Supplemental Materials
This article contains the following supporting material:
- Supplemental Materials
- Movie 1 - Supplemental Movie 1: Actin bundle alignment is proportional to cell stretch. Confocal maximum intensity projections of two ovulation movies showing spermathecal actin labeled with GFP (GFP::ACT-1) in a slightlystretched spermatheca (top) and normally stretched spermatheca (bottom). Time is indicated in sec. Movie frame rate is 10 frames/sec. Scale bar: 20 μm.
- Movie 2 - Supplemental Movie 2: Actin bundle alignment coincides with cell contraction during ovulation.Confocal maximum intensity projections of an ovulation movie of a wild-type spermathecaexpressing GFP labeled actin (GFP::ACT-1). The spermatheca is compact and becomes stretched upon oocyte entry (∼350 sec). At this time the webby immature actin network is present. Contraction and actin reorganization can be seen at ∼896 sec. By ∼1246 sec prominent parallel bundles are present. These bundles become clearer as the movie progresses. Time is indicated in sec. Movie frame rate is 10 frames/sec. Scale bar: 20 μm.
- Movie 3 - Supplemental Movie 3: Loss of contraction in phospholipase C null animals delays actin bundle production and alignment.Confocal maximum intensity projections of an ovulation movie of a plc-1(rx1) null spermathecaexpressing GFP labeled actin (GFP::ACT-1). The spermatheca is compact and becomes stretched upon oocyte entry (∼42 sec). At this time the webby immature actin network is present. This network is maintained throughout the time observed and parallel, prominent actin bundles do not develop.Time is indicated in sec. Movie frame rate is 10 frames/sec. Scale bar: 20 μm.
- Movie 4 - Supplemental Movie 4: Depletion of myosin phosphataserescues timely actin bundle formation but not orientation in phospholipase C null spermathecae. Confocal maximum intensity projections of an ovulation movie of a plc-1(rx1) null spermatheca treated with RNAi against myosin phosphatase, mel-11, expressing GFP labeled actin (GFP::ACT-1). The spermatheca is compact and becomes stretched upon oocyte entry (∼266 sec). At this time the webby immature actin network is present. Contraction and actin reorganization can be seen at ∼630 sec. During contraction, prominent bundles become apparent. These bundles become clearer as the movie progresses. However, bundles that are not properly aligned parallel to the long axis of the cell are maintained throughout the time observed. Time is indicated in sec. Movie frame rate is 10 frames/sec. Scale bar: 20 μm.
- Movie 5 - Supplemental Movie 5: Myosin is highly dynamic during ovulation in wild-type animals. Confocal maximum intensity projections of an ovulation movie of a wild-type spermathecaexpressing moeABD::mCherry to labeled actin, red (top), and GFP labeled myosin, GFP::NMY-1, green (middle). Yellow indicates colocalization of actin and myosin (bottom). The spermatheca is compact and becomes stretched upon oocyte entry (∼40 sec). At this time GFP myosin is diffuse throughout the cytosol. A subpopulation of GFP::NMY-1 is rapidly recruited to actin bundles as cells contract and the oocyte is expelled from the spermatheca (∼300 sec). The spermatheca returns to a compact conformation once the oocyte is expelled (∼540 sec).Time is indicated in sec. Movie frame rate is 10 frames/sec. Scale bar: 20 μm.
- Movie 6 - Supplemental Movie 6: Elevated myosin activity alters myosin recruitment to and organization within actomyosinbundles. Myosin activity was elevated by RNAi knock down of the myosin phosphatase, mel-11. Confocal maximum intensity projections of an ovulation movie of a mel-11(RNAi) spermathecaexpressing moeABD::mCherry to labeled actin, red (top), and GFP labeled myosin, GFP::NMY-1, green (middle). Yellow indicates colocalization of actin and myosin (bottom). The spermatheca is compact and becomes stretched upon oocyte entry (∼60 sec). At this time,GFP::NMY-1 is rapidly recruited to actin bundles and begins to form clusters along actin bundles that are clearly visible by 320 sec. These myosin clusters increase in intensity and decrease in frequency as smaller clusters fuse together (compare myosin clusters at 320 sec to 1280 sec). Contraction continues and disruption of basal cell-cell contacts is evident by 1260 sec (see small bleb at the ventral side of the spermatheca). The basal layer pulls back (1340 sec) and tissue integrity is maintained until cell-cell contacts rupture completely (1520 sec). Time is indicated in sec. Movie frame rate is 10 frames/sec. Scale bar: 20 μm.