Spatiotemporal control of mitosis by the conserved spindle matrix protein Megator

Abstract

A putative spindle matrix has been hypothesized to mediate chromosome motion, but its existence and functionality remain controversial. In this report, we show that Megator (Mtor), the Drosophila melanogaster counterpart of the human nuclear pore complex protein translocated promoter region (Tpr), and the spindle assembly checkpoint (SAC) protein Mad2 form a conserved complex that localizes to a nuclear derived spindle matrix in living cells. Fluorescence recovery after photobleaching experiments supports that Mtor is retained around spindle microtubules, where it shows distinct dynamic properties. Mtor/Tpr promotes the recruitment of Mad2 and Mps1 but not Mad1 to unattached kinetochores (KTs), mediating normal mitotic duration and SAC response. At anaphase, Mtor plays a role in spindle elongation, thereby affecting normal chromosome movement. We propose that Mtor/Tpr functions as a spatial regulator of the SAC, which ensures the efficient recruitment of Mad2 to unattached KTs at the onset of mitosis and proper spindle maturation, whereas enrichment of Mad2 in a spindle matrix helps confine the action of a diffusible "wait anaphase" signal to the vicinity of the spindle.

Figure 1.

Mtor is part of a dynamic nuclear derived spindle matrix distinct from MTs. (A) An S2 cell stably expressing Mtor-mCherry (red) and GFP–α-tubulin (green). (A′) The corresponding Mtor-mCherry channel alone. (B) S2 cell stably expressing Mtor-mCherry and GFP–α-tubulin upon colchicine addition (time = 0). (C–E) Live cell analysis of GFP–α-tubulin, Mtor-mCherry, and Jupiter-GFP after colchicine treatment. (C′) Loss of GFP–α-tubulin fluorescence in the spindle is accompanied with equivalent fluorescence gain in the cytoplasm. (D′) Mtor-mCherry fluorescence in the spindle is not affected by MT depolymerization. (E′) Jupiter-GFP fluorescence is lost from the spindle to the cytoplasm after MT depolymerization. (F–F″) Endogenous Mtor after cold-induced MT depolymerization. (G and G′) Lamin B localization around the spindle. F.I., fluorescence intensity. Time is shown in minutes/seconds. Bars, 5 µm.

Figure 2.

Analysis of Mtor-mCherry dynamics by FRAP. (A and A′) FRAP of Mtor-mCherry in interphase nuclei. The white ROI shows fluorescence recovery within a bleached region; the red ROI shows fluorescence loss from an equivalent unbleached region; and the green ROI shows the entire nuclear area bleached in a neighboring cell. (B and B′) FRAP of Mtor-mCherry in one half-spindle (white ROI) and respective fluorescence loss in the other half-spindle (red ROI). The green ROI shows cytoplasmic fluorescence decay. (C and C′) FRAP of Mtor-mCherry in the entire mitotic spindle (white circle). Fluorescence decay in an equivalent area in the cytoplasm is indicated in the graph. (D–D″) Simultaneous FRAP of Mtor-mCherry (red) and GFP–α-tubulin (green) in the mitotic spindle. The corresponding FRAP of Mtor-mCherry and GFP–α-tubulin (white ROI) in the half-spindle was measured and compared with fluorescence loss in the unbleached half-spindle (red ROI) and cytoplasm (green ROI). (E and E′) Cell with two spindles in which Mtor-mCherry was photobleached in one half-spindle (white ROI). FRAP of Mtor-mCherry in this half-spindle was measured and compared with fluorescence loss in the unbleached half-spindle (red ROI), in the entire unbleached spindle (blue ROI), and cytoplasm (green ROI). Time = 0 at first frame after photobleaching. Relative fluorescence int, relative fluorescence intensity. Bars, 5 µm.

Figure 3.

Mtor is required for proper mitotic timing and SAC response. (A–C) S2 cells stably expressing GFP–α-tubulin (green) and CID-mCherry (red) were used for live imaging of mitotic progression in control (A), Mtor RNAi (B), and Mad2 RNAi (C). (D) Respective quantification of the time from NEB to anaphase. Mtor and Mad2 RNAi are statistically different from controls (P < 0.05; Dunn's test). Mtor is also statistically different from controls in a pairwise comparison (P = 0.003; Mann-Whitney test). (E) Mitotic index under physiological conditions or after colchicine treatment. Error bars represent SD from the mean obtained from three independent experiments. (F) Western blot analysis of Mtor. (left to right) Control, Mtor RNAi (75% depletion), stable expression of Mtor-mCherry without induction, stable expression of Mtor-mCherry after induction, RNAi using the 3′ UTR region of Mtor as target (86% depletion), and stable expression of Mtor-mCherry after induction and RNAi using the 3′ UTR region of Mtor as target. Chromator was used as loading control. (G–I) Analysis of chromosome and spindle dynamics during anaphase in control (G), Mtor RNAi (H), and Mad2 RNAi cells (I). (G′–I′) The corresponding kymograph analyses are shown. (G″–I″) Half-spindle elongation (spindle elong) and chromosome segregation (chrom segreg) velocities in control, Mtor RNAi, and Mad2 RNAi cells. Black lines indicate reference mean values for control cells. Spindle elongation in Mtor and Mad2 RNAi is statistically different from controls (P < 0.05; Student-Newman-Keuls Method). Time is shown in minutes/seconds. Bars, 5 µm.

Figure 4.

Mad2 associates with and requires Mtor to localize to unattached KTs. (A and B) S2 cells treated with colchicine and processed for immunofluorescence with Mad2 (red) and CID (green) antibodies. DNA (blue) was counterstained with DAPI. (C) Quantification of Mad2/CID pixel intensity at KTs for control (median = 0.946, range = 0–5.52, n = 571 KTs/20 cells) and Mtor RNAi (median = 0.357, range = 0–3.95, n = 515 KTs/20 cells). The two populations are statistically different (P < 0.001; Mann-Whitney test). (D–E′) Mitotic progression in S2 cells stably expressing GFP–α-tubulin (green) and mRFP-Mad2 (red). Arrows indicate KTs. Red cytoplasmic aggregates likely correspond to misfolded mRFP-Mad2. (F and G) Control and Mtor RNAi cells were treated with colchicine and processed for immunofluorescence with Mps1 (red) and CID (green) antibodies. (H) Quantification of Mps1/CID pixel intensity at KTs for control (median = 2.06, range = 0.04–13.1, n = 384 KTs/20 cells) and Mtor RNAi (median = 0.91, range = 0–8.9, n = 391 KTs/20 cells). The two populations are statistically different (P < 0.001; Mann-Whitney test). (I) Co-IP of Mad2 with Mtor in lysates obtained from Drosophila embryos harvested between 0–3 h after egg laying. Time is given in minutes/seconds. Bars, 5 µm.

Figure 5.

Human Tpr shares functional conservation with Drosophila Mtor. (A) Immunodetection of endogenous Tpr with a mouse mAb (red), ACA (green), and MTs (blue) in HeLa cells. (B and C) Luciferase (control) and Tpr RNAi cells were treated with nocodazole and processed for immunofluorescence with Mad2 (red) and ACA (green) antibodies. DNA (blue) was counterstained with DAPI. White boxed regions indicate the chromosome that is shown at a higher magnification on the right. (D) Quantification of Mad2/ACA pixel intensity at KTs for luciferase (median = 1.039, range = 0.44–4.45, n = 486 KTs/13 cells) and Tpr RNAi (median = 0.46, range = 0.09–1.69, n = 528 KTs/14 cells). The two populations are statistically different (P < 0.001; Mann-Whitney test). (E) Mitotic index in HeLa cells after luciferase and Tpr RNAi under physiological conditions or 16 h nocodazole treatment. Error bars represent SD from the mean obtained from three independent experiments. (F) IP from mitotic enriched parental HeLa cells (En) or HeLa cells stably expressing EGFP-Tpr (GFP). Load indicates total protein extracts. Purified beads (IP) were subjected to Western blot analysis for detection of interacting proteins. (G and H) IP from mitotic enriched HeLa extracts using an unspecific rabbit IgG (Un), rabbit anti-Mad2, or anti-Mad1 IgGs. (I) Colocalization of Mad1 (red) with Tpr (green) at nuclear pores but not at KTs (blue) during early prometaphase. MTs are shown in white. Inset shows a higher magnification of one KT pair without depicting MTs. (J) Proposed model for the role of Mtor/Tpr in the recruitment of Mad2 and Mps1 to unattached KTs after NEB. Ab, antibody; APC/C, anaphase-promoting complex/cyclosome; P, phosphorylation; Promet, prometaphase. Bars, 5 µm.