Supplemental Material

Time-lapse videos of spindle and nuclear division defects caused by disruption of Ran pathway are provided as supplemental videos. Videos 1 and 2 are control embryos that show normal spindle assembly and nuclear division respectively. Videos 3 and 4 show defects on spindle assembly upon injection of RanT24N. Video 5a shows defects in spindle assembly upon injections of importins compared to video 5b that shows a control injected embryo, and Video 6 shows defects on spindle assembly upon injection of RanBP1. Videos 7 and 8 show defects on nuclear division upon injection of RanT24N and RCC1 respectively, and video 9 shows defects on midbody assembly upon injection of Ran T24N. QuickTime movies were produced using MetaMorph software.

Supplemental figure 1 shows the specificity of anti-Aurora A antibody and the quantification of Ran in Drosophila embryos. The detailed phenotype frequency induced by different injections that disrupt Ran pathway are presented in supplemental Tables. Table S1 contains effects on spindles, Table S2 contains effects on nuclear division, and Table S3 contains effects on midbodies.

This article contains the following supporting material:

• Figure 1 - (A) Western blot for the anti-aurora A kinase antibody shows a single band of 50 kDa after it was affinity purified. (B) Ran represents 0.06% of total Drosophila embryo proteins. Different concentrations of RanL43E and Drosophila embryo extracts were probed with anti-Ran antibody by Western blot.

• Movie 1 - Microtubule organization in a control Drosophila embryo expressing GFP-tubulin.
  Frames collected every 8 s are played at 6 frames/s. Figure 2A contains images from Video 1.

• Movie 2 - Chromatin organization in a control Drosophila embryo expressing GFP-histone.
  Frames collected every 8s are played at 6 frames/s. Figure 5B contains images from Video 2.

• Movie 3 - Microtubule organization in a GFP-tubulin expressing Drosophila embryo injected with 32 mg/ml RanT24N.
  Microtubules grow outward probing the environment. Frames collected every 8s are played at 6 frames/s.

• Movie 4 - Microtubule organization in a GFP-tubulin expressing Drosophila embryo injected with 32 mg/ml RanT24N.
  Multipolar spindles. Frames collected every 8s are played at 6 frames/s.

• Movie 5a - Microtubule organization in a GFP-tubulin expressing Drosophila embryo injected with importin α+ΔN importin β.
  Spindles start to form from around chromosomes. Frames collected every 8s are played at 6 frames/s. Figure 3C contains images from Video 5a.

• Movie 5b - Microtubule organization in a GFP-tubulin expressing Drosophila control embryo.
  Spindles start to form from centrosomes. Frames collected every 8s are played at 6 frames/s. Figure 3C contains images from Video 5b.

• Movie 6 - Microtubule organization in a GFP-tubulin expressing Drosophila embryo injected with RanBP1.
  Spindles exhibited multiple successive defects: centrosome release, MT probing the environment rather than rapidly growing towards the chromosomes as seen in control embryos, and spindle shortening. Frames collected every 8s are played at 6 frames/s. Figure 3D contains images from Video 6.

• Movie 7 - Chromatin organization in a GFP-histone expressing Drosophila embryo injected with 32 mg/ml RanT24N.
  Chromosomes from adjacent nuclei fuse. Frames collected every 8s are played at 6 frames/s. Figure 5F contains images from Video 7.

• Movie 8 - Chromatin organization in GFP-histone expressing Drosophila embryo injected with RCC1.
  Metaphase chromosomes do not disjoin, they decondense. Frames collected every 8s are played at 6 frames/s.

• Movie 9 - Post-metaphase microtubule organization in a GFP-tubulin expressing Drosophila embryo injected with 32 mg/ml RanT24N.
  After the formation of morphologically normal spindles, the midbodies fail to bundle correctly and become O shaped. Frames collected every 8s are played at 6 frames/s.

• Table 1
• Table 2
• Table 3