The FHA domain determines Drosophila Chk2/Mnk localization to key mitotic structures and is essential for early embryonic DNA damage responses

Supplemental Materials

This article contains the following supporting material:

• Supplemental Materials
• Movie 3 - Movie S3. Wild type DNA damage response in a w1118; PmTb-GAL4VP16 embryo. Rhodamine-Tubulin and bleomycin were injected. Rhodamine-Tubulin signal was captured by laser scanning confocal microscopy with 10 sec. interval. 7 frames/sec. Selected frames are shown in Figure 2B.
• Movie 20 - Movie S20. EGFP-RSK does not restore DNA damage responses in mnk mutant embryo. It does not form foci on mitotic chromosomes with DNA damage. Rhodamine-Tubulin and bleomycin were injected. Rhodamine (red) and EGFP (green) signals were captured by laser scanning confocal microscopy with 10 sec. interval. 7 frames/sec. Selected frames are shown in Figure 7C.
• Movie 18 - Movie S18. EGFP-RSK localization without DNA damage. EGFP signal was captured by laser scanning confocal microscopy with 10 sec. interval. 7 frames/sec. Selected frames are shown in Figure 7B, EGFP-RSK.
• Movie 7 - Movie S7. EGFP-D303A localization without DNA damage. EGFP signal was captured by laser scanning confocal microscopy with 10 sec. interval. 7 frames/sec. Selected frames are shown in Figure 4B.
• Movie 1 - Movie S1. EGFP-Mnk localization during the syncytial blastoderm stage without DNA damage. EGFP-Mnk signal was captured by spinning-disk confocal microscopy with 5 sec. interval. 14 frames/sec. Selected frames are shown in Figure 1A.
• Movie 2 - Movie S2. Normal spindle formation and cell cycle progression in a w1118; mnk embryo injected with Rhodamine-Tubulin. Rhodamine signal was captured by laser scanning confocal microscopy with 10 sec. interval. 7 frames/sec. Selected frames are shown in Figure 2A.
• Movie 15 - Movie S15. EGFP-Δ47-163 does not restore DNA damage responses in mnk mutant embryo. It does not localize to centrosomes and other mitotic structures, but localizes to the interphase nucleus. Rhodamine-Tubulin and bleomycin were injected. Rhodamine (red) and EGFP (green) signals were captured by laser scanning confocal microscopy with 10 sec. interval. 7 frames/sec. Selected frames are shown in Figure 6A.
• Movie 10 - Movie S10. EGFP-Δ26-46 localization without DNA damage. EGFP signal was captured by laser scanning confocal microscopy with 10 sec. interval. 7 frames/sec. Selected frames are shown in Figure 4E.
• Movie 9 - Movie S9. EGFP-Δ470-476 localization without DNA damage. EGFP signal was captured by laser scanning confocal microscopy with 10 sec. interval. 7 frames/sec. Selected frames are shown in Figure 4D.
• Movie 5 - Movie S5. EGFP-Mnk is functional to restore DNA damage responses in mnk mutant embryo. It localizes to the nucleus and centrosomes, and forms numerous foci/aggregates on mitotic chromosomes with DNA damage. Rhodamine-Tubulin and bleomycin were injected. EGFP-Mnk Rhodamine (red) and EGFP (green) signals were captured by laser scanning confocal microscopy with 10 sec. interval. 7 frames/sec. Selected frames from Rhodamine channel are shown in Figure 2D and selected frames of a representative mitosis are shown in Figure 5A.
• Movie 19 - Movie S19. EGFP-R73A does not restore DNA damage responses in mnk mutant embryo. It does not form foci on mitotic chromosomes with DNA damage. Rhodamine-Tubulin and bleomycin were injected. Rhodamine (red) and EGFP (green) signals were captured by laser scanning confocal microscopy with 10 sec. interval. 7 frames/sec. Selected frames are shown in Figure 7C.
• Movie 13 - Movie S13. EGFP-Δ1-25 does not restore DNA damage responses in mnk mutant embryo. It localizes similarly to EGFP-Mnk. Rhodamine-Tubulin and bleomycin were injected. Rhodamine (red) and EGFP (green) signals were captured by laser scanning confocal microscopy with 10 sec. interval. 7 frames/sec. Selected frames are shown in Figure 6A.
• Movie 11 - Movie S11. EGFP-Δ47-163 localization without DNA damage. EGFP signal was captured by laser scanning confocal microscopy with 10 sec. interval. 7 frames/sec. Selected frames are shown in Figure 4F.
• Movie 16 - Movie S16. EGFP-Δ470-476 does not restore DNA damage response in mnk mutant embryo. It localizes to centrosomes, and forms numerous foci/aggregates on mitotic chromosomes with DNA damage. It is not actively imported into the nucleus. Rhodamine-Tubulin and bleomycin were injected. Rhodamine (red) and EGFP (green) signals were captured by laser scanning confocal microscopy with 10 sec. interval. 7 frames/sec. Selected frames of a representative mitosis are shown in Figure 5C.
• Movie 22 - Movie S22. H2B-EGFP-Mnk localization without DNA damage. It is tethered to chromatin throughout the cell cycle. EGFP signal was captured by by laser scanning confocal microscopy with 10 sec. interval. 7frames/sec. Selected frames are shown in Supplemental Figure S2, B.
• Movie 6 - Movie S6. EGFP-D303A (KD) does not restore DNA damage response in mnk mutant embryo. It localizes to the nucleus and centrosomes, and forms numerous foci/aggregates on mitotic chromosomes with DNA damage. Rhodamine-Tubulin and bleomycin were injected. Rhodamine (red) and EGFP (green) signals were captured by laser scanning confocal microscopy with 10 sec. interval. 7 frames/sec. Selected frames from Rhodamine channel are shown in Figure 2E and selected frames of a representative mitosis are shown in Figure 5B.
• Movie 4 - Movie S4. mnk mutant embryo does not show normal DNA damage response. Rhodamine-Tubulin and bleomycin were injected. Rhodamine-Tubulin signal was captured by laser scanning confocal microscopy with 10 sec. interval. 7 frames/sec. Selected frames are shown in Figure 2C.
• Movie 8 - Movie S8. EGFP-Δ1-25 localization without DNA damage. EGFP signal was captured by laser scanning confocal microscopy with 10 sec. interval. 7 frames/sec. Selected frames are shown in Figure 4C.
• Movie 21 - Movie S21. EGFP-Mnk and mRFP-Rod localization without DNA damage. EGFP (green) and mRFP (red) signals were captured by laser scanning confocal microscopy with 5 sec. interval. 14 frames/sec. Selected frames of a representative mitosis are shown in Figure 1E.
• Movie 17 - Movie S17. EGFP-R73A localization without DNA damage. EGFP signal was captured by laser scanning confocal microscopy with 10 sec. interval. 7 frames/sec. Selected frames are shown in Figure 7B, EGFP-R73A.
• Movie 12 - Movie S12. EGFP-26-163 localization without DNA damage. EGFP signal was captured by laser scanning confocal microscopy with 10 sec. interval. 7 frames/sec. Selected frames are shown in Figure 4G.
• Movie 14 - Movie S14. EGFP-Δ26-46 does not restore DNA damage responses in mnk mutant embryo. It localizes to the nucleus and centrosomes, but excluded from the vicinity of mitotic chromosomes. Rhodamine (red) and EGFP (green) signals were captured by laser scanning confocal microscopy with 10 sec. interval. 7 frames/sec. Selected frames of a representative mitosis is shown in Figure 5D.