JCS041798 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Kinetochore localization of NudE in larval brain cells. In the merged images, the centromere marker CID is red, NudE is green, and DNA is blue. In wild type (Oregon-R), NudE signals are found adjacent to sites of CID staining during prometaphase and metaphase, and also in ‘c-metaphase’ cells treated with colchicine. The approximate location of the spindle poles is indicated with asterisks in the metaphase figures; the insert shows that the NudE signals are located closer to the poles than the CID-staining loci. Streaming of NudE onto kMTs is seen in the second and third rows of metaphase figures (the third row is from a separate experiment in which the localization of CID was not investigated). NudE localization at either the kinetochores or the kMTs is not observed in brain cells from nudE^39A homozygous mutant larvae, demonstrating the specificity of the immunofluorescence signals. This is true even when the brains have been cultured with colchicine, a treatment that often greatly increases the association of kinetochore components with the kinetochores (Williams et al., 1992). Scale bar: 5 µm.

• Supplemental Figure S2 -

  Fig. S2. Chromosome and mitochondrial segregation problems in nudE^39A mutant larval testes. Top: DNA (Hoechst stain) in wild-type (OR) and nudE mutant ana/telophase I figures. The mutant meioses display unequal segregation of DNA to the two poles or chromosomal bridges/lagging DNA. Bottom: Unequal-sized nuclei and Nebenkern in ‘onion stage’ spermatids of nudE^39A mutant larval testes. In the wild-type control, each nucleus is associated with an equal-sized Nebenkern (mitochondrial derivative). Note the uniformity in the sizes of nuclei and Nebenkern. By contrast, the sizes of nuclei and Nebenkern are irregular in nudE^39A spermatids; see for example the micronucleus indicated with the arrowhead in the panel at the bottom right. Some nuclei are associated with more than one Nebenkern. Scale bar: 10 µm.

• Supplemental Figure S3 -

  Fig. S3. TAP-tagging with NudE. TAP-tagged NudE protein was expressed in Drosophila tissue culture cells, and the tagged protein and its interactors were purified as described in Materials and Methods. In the experiment at the right, the cells were treated with colchicine for 90 minutes prior to harvesting, so as to arrest the cells in M phase. This figure shows an SDS-PAGE analysis of the final elution from each experiment. The tagged form of NudE comigrates with Lis1, whose presence in the most prominent band was verified by mass spectrometery and western blotting. Some NudE degradation products are also observed in both experiments (NudE*). All other visible bands in the eluates have been observed in a similar experiments performed with other TAP-tagged proteins (Williams et al., 2007), and are thus not specific to NudE. Zipper is the Drosophila nonmuscle myosin heavy chain.

• Supplemental Figure S4 -

  Fig. S4. NudE associates with kinetochores in prometaphase/ metaphase figures in larval brains of asp¹, dynein heavy chain (Dhc64C^6-10), nudC^9jE8, and bubR1¹ mutants. In all merged images, CID is red, NudE is green, and DNA is blue. See supplemental material Fig. S1 for wild-type controls. Scale bar: 5 µm.

• Supplemental Figure S5 -

  Fig. S5. Kinetochore targeting of NudE during male meiosis requires Cenp-meta and Lis1. In the merged images, NudE is red, alpha-tubulin is green, and DNA is blue. NudE fails to accumulate at kinetochores in cenp-meta (cmetΔ) or in lis1^K13201 mutant spermatocytes; this is particularly evident in prometaphase I and metaphase I figures. NudE accumulates at the spindle poles in the absence of Cenp-meta. NudE is also found in the vicinity of the spindle envelope in cmetΔ mutant spermatocytes, but the levels are much reduced relative to that seen in wildtype (compare with the wildt-ype images in Fig. 2). The protein also strongly associates with the reforming nuclear envelopes during telophase. In lis1 mutant spermatocytes, NudE is not found at the kinetochores, but instead accumulates along what appears to be the spindle envelope. Note that lis1 spermatocytes exhibit the same defects in centrosome migration seen in nudE mutant meiotic figures (see also Fig. 4). Scale bars: 10 µm.

• Supplemental Figure S6 -

  Fig. S6. Localization of dynein heavy chain to kinetochores in wild type and nudE^39A mutant neuroblasts treated with colchicine. These merged images show dynein heavy chain in red, and DNA in blue. Scale bar: 5 µm.

• Supplemental Figure S7 -

  Fig. S7. Distribution of Cenp-meta protein in wildtype spermatocytes. In the merged images, Cenp-meta is red, α-tubulin is green, and DNA is blue. As with NudE (see Fig. 2 in the text), Cenp-meta is mostly localized to the nuclear envelope during meiosis, except when the nuclear envelope is disrupted. Cenp-meta accumulates strongly on the kinetochores during meiotic prometaphase/metaphase; in contrast with NudE, ‘streaming along kMTs is not observed. During meiotic anaphase, Cenp-meta staining is restricted to the spindle envelope region, as is also true of NudE (see Fig. 2). Scale bar: 10 µm.

• Supplemental Figure S8 -

  Fig. S8. Disruption of Cenp-meta association with kinetochores in colchicine-treated spermatocytes from nudE^39A homozygous mutant larvae. In the merged images, Cenp-meta is in red, while DNA is shown in blue. Cenp-meta accumulates at high levels on the kinetochores of chromosomes in wild type larvae treated with the MT-disrupting drug colchicine. Although this drug increases the kinetochore targeting of many proteins, 70% of nudE^39A spermatocytes fail to show any Cenp-meta at kinetochores (bottom row), while a weak kinetochore signal is seen in 30% of spermatocytes (third row). The resolution in some meiotic figures is sufficient to discriminate XY from 4^th chromosome from autosomal (A=2^nd or 3^rd chromosome) bivalents. Scale bar: 10 µm.

• Supplemental Figure S9 -

  Fig. S9. Association of Asp with MT minus ends in nudE^39A neuroblasts. (a-c) Wild type; (d-f) nudE^39A. In the merged panels, the MT-associated protein Asp is red, tubulin is green, and DNA is blue. Asp accumulates at the MT minus ends in both wild type and mutant brain cells. This localization is generally in the vicinity of the centrosomes, but in nudE mutants, particularly during metaphase, the distribution of Asp is more diffuse because the spindle is splayed into separate bunches that apparently connect with individual chromosomes. Scale bar: 10 µm.

• Supplemental Figure S10 -

  Fig. S10. Association of Asp with MT minus ends in nudE^39A spermatocytes. In the merged panels, Asp is red, tubulin is green, and DNA is blue. Asp accumulates at the spindle poles in wild type and nudE mutant spermatocytes. Note in addition the ‘spindle-in-the-spindle’ phenotype characteristic of nudE spermatocytes that was previously presented in Figs. 4 and 5. In some cells, Asp can be found at the poles of the inner of the two spindles (middle row). Scale bar: 10 µm.