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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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Dynein/dynactin inhibition induces a Mad2-dependent mitotic arrest in PtK1 cells, but does not block chromosomes congression to the spindle equator or anaphase chromosome segregation and cytokinesis after the checkpoint is inactivated by microinjection of Mad2 antibody. (A) Time-lapse of early prometaphase cell microinjected with p50 (time, 00:00) shows chromosome congression and metaphase arrest. (B) Time-lapse of a prometaphase PtK1 microinjected with p50 (time, 00:02) to induce a mitotic block, and then further injected with Mad2 antibodies to inactivate the checkpoint (00:56), inducing anaphase chromosome segregation and cytokinesis. Time, hrs:min. (C) Graph of anaphase A kinetochore to pole movements for four chromosomes measured (K-P, ♦, ⋄) and anaphase B spindle pole elongation (P-P, ▪) for cell in (B). Not all measured chromosomes are plotted for clarity. Bar, 10 μm.
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fig4: Dynein/dynactin inhibition induces a Mad2-dependent mitotic arrest in PtK1 cells, but does not block chromosomes congression to the spindle equator or anaphase chromosome segregation and cytokinesis after the checkpoint is inactivated by microinjection of Mad2 antibody. (A) Time-lapse of early prometaphase cell microinjected with p50 (time, 00:00) shows chromosome congression and metaphase arrest. (B) Time-lapse of a prometaphase PtK1 microinjected with p50 (time, 00:02) to induce a mitotic block, and then further injected with Mad2 antibodies to inactivate the checkpoint (00:56), inducing anaphase chromosome segregation and cytokinesis. Time, hrs:min. (C) Graph of anaphase A kinetochore to pole movements for four chromosomes measured (K-P, ♦, ⋄) and anaphase B spindle pole elongation (P-P, ▪) for cell in (B). Not all measured chromosomes are plotted for clarity. Bar, 10 μm.

Mentions: To examine the effect of dynein/dynactin inhibition on mitotic progression in PtK1 cells, prometaphase cells containing an established bipolar spindle were microinjected with either p50 protein or 70.1 antibody and imaged by phase-contrast microscopy. Chromosome congression and oscillations continued in dynein-inhibited cells, yet cells arrested in metaphase, i.e., p50 or 70.1 antibody– injected cells never entered anaphase during the time of observation (up to 3.5 h after metaphase arrest; n = 12) (Fig. 4 A; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200105093/DC1).


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)

Dynein/dynactin inhibition induces a Mad2-dependent mitotic arrest in PtK1 cells, but does not block chromosomes congression to the spindle equator or anaphase chromosome segregation and cytokinesis after the checkpoint is inactivated by microinjection of Mad2 antibody. (A) Time-lapse of early prometaphase cell microinjected with p50 (time, 00:00) shows chromosome congression and metaphase arrest. (B) Time-lapse of a prometaphase PtK1 microinjected with p50 (time, 00:02) to induce a mitotic block, and then further injected with Mad2 antibodies to inactivate the checkpoint (00:56), inducing anaphase chromosome segregation and cytokinesis. Time, hrs:min. (C) Graph of anaphase A kinetochore to pole movements for four chromosomes measured (K-P, ♦, ⋄) and anaphase B spindle pole elongation (P-P, ▪) for cell in (B). Not all measured chromosomes are plotted for clarity. Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199338&req=5

fig4: Dynein/dynactin inhibition induces a Mad2-dependent mitotic arrest in PtK1 cells, but does not block chromosomes congression to the spindle equator or anaphase chromosome segregation and cytokinesis after the checkpoint is inactivated by microinjection of Mad2 antibody. (A) Time-lapse of early prometaphase cell microinjected with p50 (time, 00:00) shows chromosome congression and metaphase arrest. (B) Time-lapse of a prometaphase PtK1 microinjected with p50 (time, 00:02) to induce a mitotic block, and then further injected with Mad2 antibodies to inactivate the checkpoint (00:56), inducing anaphase chromosome segregation and cytokinesis. Time, hrs:min. (C) Graph of anaphase A kinetochore to pole movements for four chromosomes measured (K-P, ♦, ⋄) and anaphase B spindle pole elongation (P-P, ▪) for cell in (B). Not all measured chromosomes are plotted for clarity. Bar, 10 μm.
Mentions: To examine the effect of dynein/dynactin inhibition on mitotic progression in PtK1 cells, prometaphase cells containing an established bipolar spindle were microinjected with either p50 protein or 70.1 antibody and imaged by phase-contrast microscopy. Chromosome congression and oscillations continued in dynein-inhibited cells, yet cells arrested in metaphase, i.e., p50 or 70.1 antibody– injected cells never entered anaphase during the time of observation (up to 3.5 h after metaphase arrest; n = 12) (Fig. 4 A; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200105093/DC1).

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