitor treatment. Astral microtubule assembly was enhanced after inhibitor treatment as reported previously for BSC1 cells (Fig. 2 A; Wadsworth and Salmon, 1988). Cells progressed normally through mitosis after inhibitor washout (DeBrabender et al., 1981; unpublished data).

Bottom Line: These proteins include the microtubule motors CENP-E and cytoplasmic dynein, and proteins involved with the mitotic spindle checkpoint, Mad2, Bub1R, and the 3F3/2 phosphoantigen.Depletion of these components did not disrupt kinetochore outer domain structure or alter metaphase kinetochore microtubule number.Thus, a major function of dynein/dynactin in mitosis is in a kinetochore disassembly pathway that contributes to inactivation of the spindle checkpoint.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina, Chapel Hill, NC 27599, USA. Bhowell@email.unc.edu

ABSTRACT
We discovered that many proteins located in the kinetochore outer domain, but not the inner core, are depleted from kinetochores and accumulate at spindle poles when ATP production is suppressed in PtK1 cells, and that microtubule depolymerization inhibits this process. These proteins include the microtubule motors CENP-E and cytoplasmic dynein, and proteins involved with the mitotic spindle checkpoint, Mad2, Bub1R, and the 3F3/2 phosphoantigen. Depletion of these components did not disrupt kinetochore outer domain structure or alter metaphase kinetochore microtubule number. Inhibition of dynein/dynactin activity by microinjection in prometaphase with purified p50 "dynamitin" protein or concentrated 70.1 anti-dynein antibody blocked outer domain protein transport to the spindle poles, prevented Mad2 depletion from kinetochores despite normal kinetochore microtubule numbers, reduced metaphase kinetochore tension by 40%, and induced a mitotic block at metaphase. Dynein/dynactin inhibition did not block chromosome congression to the spindle equator in prometaphase, or segregation to the poles in anaphase when the spindle checkpoint was inactivated by microinjection with Mad2 antibodies. Thus, a major function of dynein/dynactin in mitosis is in a kinetochore disassembly pathway that contributes to inactivation of the spindle checkpoint.

Show MeSH
Related in: MedlinePlus