NuMA is required for proper spindle assembly and chromosome alignment in prometaphase

Abstract

Background: NuMA is a protein that has been previously shown to play a role in focusing microtubules at the mitotic spindle poles. However, most previous work relies on experimental methods that might cause dominant side effects on spindle formation, such as microinjection of antibodies, overexpression of mutant protein, or immunodepletion of NuMA-containing protein complexes.

Findings: To circumvent these technical problems, we performed siRNA experiments in which we depleted the majority of NuMA in human cultured cells. Depleted mitotic cells show a prolonged duration of prometaphase, with spindle pole defects and with unattached, unaligned chromosomes.

Conclusion: Our data confirm that NuMA is important for spindle pole formation, and for cohesion of centrosome-derived microtubules with the bulk of spindle microtubules. Our findings of NuMA-dependent defects in chromosome alignment suggest that NuMA is involved in stabilizing kinetochore fibres.

Figure 1

Depletion of NuMA from HeLa cells by siRNA. (A) HeLa cells were treated with control dsRNA, or two specific siRNAs against NuMA (siRNA 1 and 2; see main text for description of targeting sequences). Cells were lysed after 72 hours, and lysates were analysed by immunoblot, using antibodies against NuMA (monoclonal antibody NA09L, Calbiochem) or against alpha-tubulin (monoclonal antibody DM1a, Sigma-Aldrich). (B) Immunoblots of decreasing amounts of HeLa lysates from control cells, or cells treated with NuMA siRNA1 for 72 or 96 hours, respectively. Amounts were loaded as indicated. The blots were probed with antibodies against NuMA, or nucleolin as a loading control. Depletion efficiency was determined by scanning of the blots and comparing the intensity of NuMA from depletion experiments to the dilution series of control lysate. (C) Testing of the depletion efficiency of NuMA by immunofluorescence. Left, cells treated with control RNA for 72 hours; right, cells treated with NuMA siRNA1. Bar, 20 μm.

Figure 2

Unaligned chromosomes and spindle abnormalities in NuMA-depleted cells. Depletion of NuMA was performed with siRNA2, as described in Fig. 1. Immunofluorescence of microtubules (green) and NuMA (red) was performed using monoclonal anti-NuMA, combined with a rabbit antibody against tubulin. DNA was stained with 4',6-diamidino-2-phenylindole (DAPI, blue). Arrows depict centrosomes that are slightly disconnected from the main body of the spindle in NuMA-depleted cells. The top row shows a cell treated with control RNA, for comparison. Micrographs were taken with a Leica TCS SP confocal microscope, equipped with a PlanApo 100x/1.4NA objective lens (Leica Microsystems). Bar, 5 μm.

Figure 3

Silencing of NuMA activates the spindle assembly checkpoint. (A) Cells treated with NuMA siRNA1 and control cells were processed for immunofluorescence of centromeres with human CREST autoimmune serum (top, green), or with specific antibodies against the checkpoint proteins BubR1 (middle, green), and Mad2 (bottom, green). Antibody against BubR1 was provided by Dr Tim Yen (Fox Chase Center, Philadelphia, PA); antibody against Mad2 was from Berkeley Antibody Company. DNA was stained with DAPI (blue). Bar, 10 μm. (B) The percentage of mitotic cells in different phases is indicated for controls (blue) and cultures treated with NuMA siRNA1 for 72 hours (red). Standard deviations were calculated from n = 706 mitotic control cells, and from n = 601 depleted mitotic cells (data obtained in three independent experiments). The percentages of those prometaphases that show unaligned chromosomes besides mostly aligned chromosomes (as seen in A) are indicated separately on the left. (C) Growth curve of HeLa cells subjected to control treatment or NuMA siRNA1.

Figure 4

Spindle microtubule re-growth is delayed in NuMA-depleted cells. (A) Microtubules were depolymerised on ice for 1.5 hours, and re-polymerised at 37°C. At the indicated time points, cells were fixed and subjected to immunofluorescence of NuMA (red), tubulin (green), and staining of DNA (blue). Spindles in NuMA-depleted cells have a lower microtubule density and are less 'compact'. Bar, 5 μm. (B) Graph, indicating the percentage of regular, compact spindles at the indicated time points of microtubule re-growth (data from four independent experiments; between 50 and 100 cells were counted for each point).

Figure 5

NuMA depletion induces reduced tension at kinetochores and lagging chromosomes. (A) Confocal series (frames 1–3) of a NuMA-depleted mitotic cell, showing immunofluorescence staining of kinetochores (using a human CREST autoimmune serum, red), and microtubules (green). On the left, a projection of all sections of the confocal series through the cell is shown. Interkinetochore distances of lagging (#) and aligned (←) chromosomes were quantified and plotted in (B). Error bars represent the SEM (standard error of the mean). Mean values for the aligned chromosomes were significantly different between depleted and control cells (two-tailed τ test, * p < 0.001) (C) NuMA-depleted cell in late anaphase, stained for tubulin (green), NuMA (red), and DNA (blue). Chromosomes are separated to the respective poles, but single lagging chromosomes can be seen, as indicated by the arrow. Bars in A and C, 5 μm.