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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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Model of dynein/dynactin-driven poleward transport of kinetochore proteins along spindle microtubules, and the role of this transport in inactivation of the spindle checkpoint activity at kinetochores. (A) Mad2 + Mad2 complexes (blue oval) and other motor and checkpoint proteins (corona filament) assemble from cytoplasmic pools onto unattached kinetochores where the checkpoint proteins catalyze formation of Mad2-Cdc20 inhibitory complexes. (B) Dissociation of Mad2 and other outer domain components occurs either by direct exchange with cytoplasmic pools or through dynein/dynactin-interactions with non-kinetochore (nkMT) or kinetochore (kMT) microtubules. Motor and checkpoint protein complexes are transported poleward by dynein/dynactin where they dissociate into the cytoplasm. (C) Full kinetochore microtubule occupancy on metaphase-aligned chromosomes prevents association of outer domain components, thereby blocking formation of Mad2–Cdc20 inhibitory complexes and allowing for spindle checkpoint inactivation.
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fig7: Model of dynein/dynactin-driven poleward transport of kinetochore proteins along spindle microtubules, and the role of this transport in inactivation of the spindle checkpoint activity at kinetochores. (A) Mad2 + Mad2 complexes (blue oval) and other motor and checkpoint proteins (corona filament) assemble from cytoplasmic pools onto unattached kinetochores where the checkpoint proteins catalyze formation of Mad2-Cdc20 inhibitory complexes. (B) Dissociation of Mad2 and other outer domain components occurs either by direct exchange with cytoplasmic pools or through dynein/dynactin-interactions with non-kinetochore (nkMT) or kinetochore (kMT) microtubules. Motor and checkpoint protein complexes are transported poleward by dynein/dynactin where they dissociate into the cytoplasm. (C) Full kinetochore microtubule occupancy on metaphase-aligned chromosomes prevents association of outer domain components, thereby blocking formation of Mad2–Cdc20 inhibitory complexes and allowing for spindle checkpoint inactivation.

Mentions: Our results indicate that outer domain kinetochore motors CENP-E and cytoplasmic dynein and the checkpoint-related proteins Mad2, BubR1, and the 3F3/2 antigen, are in a dynamic assembly at kinetochores with the rate of association from cytoplasmic pools balanced by either the rate of dissociation driven by dynein/dynactin interactions with spindle microtubules, or by direct dissociation into the cytoplasm (Fig. 7). Our ATP reduction assay should be useful for identifying other kinetochore proteins which undergo dynamic assembly at kinetochores, which proteins are more stable (e.g., like the inner core CREST antigens), and which outer plate proteins are responsible for attachment to the plus ends of kinetochore microtubules (Fig. 2 B).


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)

Model of dynein/dynactin-driven poleward transport of kinetochore proteins along spindle microtubules, and the role of this transport in inactivation of the spindle checkpoint activity at kinetochores. (A) Mad2 + Mad2 complexes (blue oval) and other motor and checkpoint proteins (corona filament) assemble from cytoplasmic pools onto unattached kinetochores where the checkpoint proteins catalyze formation of Mad2-Cdc20 inhibitory complexes. (B) Dissociation of Mad2 and other outer domain components occurs either by direct exchange with cytoplasmic pools or through dynein/dynactin-interactions with non-kinetochore (nkMT) or kinetochore (kMT) microtubules. Motor and checkpoint protein complexes are transported poleward by dynein/dynactin where they dissociate into the cytoplasm. (C) Full kinetochore microtubule occupancy on metaphase-aligned chromosomes prevents association of outer domain components, thereby blocking formation of Mad2–Cdc20 inhibitory complexes and allowing for spindle checkpoint inactivation.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Model of dynein/dynactin-driven poleward transport of kinetochore proteins along spindle microtubules, and the role of this transport in inactivation of the spindle checkpoint activity at kinetochores. (A) Mad2 + Mad2 complexes (blue oval) and other motor and checkpoint proteins (corona filament) assemble from cytoplasmic pools onto unattached kinetochores where the checkpoint proteins catalyze formation of Mad2-Cdc20 inhibitory complexes. (B) Dissociation of Mad2 and other outer domain components occurs either by direct exchange with cytoplasmic pools or through dynein/dynactin-interactions with non-kinetochore (nkMT) or kinetochore (kMT) microtubules. Motor and checkpoint protein complexes are transported poleward by dynein/dynactin where they dissociate into the cytoplasm. (C) Full kinetochore microtubule occupancy on metaphase-aligned chromosomes prevents association of outer domain components, thereby blocking formation of Mad2–Cdc20 inhibitory complexes and allowing for spindle checkpoint inactivation.
Mentions: Our results indicate that outer domain kinetochore motors CENP-E and cytoplasmic dynein and the checkpoint-related proteins Mad2, BubR1, and the 3F3/2 antigen, are in a dynamic assembly at kinetochores with the rate of association from cytoplasmic pools balanced by either the rate of dissociation driven by dynein/dynactin interactions with spindle microtubules, or by direct dissociation into the cytoplasm (Fig. 7). Our ATP reduction assay should be useful for identifying other kinetochore proteins which undergo dynamic assembly at kinetochores, which proteins are more stable (e.g., like the inner core CREST antigens), and which outer plate proteins are responsible for attachment to the plus ends of kinetochore microtubules (Fig. 2 B).

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