JCS012112 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig S1. mitch RNAi results in mitotic defects. Drosophila S2 tissue culture cells were stained for microtubules (green), DNA (blue) and centromeres (CID; red). Chromosomes normally align at the metaphase plate in untreated controls (A), but in cells treated with mitch dsRNA (B-D), chromosomes fail to congress and instead are scattered across the spindle and are often found near the poles. Bar, 5 μm.

• Supplemental Figure S2 -

  Fig S2. PSCS in mitch RNAi cells. Either untreated (A-B) or mitch dsRNA-treated S2 cells (C-E) were treated with colchicine and hypotonic solution and stained with Hoechst 33248 to visualize the chromosomes. mitch RNAi cells display high levels of PSCS. Bar, 5 μm.

• Supplemental Figure S3 -

  Fig S3. Identification of the mitch gene. Molecular map of conceptual genes in the mitch region, according to (http://flybase.bio.indiana.edu/). Left-to-right corresponds to the direction from the centromere to the telomere of chromosome 3R; genes above the hatched line indicating distance along the DNA are transcribed from left-to-right; genes below the line are transcribed in the opposite direction. Coding regions are represented by thick black bars, 5′- and 3′-untranslated regions by gray thick bars, and introns by thin lines. The sequences deleted by Df(3R)ry614 and Df(3R)ry1608 are shown by black lines at the top; sequences deleted in several imprecise excisants of the P element insertion CK4A are shown by black lines at the bottom (see Materials and Methods for details). TE refers to a BS transposable element (Udomkit et al., 1995) that was found on the chromosome bearing the l(3)S125006a marked P element insertion (Deak et al., 1997) but is not found in the Flybase annotation of the Drosophila genome (http://flybase.bio.indiana.edu/). Neither the P element nor the BS transposon is responsible for the lethality among homozygotes for the l(3)S125006a chromosome; this is due to a secondary lesion elsewhere on the third chromosome (our unpublished results).

• Supplemental Figure S4 -

  Fig S4. Mitch targeting to the kinetochore is dependent on CID and CENP-C. Kc tissue culture cells were either untreated or treated with cid or cenp-c dsRNA. In dsRNA-treated cultures, cells lacking detectable CID or CENP-C also lack Mitch at the kinetochores. RNAi cells that still possess detectable CID or CENP-C also stain positive for Mitch (data not shown). The presence or absence of Mitch at the kinetochore is thus correlated with the association of CID and CENP-C with the centromere.

• Supplemental Figure S5 -

  Fig S5. mitch mutants do not affect the localization of other kinetochore components in mitosis. Untreated mitch¹ mutant brains were stained to examine (A) BubR1, (B) Bub3, (C) Zwilch, (D) Cid, (E) Klp10A, (F) Dynein heavy chain, (G) Cenp-meta, (H) Polo and (I) CENP-C (red) in metaphase cells containing misoriented chromosomes (blue). All these proteins are still able to localize to the kinetochore, indicating that mitch mutants do not grossly affect kinetochore structure. Note that the misoriented chromosomes (arrows) contain higher levels of Bub3 (B), KLP10A (E), and dynein (F) than do chromosomes at the metaphase plate. Bar, 5 μm.

• Supplemental Figure S6 -

  Fig S6. Alignment of Mitch protein sequences. (A) The predicted Mitch amino acid sequences were aligned using MegAlign (DNAstar). The species name is presented to the right of each sequence. Amino acids conserved between D. melanogaster and other species are colored blue; those conserved with D. virilis are colored red. Other homologies between the intermediate sequences can also be observed (see uncolored blocks). Corresponding DNA sequences not already in databases have been deposited in GenBank under accession numbers AY714306-AY714314. (B) Maximum parsimony phylogeny for the Mitch protein constructed using PAUP* (Swofford, 2002). The alignment corresponds to amino acids 1-233 in part A. This tree matches well with accepted phylogenies for the Drosophilids (Ko et al., 2003). (C) Sliding window plot of nonsynonymous divergence between D. melanogaster and D.simulans demonstrates the pronounced elevation of Ka in the predicted coiled-coil domain, supporting the suggestion that the coiled-coil domain is the major contributor to the high divergence of Mitch.

• Movie 1 -

  Movie 1. Time-lapse DIC microscopy of a living control Drosophila neuroblast undergoing mitosis. Note the clarity of the chromosomes and the organelle exclusion zone that demarks the spindle. Time between NEB and anaphase is 12 minutes.

• Movie 2 -

  Movie 2. Time-lapse DIC microscopy of a living mitch mutant neuroblast undergoing mitosis. Two chromosomes are mono-oriented; note also the location of the spindle and centrosomes. Time between NEB and anaphase is approximately 1 hour.

• Movie 3 -

  Movie 3. Time-lapse DIC microscopy of a living mitch mutant neuroblast undergoing mitosis. In this case, chromosome misalignment was persistent, and the cell did not exit mitosis even after more than 2 hours after NEB.

• Movie 4 -

  Movie 4. Time-lapse DIC microscopy of a living wild type neuroblast entering mitosis in the presence of colchicine. As the cell entered mitosis in the absence of microtubules, an active spindle checkpoint prevented anaphase and the cell remained in c-mitosis for more than 2 hours after NEB. Note the overcondensation of the chromosomes.

• Movie 5 -

  Movie 5. Time-lapse DIC microscopy of a living mitch mutant neuroblast entering and exiting mitosis in the presence of colchicine. In this case, the absence of microtubules was not sufficient to prevent the exit of mitosis without chromosome segregation. Chromosomes decondense and nuclear envelope reforms 13 minutes after NEB.