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First published online 25 September 2007
doi: 10.1242/jcs.012112

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Mitch – a rapidly evolving component of the Ndc80 kinetochore complex required for correct chromosome segregation in Drosophila

Byron Williams¹, Garmay Leung¹, Helder Maiato^2,*, Alex Wong¹, ZeXiao Li¹, Erika V. Williams¹, Catherine Kirkpatrick³, Charles F. Aquadro¹, Conly L. Rieder^2,4 and Michael L. Goldberg^1,

¹ Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853-2703, USA
² Division of Molecular Medicine, Wadsworth Center, New York State Dept. of Health, Albany, NY 12201-0509, USA
³ Division of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455, USA
⁴ Department of Biomedical Sciences, State University of New York, Albany, NY 12222, USA

View larger version (121K): [in this window] [in a new window]   Fig. 1. Mitotic defects in mitch mutants. (A-I) Larval brain neuroblasts from wild-type (A,G), mitch1 (B,D,F,H), and mitch2/mitch19B (C,E,I) were stained with orcein to visualize chromosomes. (A-E) Brains treated with colchicine and hypotonic solution. In contrast with normal 6A,XX or 6A,XY chromosome complements in wild-type (A; here from a male), mitch mutants contain many aneuploid cells, as seen in (B) 6A,XXX and (C) 7A,XY. (A indicates the number of autosomes, including the dot-like fourth chromosomes.) (D,E) Many mitch mutant cells treated with colchicine also exhibit PSCS in which the sister chromatids have become detached from each other even at the centromere. (F-I) Untreated brains. (F) Many mitotic figures in untreated mitch mutant brains have overcondensed chromosomes. During anaphase, when chromatids in wild type normally migrate equally to the poles (G), lagging chromatids are visible in mitch mutants (arrows; H,I). Bar, 5 µm.

View larger version (109K): [in this window] [in a new window]   Fig. 2. Chromosome misalignment in mitch mutants. (A-D) Brain neuroblasts from wild-type (A,B) and mitch1 (C,D) were stained for chromosomes (blue) and microtubules (green). At metaphase (A,C), chromosomes are correctly aligned at the metaphase plate in wild-type (A), whereas the chromosomes are mis-oriented in mitch mutants (C) with some chromosomes abnormally situated near the pole (arrow). At anaphase (B,D), the normal even distribution of chromatids (B) is disrupted in mitch mutants (D) so as to produce lagging chromosomes (arrow); moderate chromosome overcondensation is also apparent in this mutant anaphase. In all mitch mutant mitotic figures, the spindles are morphologically normal. Bar, 5 µm.

View larger version (193K): [in this window] [in a new window]   Fig. 3. Mitotic progression in living control and mitch neuroblasts. (A) Time-lapse series of an untreated wild-type neuroblast. At the start of the recording, the chromosomes are beginning to condense during prophase. Nuclear envelope breakdown (NEB; time 0) occurs 2.5 minutes later, and the chromosomes rapidly align at the metaphase plate within the next 7 minutes. The cell entered anaphase 12 minutes after NEB, which was followed by chromosome decondensation and reformation of the nuclear envelope during telophase and organization of the cleavage furrow during cytokinesis. (B) Time-lapse series of an untreated mitch neuroblast in which two chromosomes (arrows) remained mono-oriented for extended periods. Anaphase was much delayed, beginning only 1 hours after NEB; telophase and cytokinesis soon followed. (C) Time-lapse series of another untreated mitch neuroblast in which the chromosomes were distributed along the longitudinal axis of the spindle and remained misaligned in prometaphase for more than 2 hours, without entering anaphase. Note the overcondensation of the chromosomes due to prolonged mitotic arrest. Asterisks indicate the position of the centrosomes inferred by DIC as the focus center of a clear zone; these asterisks thus define the virtual longitudinal axis of the spindle. (D) Time-lapse series of a wild-type neuroblast entering mitosis in the presence of 50 µM colchicine. This cell remained in c-mitosis for more than 2 hour (when the recording was stopped) and consequently the chromosomes look overcondensed. (E) Time-lapse series of a mitch neuroblast entering and exiting mitosis in only 13 minutes in the presence of 50 µM colchicine. The chromosomes appear to disjoin at 6.5 minutes, but no anaphase movement was subsequently observed. The chromosomes decondensed and the nuclear envelope completely reformed around a single nucleus by 17 minutes. See movies in the supplementary material for clearer visualization. Bar, 5 µm.

View larger version (101K): [in this window] [in a new window]   Fig. 4. mitch mutations disrupt chromosome segregation in both male meiotic divisions. (A-L) Testes from wild-type (A,D,E,H,J), mitch1 (B,F,L), and mitch1/mitch2 (C,G,I,K) were stained to visualize chromosomes (blue) and microtubules (green). At metaphase I (A,B,C), bivalents in the mitch mutants are positioned away from the center of the cell, either off the main axis of the spindle (arrowhead) or, more frequently, on the spindle axis but closer to one pole than the other (arrows). At anaphase/telophase I (D-G), mitch bivalents often migrate only to one pole (arrows in F,G), though sometimes lagging chromosomes are observed along with unequal segregation (arrowhead, G). These phenotypes are reiterated in the second meiotic division (H-L): chromosome mis-alignment during metaphase (I; see arrow) followed by complete nondisjunction (K; arrow). Telophase II figures lacking chromosomes are also observed (L). Bar, 5 µm.

View larger version (89K): [in this window] [in a new window]   Fig. 5. The spindle checkpoint is compromised in mitch mutant mitotic cells treated with microtubule poisons. Brains from mitch1 were treated with colchicine and hypotonic solution and examined for cyclin B (A,B; red), Bub3 (C; red), or Mad2 (D,E; red) and chromosomes (blue.) High levels of cyclin B are present in cells containing attached sister chromatids (A) whereas they are drastically lowered in PSCS cells (B). Two cells next to each other in the same field (C) show a difference in the distribution of Bub3 with respect to sister chromatid separation (PSCS). In cell 1, high levels of Bub3 are present at the kinetochores of the attached sister chromatids, but very low levels of Bub3 exist at kinetochores of PSCS cells (cell 2). Mad2 is present at much higher levels at the kinetochores of attached chromosomes (D) than at the kinetochores in PSCS cells (E). Bar, 5 µm.

View larger version (64K): [in this window] [in a new window]   Fig. 6. Mitch localizes to the kinetochore during mitosis. (A) Wild-type (top row) and mitch-mutant (bottom row) brains were stained with anti-Mitch antibodies (red). In wild-type, Mitch localizes to kinetochores from prometaphase through late anaphase and likewise in chromosome spreads of cells arrested in metaphase by colchicine (c-metaphase). The kinetochore staining of anti-Mitch antibodies is absent in mitotic figures from mitch mutants. (L-R on the bottom row: mitch1, mitch2/Df, mitch2/Df, mitch1). (B) Epitope-tagged Mitch. Western blot (left panel) of Drosophila tissue culture cells non-transfected (NT) or transfected with mitch-V5, and probed with anti-V5 antibody, identifying the 30 kd Mitch-V5 protein. (Right panels) Indirect immunofluorescence of cells incubated with anti-V5 antibody. Mitch-V5 was absent in non-transfected cells (NT), but localized to the kinetochore in transfected cells (here seen in c-metaphase). Bar, 5 µm.

View larger version (38K): [in this window] [in a new window]   Fig. 7. Mitch is located between CID and Zwilch at the kinetochore. Wild-type brains treated with colchicine and hypotonic solutions were examined simultaneously for DNA, Mitch, and another kinetochore antigen (CID, Bub3, or Zwilch). The colocalization of Mitch (red) with these other kinetochore proteins (green) was observed in the merged images (far right column; also see magnified insets). Mitch appears to be distal to CID localization, but proximal to Zwilch. Mitch approximately colocalizes with the Bub3 protein (center row). Bar, 5 µm.

View larger version (54K): [in this window] [in a new window]   Fig. 8. Mitch is a component of the Ndc80 complex. (A) Localization of TAP-tagged Mitch protein expressed in Drosophila Kc tissue culture cells. Mitch fused with protein-A–CBP either at the N-terminus (NTAP) or at the C-terminus (CTAP) localizes to the kinetochores of the chromosomes (blue). Bar, 10 µm. (B) Mitch co-purifies with Hec1, Nuf2, Mis-12 and CG1558 (arrows). SDS-PAGE gels were stained with Coomassie Blue and protein bands were identified by mass spectrometry. Other bands on the gel represent proteins that are either highly abundant (tubulin, actin), also present in the control (Kc cells without the tagged construct), or breakdown products of the proteins present in the complex.