Supplemental Material

This article contains the following supporting material:

• Movie 1 - Membrane dynamics in WT embryo. Membrane has been labeled with the fluorescent dye FM 2-10. The plasma membrane is labeled and this label is also endocytosed. The cytokinetic furrow ingresses between the daughter nuclei and an accumulation of membrane can be observed at the site of scission, after the two sides of the ingressing furrow have met. Anterior is left. Images were collected at 4.4 sec intervals and played back at 8 frames per second.
• Movie 2 - Membrane dynamics in car-1(RNAi) embryo. Membrane has been labeled with the fluorescent dye FM 2-10. The cytokinetic furrow ingresses between the daughter nuclei. The two sides of the furrow meet then ultimately regress with no extensive accumulation of membrane at the scission site. Following regression, the nuclei drift back together. Anterior is left. Images were collected at 4.4 sec intervals and played back at 8 frames per second.
• Movie 3 - Microtubule dynamics in control embryo. Microtubules are labeled with GFP::tubulin. As the spindle sets up and elongates, spindle microtubules are clearly visible between the two centrosomes. As the cell cycle progresses, microtubules in the path of the ingressing cytokinetic furrow form a tight bundle (midbody) which persists for some time. Anterior is left. Images were collected at 4.4 sec intervals and played back at 8 frames per second.
• Movie 4 - Microtubule dynamics in car-1(RNAi) embryo. Microtubules are labeled with GFP::tubulin. As the spindle sets up and elongates, the spindle microtubules collapse somewhat compared to the wild type. As the spindle elongates, there appears to be very few, if any, microtubules extending the full length between the centrosomes. As cytokinesis initiates, microtubules are present in the path of the ingressing furrow but they are not swept into a bundle as in the wild type. Anterior is left. Images were collected at 4.4 sec intervals and played back at 8 frames per second.
• Movie 5 - GFP::CAR-1 dynamics in the early embryo. An embryo expressing a GFP::CAR-1 fusion protein shows the developmental changes in the localization of this protein. During pronuclear migration, puncta can be seen in the posterior region near the male pronucleus. As the female pronucleus moves toward the posterior, occasional small puncta of GFP::CAR-1 can be seen. The size of the puncta in the posterior increase as the pronuclear pair centrates and rotates, again with the occasional transient small puncta appearing in the more anterior region. As the spindle forms it is lightly decorated with GFP::CAR-1 signal. During the 2 cell stage, the majority of the GFP::CAR-1 label is found in large puncta in the posterior P1 cell. However, as the spindle forms and elongates with the division of the anterior AB cell, there is an increase in the number of small puncta, which are quite distinct at the 4 cell stage. Anterior is left. Images were collected at 5 sec intervals and played back at 8 frames per second.
• Movie 6 - Spindle association of GFP::CAR-1. The spindle region in an embryo expressing a GFP::CAR-1 fusion protein illustrating the accumulation of CAR-1 onto the spindle midzone during mitosis. Anterior is left. Images were collected at 4.5 sec intervals and played back at 8 frames per second.
• Movie 7 - ER dynamics in control embryo. ER exhibits a dispersed organization during pronuclear migration but becomes highly ordered at the onset of mitosis. The ER outlines the spindle during metaphase then moves into the spindle region as the spindle elongates. When cytokinesis begins, the reticulate organization rapidly returns to the dispersed state that is maintained during interphase. Anterior is left. Images were collected 5 sec intervals and played back at 8 frames per second.
• Movie 8 - ER dynamics in car-1(RNAi) embryo. In this embryo the ER exhibits an increase in the amount of ER in patches and thick strands, both in interphase and in mitosis. The ER attempts to form a higher order reticulate structure during mitosis but is unable to form the same structure observed in the control. Furthermore, the ER is not well maintained around the mitotic spindle nor in the spindle midzone. The patches and thick strands of ER are still observed when the embryo is in interphase following the failure of cytokinesis. Anterior is left. Images were collected 5 sec intervals and played back at 8 frames per second.
• Figure 1 - Probes for CAR-1 and DCAP-1 exhibit specificity. (A) GFP signal was eliminated from GFP::CAR-1 expressing embryos exposed to car-1 RNAi. (B) αCAR-1 antibody label was reduced in car-1(RNAi) embryos. Upper row shows αCAR-1 labeling while the lower row shows αtubulin antibody labeling of the same embryo as an antibody penetration control. Western blot showing the absence of a CAR-1 band in car-1(RNAi) worms. Actin served as a loading control. (C) αDCAP-1 labeling was reduced in dcap-1(RNAi) embryos. Upper row shows αDCAP-1 labeling while the lower row shows αtubulin antibody labeling of the same embryo as an antibody penetration control. The Western blot shows the absence of a DCAP-1 band in dcap-1(RNAi) worms. Actin served as a loading control. Scale bar = 10 microns. Western blots: Western blots were performed by boiling 30 adult worms per sample in Laemmli Sample Buffer plus 5% βmercaptoethanol (BioRad, Hercules CA) for 5 min after rinsing 3 times with M9 medium (Sulston and Hodgkin, 1988). The samples were subjected to standard SDS-PAGE (10%) electrophoresis and transfer blotting (Sambrook and Russell, 2001) using a BioRad (Hercules, CA) Mini-Protean 3 apparatus. The blot was labeled with either αCAR-1 or αDCAP-1 antibody (1:500 or 1:5000, respectively). The antibody on the blot was detected using the Pierce chemiluminescent kit (Pierce, Rockford IL). The blots were stripped (Pierce, Rockford IL) and re-probed with an αactin antibody (1:10,000) (C4: ICN Biomedical, Costa Mesa CA) as a loading control. The developed films from the sequential blots were scanned and super-imposed for presentation.
• Figure 2 - CAR-1 puncta associate with ER structures. Multiphoton images show that CAR-1 puncta (red) exhibit an association with strands of ER (green). Scale bar = 10 microns. We found that the small CAR-1 puncta are generally situated on or next to ER structures (Fig 4A) (76% of CAR-1 puncta associated with ER; N=606 puncta in 20 cells of 9 embryos). The ER in these cells occupied an average of 59% of the cross-sectional area of the cell. The significantly higher percentage of associated puncta compared to the amount of ER (p<0.001 by Chi Squared analysis) suggests that this association was not merely random. The extent of CAR-1 puncta association with ER structures was determined in fixed GFP::SP12 expressing embryos (4 to 16 cell stage) labeled with αCAR-1 antibody. The CAR-1 puncta were categorized as either associated (>10% overlap) or not associated (no overlap) with ER structures. The area of the cell occupied by the ER was determined by thresholding the image in the green emission channel using ImageJ, determining the number of ER-positive pixels and dividing it by the total number of pixels in that cell (excluding the nucleus). P cells were not included in the analysis to minimize confusion with P granules.
• Figure 3 - The localization of CAR-1 is not BFA-sensitive. Multiphoton time series showing the localization of GFP::CAR-1 in an embryo treated with BFA. Top row shows fluorescence while lower row shows corresponding brightfield. Embryo was ablated in 150 μg/ml BFA (Molecular Probes, Eugene, OR) during the first division. Times are from initiation of imaging, which is within 5 min following permeablization of the eggshell by laser ablation. CAR-1 localized in a manner similar to that observed in untreated embryos (see Figure 3): to P granules (arrowhead), small puncta (arrow) and the spindle (white bracket). The AB and P1 cytokinetic furrows appear to complete (middle panels) but ultimately regress (last panel), indicating the drug treatment was effective. Scale bar = 10 microns.