Supplemental Materials

This article contains the following supporting material:

• Figure S1 - Inhibition of Dynein Intermediate Chain (DIC) disrupts Nuf localization at the MTOC. Affinity purified anti-DIC was injected into a GFP-Nuf (green) bearing embryo 5 minutes after the start of cellularization (interphase cycle 14). Rhodamine-labeled tubulin (red) was co-injected to visualize the MTOC. 20 minutes into cellularization, GFP-Nuf fails to concentrate at the MTOC, similar to the results seen for the anti-Dynein Heavy Chain (DHC) shown in Figure 6.
• Figure S2 - Control injection of 20% DMSO into GFP-Nuf bearing embryos at cellularization. GFP-Nuf bearing embryos were injected with 20% DMSO at the start of cellularization and examined for any effects on Nuf localization. At 5 minutes after injection, there was no change in Nuf levels at the MTOC. No reduction in Nuf levels were observed at later time points of 2 and 5 minutes post-injection. This result contrasts with our colchicine injections which greatly reduce Nuf levels at the MTOC (see Figures 1 and 2). Scale bar, 5µm
• Figure S3 - Control injection of 20% DMSO does not affect furrow initiation and elongation. GFP-Moesin embryos were injected with 20% DMSO precisely at anaphase and assayed for proper actin recruitment at the following prophase. At prophase actin was recruited properly to the site of furrow formation (top panels) and nuclear division proceeded normally. Scale bar, 5µm
• Figure S4 - Injection of PBS / 40% glycerol does not affect Nuf localization at the MTOC. GFP-Nuf bearing embryos were injected with PBS / 40% glycerol as a control for the antibody injections (Figures 5 and 6). Embryos were injected at the start of cellularization and Nuf localization was examined. At 0.5, 5, 10 and 20 minutes post-injection, we did not observe reductions in Nuf levels at the MTOC. Scale bar, 5µm
• Figure S5 - Actin cap formation is independent of microtubules. Colchicine was injected into a live GFP-Moesin bearing embryo at either anaphase or telophase of nuclear cycle 12 and assayed for proper actin cap formation and furrow initiation (see Figure 8A, B). Rhodamine-labeled tubulin (red) was also injected to monitor the effects of colchicine on the microtubule network. Panel A is an image of the actin caps (green) and B is an image of the furrow network in an uninjected GFP-Moesin bearing embryo at cycle 13 prophase. Injection of colchicine at anaphase of nuclear cycle 12 has no effect on proper actin cap formation at prophase of nuclear cycle 13 (panels C-D). Injecting colchicine at telophase of nuclear cycle 12 also has no effect on proper actin cap formation (panels E-F).
• Figure S6 - Dynein is necessary for furrow elongation: affinity purified anti-DHC antibodies were injected into a RLC-GFP bearing embryo, 20 minutes after the start of nuclear cycle 14. This kymograph represents a time lapse of a portion of an embryo at the site of injection (bottom panel) compared to normal furrow progression (top panel). Furrow progression is halted upon inhibition of cytoplasmic Dynein, however myosin ring organization is not altered and the rings are still able to pinch off at the end of cellularization (data not shown). Top panel bar: 30µm, Bottom panel bar: 13µm
• Movie M1 - Microtubules are required for the maintenance of Nuf at the MTOC. Movie depicting a GFP-Nuf bearing embryo (green) injected with the microtubule depolymerizing agent colchicine at the start of cellularization. Rhodamine-labeled tubulin (red) was also injected prior to the colchicine injection to mark the position of the MTOC. Images were recorded every 30 seconds.
• Movie M2 - Dynein is required for the proper localization of Nuf at the MTOC. Movie depicting a GFP-Nuf bearing embryo (green) injected with anti-DHC antibodies upon entry into cellularization. Rhodamine-labeled tubulin (red) was also injected prior to antibody injection to mark the position of the MTOC. Images were recorded every 30 seconds.
• Movie M3 - Microtubules are required at anaphase for the recruitment of actin to the site of furrow formation. Movie depicting a GFP-Moesin bearing embryo (green) injected with the microtubule depolymerizing agent colchicine at the beginning of anaphase. Rhodamine- labeled tubulin (red) was also injected prior to the colchicine injection the to mark the position of the spindle and the timing of the cell cycle. Images were recorded every 30 seconds.
• Movie M4 - Microtubules are not required at telophase for proper actin recruitment to the site of furrow formation. Movie depicting a GFP-Moesin bearing embryo (green) injected with the microtubule depolymerizing agent colchicine at the beginning of telophase. Rhodamine- labeled tubulin (red) was also injected prior to the colchicine injection to mark the position of the spindle and the timing of the cell cycle. Images were recorded every 30 seconds.
• Movie M5 - A RLC-GFP expressing embryo was injected with anti-DHC antibodies 10 minutes after the onset of cellularization and monitored by time-lapse confocal microscopy. The time-lapse started within 20 seconds following injection and lasted 67 minutes with an interval of 30 seconds.