MAST/Orbit has a role in microtubule-kinetochore attachment and is essential for chromosome alignment and maintenance of spindle bipolarity

Abstract

Multiple asters (MAST)/Orbit is a member of a new family of nonmotor microtubule-associated proteins that has been previously shown to be required for the organization of the mitotic spindle. Here we provide evidence that MAST/Orbit is required for functional kinetochore attachment, chromosome congression, and the maintenance of spindle bipolarity. In vivo analysis of Drosophila mast mutant embryos undergoing early mitotic divisions revealed that chromosomes are unable to reach a stable metaphase alignment and that bipolar spindles collapse as centrosomes move progressively closer toward the cell center and eventually organize into a monopolar configuration. Similarly, soon after depletion of MAST/Orbit in Drosophila S2 cells by double-stranded RNA interference, cells are unable to form a metaphase plate and instead assemble monopolar spindles with chromosomes localized close to the center of the aster. In these cells, kinetochores either fail to achieve end-on attachment or are associated with short microtubules. Remarkably, when microtubule dynamics is suppressed in MAST-depleted cells, chromosomes localize at the periphery of the monopolar aster associated with the plus ends of well-defined microtubule bundles. Furthermore, in these cells, dynein and ZW10 accumulate at kinetochores and fail to transfer to microtubules. However, loss of MAST/Orbit does not affect the kinetochore localization of D-CLIP-190. Together, these results strongly support the conclusion that MAST/Orbit is required for microtubules to form functional attachments to kinetochores and to maintain spindle bipolarity.

Figures

Figure 1.

Characterization of mast5. (A) Schematic representation of the deletion (dotted line) associated with the mast5 allele. The gray box represents the open reading frame of mast. (B) Western blot analysis of wild-type and mast5 embryos. α-Tubulin levels are shown as a loading control. Wild-type (C–E) or mast5 (F–K) embryos were immunostained to reveal the spindle and centrosomes by antibody detection of α-tubulin (green) and centrosomin (red) respectively, and DNA was counterstained with DAPI (blue). Wild-type embryos during early (C) and late (D and E) embryogenesis show well-organized bipolar spindles with centrosomes at both poles. (F) Early syncytial mast5 embryo (eight nuclei) showing monopolar spindles. (G) mast5 embryo showing monopolar spindles with chromosomes associated with the aster. Mutant embryos also show isolated centrosomes (arrows) and a few well-organized bipolar spindles with highly condensed chromosomes. (H) mast5 embryo showing very short bipolar spindles and bipolar spindles with centrosomes in a single pole (arrow). (I) Postcellularized wild-type embryo showing a mitotic domain. (J) Late mast5 embryo showing highly abnormal mitotic domains with polyploid cells containing monopolar spindles organized from clusters of centrosomes. (K) Part of a cellularized embryo with a large polyploid cell and another cell with a bipolar spindle with centrosomin staining in a single pole (arrow). Bar, 10 μm.

Figure 2.

In vivo analysis of mitotic progression in early mast5 embryos. Control and mast5 embryos carrying the gfp–polo transgene were followed by confocal time-lapse microscopy. (A) Time-lapse series of GFP–Polo fluorescence images showing the organization of the mitotic apparatus during mitotic progression in control gfp–polo (left) and gfp–polo; mast5 (right) embryos during cycle 12. Time is shown in seconds. Arrowheads represent the positioning of centrosomes in two sample nuclei. For discussion see text. (B) Graphic representation of kinetochore distribution on the spindle of wild type and mast5 at metaphase. The position of centrosomes is labeled as c. (C) Quantification of changes in spindle length in wild-type and mast5 embryos during mitosis. Measurements are from time-lapse confocal images taken every 10 s, taking the initiation of NEB as a reference point. The times corresponding to the images represented in A are indicated by an asterisk. Spindle length data correspond to the average measurement of seven nuclei in two different embryos (14 spindles in total). Error bars represent the standard deviation of the sample.

Figure 3.

Time course analysis of MAST RNAi in Drosophila S2 cells. (A, left) MAST inactivation was monitored over time by Western blot analysis. 106 cells were loaded in each lane and α-tubulin detection was used as a loading control. (A, right) Titration of anti-MAST antibody. (B) Proliferation analysis of S2 cells after MAST RNAi. (C) Mitotic index after MAST RNAi is represented as the average percentage of mitotic cells (n > 100) in the total population. (D and D′) Quantification of the mitotic parameters during the course of the experiment in control and in cells after MAST RNAi, respectively (n > 100). (E and E′) Quantification of interphase parameters during the course of the experiment in control and in cells after MAST RNAi (n > 250). (F and F′) Low magnification views of representative optical fields by 72 h in control and MAST RNAi cells, respectively, stained with an anti–P-histone 3 antibody to detect cells undergoing mitosis or, after 144 h (G and G′), stained for actin. Note the formation of cells with a very large (four- to fivefold) nucleus after MAST RNAi. Bar, 50 μm.

Figure 4.

Organization of the mitotic apparatus after MAST RNAi. Cells after MAST RNAi were stained with an anti-CP190 antibody to reveal the centrosomes (red), an anti–α-tubulin antibody to visualize the microtubules (green), and DAPI to counterstain the DNA (blue). (A) Normal bipolar spindle in a control cell. (B and C) Monopolar and polyploid monopolar spindles with two and at least four centrosomes, respectively, observed 72 or 96 h after MAST RNAi. Centrosome staining alone can be seen in the insertion on top right of the figures. (D) Bipolar spindle with two centrosomes (arrowheads) only in one pole. (E) Anaphase-like cell displaying a bipolar spindle with centrosomes in a single pole and chromosomes distributed in two distinct populations on each side of the spindle. Unmerged images revealing the centrosomes (E′), spindle (E′′), and chromosomes (E′′′) are shown. (F) A cell with a bipolar spindle and multiple centrosomes clustered in a single pole where the chromosomes appear distributed along the spindle. (G) Abnormal telophase-like cell showing the formation of a cleavage furrow (arrows), with centrosomes in only one pole and decondensed chromatin. Separate centrosome and DNA staining can be seen in (G′) and (G′′), respectively. Bar, 5 μm. (H and H′) Quantification of the observed spindle defects in control and MAST-depleted cells, respectively (n > 100).

Figure 5.

Sister chromatid separation in cells after MAST RNAi. (A–C) Samples were collected between 96 and 144 h after MAST RNAi and processed for immunofluorescence analysis with anti-BubR1 and anti-Cid antibodies as markers for kinetochores (red). Microtubules were stained with an anti–α-tubulin antibody (green), and chromosomes were counterstained with DAPI (blue). (A) Control cells in the same optical field undergoing prometaphase (pm), metaphase (m), and anaphase (a). (B) Anaphase-like cells after MAST RNAi showing very strong BubR1 staining at both kinetochores of each chromosome (arrow; 3× amplified insert), confirming that sister chromatid separation has not taken place. Unmerged images showing the mitotic spindle (B′), chromosomes (B′′), and BubR1 (B′′′) can be seen separately. (C) Quantification of normalized BubR1 pixel intensity in the control cells shown in A and the anaphase-like cell shown in B. (D and D′) Cell after MAST RNAi, showing the monopolar spindle organization with very strong BuBR1 staining at kinetochore pairs. (E–E′′) Cid staining on anaphase-like cells revealed that >90% of these cells (n = 20) show an unequal number of chromosomes in each pole that seem to interact laterally with the spindle microtubules. Bars, 5 μm.

Figure 6.

Analysis of kinetochore– microtubule attachment in MAST- depleted cells. Control cells (A–B') as well as MAST-depleted cells by RNAi (C–H') were stained with antibodies against Cid (red) and α-tubulin (green) and counterstained with DAPI to reveal the DNA (blue). (A and A') Control cell in metaphase showing that all the kinetochores are end-on attached to microtubules. (B and B') Control cell in metaphase that was pretreated with calcium before fixation, showing that the kinetochore microtubules were selectively preserved. (C–D′) Cells with bipolar spindles after MAST RNAi with misaligned chromosomes without (C and C′) or with (D and D′) a pretreatment with calcium. (E-E' and F-F') Cells with monopolar spindles after MAST RNAi with or without a pretreatment with calcium, respectively, showing chromosomes buried close to the center of the aster, and the kinetochores cannot be found associated with microtubule plus ends. (G–H') Treatment of MAST- depleted cells with taxol leads chromosomes to be organized at the periphery of the aster clearly associated with microtubule plus ends. Insertions inside panels represent the selected area in the cells that was magnified twice. Bars, 5 μm.

Figure 7.

Ultrastructural analysis of S2 cells after MAST RNAi. (A) Control cell showing a bipolar spindle with bundles of microtubules organized from the centrosomes. (A1) Higher magnification of chromosomes being captured by bundles of microtubules in a region that corresponds to the kinetochore (A1′, arrowhead). (A2) Higher magnification of one of the centrioles. (B) Top view of a MAST-depleted cell after RNAi showing a monopolar spindle with chromosomes very close to the center of the aster where three centrioles can be seen. (C) Side view of another MAST-depleted cell showing a monopolar aster where individual microtubules can be seen on the same plane of the chromosomes, suggesting lateral interactions. Bars, 2 μm.

Figure 8.

Immunolocalization of kinetochore-associated proteins in the absence of MAST. (A–B', E, F, and H–I') Untreated control cells or (C–D′, G, G′, J, and J′) cells 72 h after MAST RNAi, were stained (red) with anti-ZW10, anti-dynein, and anti–D-CLIP-190 antibodies. α-Tubulin (green) and DNA (blue) are also shown when possible. (A, A′, E, H, and H′) Cells in prometaphase with mono-oriented chromosomes showing kinetochore-associated ZW10, dynein, and D-CLIP-190 (arrows). (B, B′, F, I, and I′) During metaphase, when all the chromosomes become bioriented, ZW10 transfers to the kinetochore microtubules (arrowheads), and dynein and D-CLIP-190 are barely detectable at the kinetochores. (C and C′) Cells with monopolar or (D and D′) bipolar spindles with misaligned chromosomes showing strong staining of ZW10 at kinetochores. Insertions in panels C′ and D′ show ZW10 staining alone. In cells with monopolar spindles, after MAST RNAi, dynein (G and G′) and D-CLIP-190 (J and J′) show strong accumulation at kinetochores. Insertions in panels H′ and J′ show magnified views of the chromosomes indicated by the arrows. Bar, 5 μm.