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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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Outer domain kinetochore components localize to spindle poles after ATP reduction if the mitotic spindle is present. PtK1 cells were incubated for 30 min in either saline alone, saline supplemented with 5 mM Az/DOG, or saline + Az/DOG for 30 min followed by a 10-min rinse in saline (Az/DOG + wash). This assay was done both in the absence (A, C, and E) and presence (B and D) of 20 μM nocodazole. Mad2, BubR1, CENP-E, dynein (A), and 3F3/2 (C) fluorescence diminished at kinetochores and concentrated at spindle poles after inhibitor treatment in the absence of nocodazole. After washout of the inhibitors, Mad2, BubR1, CENP-E, and dynein fluorescence recovered at kinetochores and diminished at spindle poles, similar to the localization pattern seen with saline alone (A) (Az/DOG + Wash). 3F3/2 fluorescence reappeared on most kinetochores and was reduced at the spindle poles after inhibitor washout (C) (Az/DOG + wash). Incubation of cells with 20 μM nocodazole for 15 min before and during inhibitor treatment blocked this redistribution pattern for Mad2, BubR1, CENP-E, dynein, and 3F3/2 (B and D). In contrast to the outer domain kinetochore components, the inner domain CREST antigens remained localized to kinetochores after inhibitor treatment (E). For each cell, phase-contrast images are on the left and the corresponding fluorescent images are on the right. Bars, 10 μm.
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fig1: Outer domain kinetochore components localize to spindle poles after ATP reduction if the mitotic spindle is present. PtK1 cells were incubated for 30 min in either saline alone, saline supplemented with 5 mM Az/DOG, or saline + Az/DOG for 30 min followed by a 10-min rinse in saline (Az/DOG + wash). This assay was done both in the absence (A, C, and E) and presence (B and D) of 20 μM nocodazole. Mad2, BubR1, CENP-E, dynein (A), and 3F3/2 (C) fluorescence diminished at kinetochores and concentrated at spindle poles after inhibitor treatment in the absence of nocodazole. After washout of the inhibitors, Mad2, BubR1, CENP-E, and dynein fluorescence recovered at kinetochores and diminished at spindle poles, similar to the localization pattern seen with saline alone (A) (Az/DOG + Wash). 3F3/2 fluorescence reappeared on most kinetochores and was reduced at the spindle poles after inhibitor washout (C) (Az/DOG + wash). Incubation of cells with 20 μM nocodazole for 15 min before and during inhibitor treatment blocked this redistribution pattern for Mad2, BubR1, CENP-E, dynein, and 3F3/2 (B and D). In contrast to the outer domain kinetochore components, the inner domain CREST antigens remained localized to kinetochores after inhibitor treatment (E). For each cell, phase-contrast images are on the left and the corresponding fluorescent images are on the right. Bars, 10 μm.

Mentions: To test for kinetochore protein transport along spindle microtubules to the poles we used the ATP reduction assay developed by Howell et al. (2000). For outer domain proteins we tested Mad2, BubR1, CENP-E, and cytoplasmic dynein (Fig. 1, A and B) and the 3F3/2 antigen (Fig. 1, C and D). Consistent with previous studies (Kallio et al., 1998; Waters et al., 1998; Howell et al., 2000; Hoffman et al., 2001), prometaphase PtK1 cells treated with saline alone showed strong staining for Mad2, cytoplasmic dynein, and 3F3/2 at unattached and partially attached prometaphase kinetochores, whereas BubR1 and CENP-E localized to all kinetochores (Fig. 1, A and C). After a 30-min incubation with 5 mM sodium azide and 1 mM 2-deoxyglucose to reduce ATP to 5–10% of normal levels (see Materials and methods), Mad2, BubR1, CENP-E, and 3F3/2 fluorescence diminished at kinetochores and concentrated at spindle poles (Fig. 1, A–C). Cytoplasmic dynein fluorescence also increased at the spindle poles, but dim fluorescence was occasionally evident on a few kinetochores and along spindle microtubules (Fig. 1 A). After a 10-min washout period in saline, ATP recovered to 50–75% normal levels, and fluorescence returned at kinetochores and diminished at the spindle poles for all these outer domain proteins (Fig. 1, A and C). Notably, Mad2 and cytoplasmic dynein fluorescence returned only to unattached or partially attached kinetochores and were not evident on fully attached, metaphase-aligned kinetochores after inhibitor washout (Fig. 1 A). Unlike Mad2 and cytoplasmic dynein, the kinetochore components CENP-E and BubR1 were evident on all kinetochores after inhibitor washout, consistent with previous findings (Fig. 1 A; Hoffman et al., 2001). After washout, most kinetochores in 3F3/2 stained cells appeared labeled, perhaps because tension is not fully regained during the brief washout period (Fig. 1 C). Quantification of kinetochore and pole fluorescence for these experiments is presented in Table I. Microtubule depolymerization with nocodazole prevented redistribution of outer domain proteins from kinetochores to the poles (Fig. 1, B and D; Table II), showing that this process depends on spindle microtubules. Quantification of kinetochore fluorescence in nocodazole cells treated ± ATP inhibitors revealed similar values between controls and inhibitor treatments, with exception of the 3F3/2 antigen which was reduced 50% after azide/deoxyglucose treatment (Table II), an effect likely due to dephosphorylation of the 3F3/2 epitope. Unlike the outer domain proteins tested, we found no loss in kinetochore staining or increase in concentration at the spindle poles for the inner domain CREST antigens after 30-min treatment of cells with ATP inhibitors (Fig. 1 E; Table I).


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)

Outer domain kinetochore components localize to spindle poles after ATP reduction if the mitotic spindle is present. PtK1 cells were incubated for 30 min in either saline alone, saline supplemented with 5 mM Az/DOG, or saline + Az/DOG for 30 min followed by a 10-min rinse in saline (Az/DOG + wash). This assay was done both in the absence (A, C, and E) and presence (B and D) of 20 μM nocodazole. Mad2, BubR1, CENP-E, dynein (A), and 3F3/2 (C) fluorescence diminished at kinetochores and concentrated at spindle poles after inhibitor treatment in the absence of nocodazole. After washout of the inhibitors, Mad2, BubR1, CENP-E, and dynein fluorescence recovered at kinetochores and diminished at spindle poles, similar to the localization pattern seen with saline alone (A) (Az/DOG + Wash). 3F3/2 fluorescence reappeared on most kinetochores and was reduced at the spindle poles after inhibitor washout (C) (Az/DOG + wash). Incubation of cells with 20 μM nocodazole for 15 min before and during inhibitor treatment blocked this redistribution pattern for Mad2, BubR1, CENP-E, dynein, and 3F3/2 (B and D). In contrast to the outer domain kinetochore components, the inner domain CREST antigens remained localized to kinetochores after inhibitor treatment (E). For each cell, phase-contrast images are on the left and the corresponding fluorescent images are on the right. Bars, 10 μm.
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Related In: Results  -  Collection

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fig1: Outer domain kinetochore components localize to spindle poles after ATP reduction if the mitotic spindle is present. PtK1 cells were incubated for 30 min in either saline alone, saline supplemented with 5 mM Az/DOG, or saline + Az/DOG for 30 min followed by a 10-min rinse in saline (Az/DOG + wash). This assay was done both in the absence (A, C, and E) and presence (B and D) of 20 μM nocodazole. Mad2, BubR1, CENP-E, dynein (A), and 3F3/2 (C) fluorescence diminished at kinetochores and concentrated at spindle poles after inhibitor treatment in the absence of nocodazole. After washout of the inhibitors, Mad2, BubR1, CENP-E, and dynein fluorescence recovered at kinetochores and diminished at spindle poles, similar to the localization pattern seen with saline alone (A) (Az/DOG + Wash). 3F3/2 fluorescence reappeared on most kinetochores and was reduced at the spindle poles after inhibitor washout (C) (Az/DOG + wash). Incubation of cells with 20 μM nocodazole for 15 min before and during inhibitor treatment blocked this redistribution pattern for Mad2, BubR1, CENP-E, dynein, and 3F3/2 (B and D). In contrast to the outer domain kinetochore components, the inner domain CREST antigens remained localized to kinetochores after inhibitor treatment (E). For each cell, phase-contrast images are on the left and the corresponding fluorescent images are on the right. Bars, 10 μm.
Mentions: To test for kinetochore protein transport along spindle microtubules to the poles we used the ATP reduction assay developed by Howell et al. (2000). For outer domain proteins we tested Mad2, BubR1, CENP-E, and cytoplasmic dynein (Fig. 1, A and B) and the 3F3/2 antigen (Fig. 1, C and D). Consistent with previous studies (Kallio et al., 1998; Waters et al., 1998; Howell et al., 2000; Hoffman et al., 2001), prometaphase PtK1 cells treated with saline alone showed strong staining for Mad2, cytoplasmic dynein, and 3F3/2 at unattached and partially attached prometaphase kinetochores, whereas BubR1 and CENP-E localized to all kinetochores (Fig. 1, A and C). After a 30-min incubation with 5 mM sodium azide and 1 mM 2-deoxyglucose to reduce ATP to 5–10% of normal levels (see Materials and methods), Mad2, BubR1, CENP-E, and 3F3/2 fluorescence diminished at kinetochores and concentrated at spindle poles (Fig. 1, A–C). Cytoplasmic dynein fluorescence also increased at the spindle poles, but dim fluorescence was occasionally evident on a few kinetochores and along spindle microtubules (Fig. 1 A). After a 10-min washout period in saline, ATP recovered to 50–75% normal levels, and fluorescence returned at kinetochores and diminished at the spindle poles for all these outer domain proteins (Fig. 1, A and C). Notably, Mad2 and cytoplasmic dynein fluorescence returned only to unattached or partially attached kinetochores and were not evident on fully attached, metaphase-aligned kinetochores after inhibitor washout (Fig. 1 A). Unlike Mad2 and cytoplasmic dynein, the kinetochore components CENP-E and BubR1 were evident on all kinetochores after inhibitor washout, consistent with previous findings (Fig. 1 A; Hoffman et al., 2001). After washout, most kinetochores in 3F3/2 stained cells appeared labeled, perhaps because tension is not fully regained during the brief washout period (Fig. 1 C). Quantification of kinetochore and pole fluorescence for these experiments is presented in Table I. Microtubule depolymerization with nocodazole prevented redistribution of outer domain proteins from kinetochores to the poles (Fig. 1, B and D; Table II), showing that this process depends on spindle microtubules. Quantification of kinetochore fluorescence in nocodazole cells treated ± ATP inhibitors revealed similar values between controls and inhibitor treatments, with exception of the 3F3/2 antigen which was reduced 50% after azide/deoxyglucose treatment (Table II), an effect likely due to dephosphorylation of the 3F3/2 epitope. Unlike the outer domain proteins tested, we found no loss in kinetochore staining or increase in concentration at the spindle poles for the inner domain CREST antigens after 30-min treatment of cells with ATP inhibitors (Fig. 1 E; Table I).

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