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

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.

Figure 1.

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.

Figure 2.

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.

Figure 3.

p50 injection prevents dynein localization to kinetochores (A) and blocks poleward transport of kinetochore outer domain components (B and C) in cells treated with Az/DOG. (A) Nocodazole-treated PtK1 cells were uninjected (top) or injected with p50 (bottom), incubated for 30 min, and then processed for dynein immunofluorescence. (B) p50 injected PtK1 cells incubated in Az/DOG for 30 min and processed for Mad2 (top), BubR1 (middle), and CENP-E + tubulin (bottom) immunofluorescence. (C) 70.1 mAb–injected PtK1 cells incubated in Az/DOG for 30 min and processed for Mad2 (top) and CENP-E (bottom) immunofluorescence. Bars, 5 μm.

Figure 4.

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.

Figure 4.

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.

Figure 5.

Dynein/dynactin inhibition prevents Mad2 depletion from attached kinetochores with normal numbers of metaphase kinetochore microtubules. (A) Phase contrast and fluorescence images of metaphase PtK1 cells were either uninjected (top) or injected with p50 in early prometaphase (bottom). Cells were fixed 60 min after p50 microinjection and processed for tubulin (α-tb, green) and Mad2 (α-Mad2, red). Note, the gluteraldhyde fixation used to preserve microtubules results in particulate Mad2 throughout the cytoplasm. Low magnification (B) and high magnification (C) electron micrographs of metaphase kinetochore fibers in cells microinjected with p50 in early prometaphase. Counts of kinetochore microtubule numbers at metaphase kinetochores in p50 injected cells (25.5 ± 6.1) were nearly identical to those in uninjected cells (24.3 ± 4.9). Bars: (A) 5 μm; (B) 1 μm; (C) 0.5 μm.

Figure 6.

Fluorescent Mad2 transport and localization to spindle poles disappears in vivo upon inhibition of dynein/dynactin. Time-lapse of a late prometaphase PtK1 cell microinjected with fluorescence Mad2 protein before (time, −00:10:25) and after further injection with p50 (time, 00:00:00). Fluorescent Mad2 localized to unattached or partially attached kinetochores, the spindle fibers in between these kinetochores and the pole, and the proximal spindle pole (arrowheads). No Mad2 fluorescence was evident on the kinetochores of metaphase-aligned chromosomes (top). Following microinjection with p50, fluorescent Mad2 disappeared at the spindle pole but remained bright on the unattached or partially attached kinetochores. Fluorescent Mad2 also accumulated over time on kinetochores of metaphase-aligned chromosomes (arrows). Bar, 5 μm.

Figure 7.

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.