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Kinetochores bind microtubules laterally in a transient fashion and stably, by insertion of plus ends. These pathways may exist to carry out distinct tasks during different stages of mitosis and likely depend on distinct molecular mechanisms. On isolated chromosomes, we found microtubule nucleation/binding depended additively on both dynein/dynactin and on the Ndc80/Hec1 complex. Studying chromosome movement in living Xenopus cells within the simplified geometry of monopolar spindles, we quantified the relative contributions of dynein/dynactin and the Ndc80/Hec1 complex. Inhibition of dynein/dynactin alone had minor effects but did suppress transient, rapid, poleward movements. In contrast, inhibition of the Ndc80 complex blocked normal end-on attachments of microtubules to kinetochores resulting in persistent rapid poleward movements that required dynein/dynactin. In normal cells with bipolar spindles, dynein/dynactin activity on its own allowed attachment and rapid movement of chromosomes on prometaphase spindles but failed to support metaphase alignment and chromatid movement in anaphase. Thus, in prometaphase, dynein/dynactin likely mediates early transient, lateral interactions of kinetochores and microtubules. However, mature attachment via the Ndc80 complex is essential for metaphase alignment and anaphase A.
Fig. 1. Dynein/dynactin and the Ndc80 complex additively contribute to microtubule binding at kinetochores of isolated chromosomes. Isolated Xenopus chromosomes in buffer were incubated in the presence or absence of dynamitin protein and anti-Ndc80 complex antibody. After addition of buffer, 10, 15, or 20 μM tubulin, microtubule assembly was initiated, then chromosomes were centrifuged onto coverslips and fixed for immunofluorescence. a Protocol for testing nucleation/binding of microtubules to kinetochores on isolated Xenopus chromosomes. b Immunofluorescence images comparing chromosomes incubated without tubulin (upper panels) to chromosomes incubated with 15 μM tubulin (lower panels). c Quantification of microtubules bound per chromosome. Addition of nonimmune control IgG was used as a control. Dynamitin protein and anti-Ndc80 complex antibodies individually cause a reduction of microtubule association. The combined addition of both dynamitin protein and anti-Ndc80 results in additive reduction in microtubule binding. In comparison to control, for dynamitin, the p value was less than 0.001 at concentrations of tubulin of 10 and 20 μM and less than 0.05 at a concentration of tubulin of 15 μM, n = 50; for the anti-Ndc80 complex antibody, the p value was less than 0.05 for tubulin at 10 μM, was equal to 0.07 at 15 μM, and was less than 0.001 at 20 μM tubulin, n = 50; for dynamitin protein and anti-Ndc80 together, the p value was less than 0.001 at all three concentrations of tubulin, n = 50
Fig. 2. Dynein/dynactin localization in cells injected with antibody to Ndc80 complex antibody and dynamitin. Cells were pretreated with MG132 (25 μM) to block mitotic exit. After injection, all cells were treated with nocodazole (50 ng/ml) for 10 min to disassemble the microtubules and enhance dynein/dynactin association with the kinetochores. The cell in the top row was injected with anti-Ndc80 complex antibodies. The cell in the bottom row was coinjected with anti-Ndc80 complex antibodies and dynamitin. Cells were labeled with anti-rabbit IgG (green) to detect the injected antibody. Cells also were labeled with mouse monoclonal antibody to the p150Glued subunit of dynactin complex (red). Chromosomes labeled with Dapi are depicted in blue in the merged image. In cells injected with only the anti-Ndc80 complex antibodies, p150Glued was found concentrated at kinetochore foci adjacent to the injected antibody signals. Coinjection of dynamitin caused loss of p150Glued concentration at kinetochores. Insets show enlarged views of selected kinetochore regions. Bar = 10 μm
Fig. 3. Chromosomes in prometaphase cells with unseparated spindle poles exhibit distinct modes of movement dependent on dynein/dynactin and the Ndc80 complex. All cells were pretreated with monastrol (35 μM) to prevent spindle pole separation and with MG132 (25 μM) to block premature mitotic exit. a Tracking of chromosomal movements (40–45-s frame intervals). White circles indicate position of the spindle poles, and white arrows indicate position of the kinetochore of a single example chromosome. For each type of injection, the movements of 29 chromosomes were quantified within five to seven injected cells. In a control cell injected with rabbit IgG (left column), the analyzed chromosome exhibits moderate oscillation near the spindle pole. In a cell injected with antibodies to Ndc80 complex (right column), the chromosome undergoes exaggerated movement toward and away from the spindle poles. Bar = 10 μm. b Plots showing the effects of inhibition of dynein/dynactin and the Ndc80 complex in several examples of individual chromosome movements in Xenopus S3 cells. Compared to IgG-injected control cells (first row), chromosomes in cells injected with dynamitin protein (second row) showed little change in overall behavior. Chromosomes in cells injected with antibodies to the Ndc80 complex (third row) exhibited greatly exaggerated movements toward and away from the spindle pole. In cells injected with both dynamitin protein and with antibodies to the Ndc80 complex, the large excursions were suppressed. Instead, most chromosomes eventually drifted away from the spindle pole. Vertical scale bar = 600 s. Horizontal scale bar = 10 μm. c Percent of total time (mean ± sem) the chromosomes spent moving toward or away from the spindle poles. Inhibition of Ndc80 complex increased the proportion of time the chromosomes spend moving away from the spindle poles indicating a loss of stable kinetochore/microtubule attachment. Simultaneous inhibition of dynein/dynactin and the Ndc80 complex and dynein diminished the increase of chromosome movement found upon inhibition of Ndc80 complex alone. d Average velocities of chromosome movement in the injected cells. Chromosomes in cells injected with anti-Ndc80 complex antibodies exhibited a significant increase in the average velocities toward the spindle pole (p < 0.001, n = 29). Cells were observed for 15 min after injection. e Percent of total time (mean ± sem) the chromosomes move rapidly (≥10 μm/min) toward and away from the spindle poles. Inhibition of dynein/dynactin led to a 50% decrease in rapid poleward movements compared with control (p < 0.05). Inhibition of Ndc80 complex led to a significant increase in rapid chromosome movements (p < 0.001). Simultaneous inhibition of Ndc80 complex and dynein/dynactin diminished these rapid movements. Movies showing chromosome behavior in injected cells are available as Movies 1, 2, 3, and 4. Compared with controls, asterisk indicates p < 0.05, and double asterisk indicates p < 0.001
Fig. 4. Simultaneous inhibition of Ndc80 complex and dynein/dynactin in cells with monopolar spindles leads to eventual loss of chromosome poleward movement and orientation toward spindle poles. Cells were pretreated with monastrol (3 μM) to prevent spindle pole separation and with MG132 (25 μM) to block premature mitotic exit. Cells were injected with anti-Ndc80 antibody and dynamitin, n = 4 cells. Chromosomes show progressive loss of movement and orientation toward the spindle poles such that by 90 min, most chromosomes become localized near the cell periphery
Fig. 5. Inhibition of the Ndc80 complex in mitotic cells with bipolar spindles induces loss of metaphase alignment and forced exit from M phase. Image sequences from two prometaphase Xenopus S3 cell microinjected with antibodies against Ndc80 protein (a) or Nuf2 protein, n = 4 (b), components of the Ndc80 complex (injection at time point 00:00). Both antibodies cause a similar phenotype. The mitotic spindle continued to mature as the spindle poles (white arrowheads) separated. At a time point 8–9 min after injection, most chromosomes became clustered loosely near the spindle equator. Approximately 15 min after injection, anaphase ensued, indicated by the separation of the two sister chromatids (black arrows). However, sister chromatids remained at the spindle equator without any poleward anaphase movement. Movies (Movies 5 and 6) corresponding to cells in a and b are available in the supplementary material. c Simultaneous inhibition of the Ndc80 complex and dynein/dynactin leads to loss of kinetochore-driven chromosome movement in prometaphase cells. Image sequence from a Xenopus S3 cell treated with 25 μM MG132 for 30 min then coinjected with anti-Ndc80 complex antibodies and dynamitin protein at time point 00:00. Major chromosome movements gradually ceased, and many chromosome became aligned parallel to the spindle axis and often took on an “accordion-like” wrinkled morphology, perhaps because of the interaction of chromosome arms with spindle microtubules (black arrows). During this time, the spindle poles (white arrowheads) moved further apart. The proteasome inhibitor, MG132, was used to inhibit anaphase onset and mitotic exit which ordinarily occurs in cells injected with anti-Ndc80 complex antibodies. Movie 9 corresponding to this cell is available in the supplementary material. Bar = 10 μm
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