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Fig. S1. Kinetochore components are recruited independently of lis-1 and dhc-1. (A-I) Wild-type (A, D, G, J), lis-1(t1550) (B, E, H) or dhc-1(RNAi) (C, F, I) embryos fixed during anaphase or equivalent stage and stained with antibodies against either the kinetochore component HCP-4 (A-C), BUB-1 (G-I) or the kinetochore-dependent chromosomal passenger protein ICP-1 (D-F) as well as a-tubulin. Upper panels show kinetochore component staining, lower panels the merge of kinetochore component (red), a-tubulin (green), and Hoechst counterstain to view DNA (blue). Images are projections of up to three consecutive 1 mm thick confocal slices. Bar=10 mm. Note that these three components are recruited to chromosomes in the absence of dhc-1 or lis-1 function. BUB-1 and ICP-1 are known to localize transiently to chromosomes in wild-type and then accumulate on the central spindle after the metaphase to anaphase transition as illustrated here for ICP-1 (D). We found that BUB-1 and ICP-1 localization to chromosomes is also transient when dhc-1 or lis-1 is inactivated although subtle timing differences in their release from chromosomes cannot be excluded.
Movie 1. DIC time-lapse movie of wild-type one-cell stage embryo. Capture rate is 1 frame per 5 seconds. Display rate is 12 frames per seconds.
Movie 2. DIC time-lapse movie of homozygous lis-1(t1550) one-cell stage embryo. Capture rate is 1 frame per 5 seconds. Display rate is 12 frames per second. Whereas the male and female pronuclei form in the correct locations, the small yolk granule-depleted regions that correspond to the two centrosomes remain at the posterior cortex (compare with wild-type, Movie 1). Migration of the male and female pronuclei does not take place and a bipolar spindle does not assemble. Note also the yolk granule-depleted region that corresponds to microtubules emanating from the centrosomes and extending towards the anterior of the embryo following breakdown of the male pronuclear envelope.
Movie 3. DIC time-lapse movie of lis-1(RNAi) one-cell stage embryo. Capture rate is 1 frame per 5 seconds. Display rate is 12 frames per second. See movie 2 for description of the sequence of events.
Movie 4. DIC time-lapse movie of wild-type one-cell stage embryo filmed between nuclear envelope breakdown and mid anaphase. Capture rate is 1 frame per 0.5 seconds. Display rate is 12 frames per second (time stamp on images is not telling). Note rapid movements of individual yolk granules towards the center of the asters.
Movie 5. DIC time-lapse movie of zyg-9(it3) mutant one-cell stage embryo filmed between nuclear envelope breakdown and mid anaphase. Capture rate is 1 frame per 0.5 seconds. Display rate is 12 frames per second (time stamp on images is not telling). Note that rapid movements of yolk granules towards the center of the asters still take place, albeit less frequently than in the wild-type, presumably due to the fact that microtubule asters are less robust in the absence of zyg-9 function.
Movie 6. DIC time-lapse movie of lis-1(RNAi) one-cell stage embryo filmed between nuclear envelope breakdown and mid anaphase. Capture rate is 1 frame per 0.5 seconds. Display rate is 12 frames per second (time stamp on images is not telling). Note that rapid movements of yolk granules towards the center of the asters are essentially abolished.
Movie 7. DIC time-lapse movie of homozygous lis-1(t1550) one-cell stage embryo filmed between nuclear envelope breakdown and mid anaphase (time stamp on images is not telling). Capture rate is 1 frame per 0.5 seconds. Display rate is 12 frames per second. Note that rapid movements of yolk granules towards the center of the asters are essentially abolished. One rare occurrence of movement can be seen (starting at 09:20).
Movie 8. Nine consecutive 1 mm confocal slices of the P1 spindle in Fig. 3E. Separate immunostaining of LIS-1(red), a-tubulin (green), Hoechst counterstain to view DNA (blue) and the merge of these, is shown. Display rate is 2 frames per second. Note relative enrichment of LIS-1 in areas with higher microtubule densities.
Movie 9. Spinning disk confocal time-lapse movie of homozygous lis-1(t1550) one-cell stage embryo expressing GFP-LIS-1. Serial 2 mm confocal slices were captured at 5 seconds intervals. A single confocal section is shown. Display rate is 10 frames per second. Note transient enrichment of GFP-LIS-1 at the nuclear periphery in late prophase and along chromosomes during prometaphase. Moreover, GFP-LIS-1 is present in a more diffuse manner in the nucleus starting prior to apparent NEBD and coincident with the spindle thereafter.
Movie 10. Spinning disk confocal time-lapse movie of homozygous lis-1(t1550) four-cell stage embryo expressing GFP-LIS-1. Serial 2 mm confocal sections were captured at 5 seconds intervals. A single confocal section is shown. Display rate is 10 frames per second. Note transient enrichment of GFP-LIS-1 at the nuclear periphery in late prophase and subsequent entry into the nucleus in the ABa and ABp blastomeres.
Movie 11. FRAP analysis; confocal time-lapse movie of four-cell stage embryo derived from homozygous lis-1(1550) adult expressing GFP-LIS-1. The interval between frames is 10 seconds, except between frames 2 and 3, where it is ~2 seconds (duration of photobleaching). Display rate is 10 frames per second. Note very rapid recovery of fluorescence in photobleached area.
Movie 12. FRAP analysis; confocal time-lapse movie of wild-type embryo injected with FITC-labeled Dextran 70 kDa. The interval between frames is 10 seconds, except between frames 2 and 3, where it is ~2 seconds (duration of photobleaching). Display rate is 10 frames per second. Note very rapid recovery of fluorescence in photobleached area.
Movie 13. Spinning disk confocal time-lapse movie of icp-1(RNAi)-treated lis-1(t1550) mutant one-cell stage embryo expressing the GFP-LIS-1 transgene. Serial 2 mm confocal slices were captured at 5 seconds intervals. A single confocal section is shown. Display rate is 10 frames per seconds. Note that the transient enrichments of GFP-LIS-1 at the nuclear periphery in late prophase and along chromosomes during prometaphase are still present. Note also that GFP-LIS-1 becomes enriched significantly earlier in the male pronucleus than in the female pronucleus, which is characteristic of icp-1(RNAi) embryos.
Movie 14. Spinning disk confocal time-lapse movie of hcp-4(RNAi)-treated lis-1(t1550) mutant one-cell stage embryo expressing the GFP-LIS-1 transgene. Serial 2 mm confocal slices were captured at 5 seconds intervals. A single confocal section is shown. Display rate is 10 frames per seconds. Note that whereas the transient enrichment of GFP-LIS-1 at the nuclear periphery is still present, no enrichment along chromosomes can be detected. However, GFP-LIS-1 is still present in a diffuse manner in the nucleus starting prior to apparent NEBD and coincident with the spindle thereafter (compare with Movie 9).
Movie 15. Spinning disk confocal time-lapse movie of tba-2(RNAi)-treated lis-1(t1550) mutant one-cell stage embryo expressing the GFP-LIS-1 transgene. Serial confocal slices of 2mm were captured at 5 seconds intervals. A single confocal section is shown. Display rate is 10 frames per seconds. Note that the transient enrichments of GFP-LIS-1 at the nuclear periphery in late prophase and along chromosomes during prometaphase occur in a more pronounced manner than in the wild-type (compare with Movie 9). Note also that a single pronucleus is visible in this focal plane.
Movie 16. Confocal time-lapse movie of dhc-1(RNAi) one-cell stage embryo expressing GFP-TUB and GFP-HIS. Serial 2 mm confocal slices were captured at 26 seconds intervals. A single confocal section is shown. Display rate is 10 frames per seconds. Note that following breakdown of the male pronucleus, microtubules emanating from the two centrosomes appear to ‘push’ chromosomes from the male gamete towards the anterior.
Movies 17-19. Spinning disk confocal time-lapse movie of dhc-1(RNAi) (17), dnc-1(RNAi) (18) and dnc-2(RNAi)-treated (19), lis-1(t1550) mutant one-cell stage embryos expressing the GFP-LIS-1 transgene. Serial 2 mm confocal slices were captured at 5 second intervals. A single confocal section is shown. Display rate is 10 frames per seconds. Note that whereas the transient enrichments of GFP-LIS-1 along chromosomes are still present, no enrichment at the nuclear periphery can be detected. However, GFP-LIS-1 is still present in a diffuse manner in the nucleus starting prior to apparent NEBD.
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