JCS03183 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Rabl orientation in eye imaginal discs. Projected image of undifferentiated nuclei from the eye imaginal disc. Nuclei with chromosomes in Rabl orientation are outlined (cyan) along with those that are not (yellow). Nuclei were categorized as being in Rabl orientation based on the clustering of centromeres (CIDGFP signal) at one pole of the nucleus and a chromosome tip (mRFP signal) at the opposite pole.

• Supplemental Figure 2 -

  Fig. S2. MSD plots for movies where images were taken every 30 seconds. The average mean square change in distance <Δd²> (±1 s.e.m.) over all possible Δt values was plotted for time-lapse images taken every 30 seconds for a total of 300-420 seconds. Image analysis was done in 3D. The plateau height on the <Δd²> graph is used to calculate the radius of confinement. The graphs for bw⁺ as well as IU bw^D nuclei did not appear to reach a plateau with an increase in the value of Δt. In contrast, the graph for IA bw^D nuclei reached a plateau and was highly constrained.

• Supplemental Figure 3 -

  Fig. S3. Movement of Drosophila centromeres is highly confined. The average mean square change in distance <Δd²> (±1 s.e.m.) over all possible Δt values was plotted for the movement of the two closest CIDGFP signals from movies with Δt=3 seconds. The graph for CID-mRFP from IA bw^D nuclei is included so that the high degree of similarity between the two plots can be noted.

• Supplemental Figure 4 -

  Fig. S4. False sense of movement upon analysis in 2D because of nuclear rotation. Distance measurements were done in 2D as well as 3D for movies where images were taken every 3 minutes. Graphs of the MSD (±1 s.e.m.) for all possible values of Δt are shown for the analysis in 2D (A) as well as in 3D (B). Although a similar trend for movement was obtained, because of nuclear rotation the magnitude of change in <Δd²> was larger in 2D as opposed to the analysis done in 3D, and in order to get the true displacement this necessitated all analysis of the longer movies in 3D.

• Movie 1 -

  Movie 1. Time-lapse movie of undifferentiated bw^D lacO/+ nuclei present anterior to the morphogenetic furrow in the eye imaginal disc. Images were taken every 3 seconds in 2D for a total of 45 seconds (15 time points). The movie plays approximately 1.5 frames per second. The background fluorescence of the unbound mRFP-LacI fusion protein marks the nucleus (purple), the bw^D region tagged with lacO repeats is the intense purple dot and the centromeres tagged by CID-GFP are the green dots.

• Movie 2 -

  Movie 2. Time-lapse movie of undifferentiated bw^D lacO/+ nuclei present anterior to the morphogenetic furrow in the eye imaginal disc. Images were taken as 3D stacks every 30 seconds for a total of 450 seconds (15 time points). The projected images are shown here. The movie plays approximately 1.5 frames per second. The background fluorescence of the unbound mRFP-LacI fusion protein marks the nucleus (purple), the bw^D region is the more intense purple dot and the centromeres tagged by CID-GFP can be visualized as the green dots.

• Movie 3 -

  Movie 3. Time-lapse movie of undifferentiated bw^D lacO/+ nuclei present anterior to the morphogenetic furrow in the eye imaginal disc. Images were taken as 3D stacks every 3 minutes for a total of 120 minutes (40 time points). The projected images are shown here. The movie plays approximately four frames per second. The background fluorescence of the unbound mRFP-LacI fusion protein marks the nucleus (purple), the bw^D locus can be visualized as the more intense purple dot and the centromeres tagged by CID-GFP can be visualized as the green dots.

• Movie 4 -

  Movie 4. Time-lapse movie of an undifferentiated bw^D lacO/+ nucleus in the bw^D IU category, from Movie 3. Images were taken as 3D stacks every 3 minutes for a total of 120 minutes (40 time points). The projected images are shown here. The movie plays approximately four frames per second. The background fluorescence of the unbound mRFP-LacI fusion protein marks the nucleus (purple), the bw^D locus is the more intense purple dot and the centromeres tagged by CID-GFP can be visualized as the green dots.

• Movie 5 -

  Movie 5. Time-lapse movie of an undifferentiated bw^D lacO/+ nucleus in the bw^D IA category, from Movie 3. Images were taken as 3D stacks every 3 minutes for a total of 120 minutes (40 time points). The projected images are shown here. The movie plays approximately four frames per second. The background fluorescence of the unbound mRFP-LacI fusion protein marks the nucleus (purple), the bw^D locus is the more intense purple dot and the centromeres tagged by CID-GFP can be visualized as the green dots.

• Movie 6 -

  Movie 6. Time-lapse movie of an undifferentiated bw^D lacO/+ nucleus undergoing nuclear rotation, from Movie 3. Images were taken as 3D stacks every 3 minutes for a total of 120 minutes (40 time points). The projected images are shown here. The movie plays approximately four frames per second. The background fluorescence of the unbound mRFP-LacI fusion protein marks the nucleus (purple), the bw^D locus can be visualized as the more intense purple dot and the centromeres tagged by CID-GFP can be visualized as the green dots.