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Supplementary Material

DEV01916 Supplementary Material

Files in this Data Supplement:

  • Supplemental Table (Adobe PDF) -
  • Supplemental Figure 1 - Fig. S1.Time course of MSP release in wild-type hermaphrodites. MSP (red) and DNA (blue) detected on the indicated days of adulthood. (A) Day 1, MSP labeling is partitioned between spermatids in the gonad arm (arrowhead) and spermatozoa (arrows) in the spermatheca (sp). Extracellular MSP is most apparent in the gonad arm (thin arrow). (B) Day 2, few spermatids remain in the gonad arm (arrowhead), and most extracllular MSP is present in the spermatheca (thin arrows). (C) Day 3, extracellular MSP is largely confined to the spermatheca (thin arrows), with only a slight haze over the �1 oocyte. (D,E) Day 4 and 5, MSP and spermatozoa are barely detectable, and oocytes stack in the gonad arm. Scale bar: 20 mm.
  • Supplemental Figure 2 - Fig. S2. MSP release in emo-1/sec-61g(oz1) hermaphrodites. (A,D) MSP staining in dissected gonads from two adult emo-1(oz1) adult hermaphrodites (24 hours post L4). Only the proximal gonad arm is shown. (B,E) DAPI-stained images. (C,F) Merged images. Punctate (arrowheads) and diffuse (arrows) extracellular MSP is observed in the proximal gonad arm. Note the MSP staining in a region of the gonad devoid of nuclei (arrow). We also noticed a quantitative and qualitative difference in intracellular MSP staining in emo-1(oz1) mutant spermatids. The mutant spermatids appeared to contain less MSP, which was largely cortically localized. Notably, in secondary spermatocytes (inset in F shows a longer exposure), the MSP was also cortically localized. In spermatocytes, the MSP did not appear to be localized to fibrous bodies as in the wild type. Whether the fibrous body-membranous organelle complex forms properly in emo-1(oz1) mutants will need further investigation, including electron microscopy. sp, spermatheca. Scale bars: 10 mm.
  • Supplemental Figure 3 - Fig. S3.A vesicle budding model for MSP release. According to this model, spermatozoa (A) and spermatids (C) bud MSP vesicles from the cell body. We propose that the stability of the MSP vesicles governs the range and duration of signaling. Spermatozoa generate long-range MSP gradients from the spermatheca for signaling at the oocyte cell surface. Spermatids provide a temporally long-acting signal locally from within the gonad arm. (B) A model, shown in cross-sectional view, for a mechanism by which MSP generates the protrusive force for its own release. MSP localizes to the plasma membrane of the cell body and in protrusions. While there is a dearth of EM data concerning the final budding stage, it must involve more than a simple fusion event.
  • Movie 1 - Movie 1.Punctate distribution of extracellular MSP in the spermatheca. QuickTime (800 KB) projection of a confocal 3D data stack of MSP (red) in a dissected gonad from a mated female (see Fig. 2F, left panel). Spermatozoa in the spermatheca and the spermathecal-uterine junction region are the round and elliptical structures. MSP staining is observed outside of spermatozoa, and is both punctate and diffuse.
  • Movie 2 - Movie 2.Punctate distribution of extracellular MSP in the uterus. QuickTime (4.4 MB) projection of confocal 3D data stack of MSP (pink) and DNA (red) visualized in a whole-mount preparation of a mated female (see Fig. 2F, right panel). Spermatozoa in the uterus are the large round or elliptical regions of MSP staining surrounding the sperm chromatin (red dot). Note the large MSP puncta in the uterus. Diffuse and punctate MSP pools can be seen around embryos in the uterus (large ovoid clear spaces). Note the absence of MSP in the distal arm of the gonad.
  • Movie 3 - Movie 3. MSP localizes on the surface of and within the �1 oocyte. QuickTime (5.8 MB) projection of 3D data stack of MSP (red) and RME-2 (green) staining in a dissected gonad from a mated female (see Fig. 3A). MSP is localized at the surface of the oocyte, both just outside and also coincident with the RME-2 signal. Some MSP staining is internal to the oocyte, suggestive of endocytosis.
  • Movie 4 - Movie 4. 3D reconstruction of RME-2 and MSP localization on �1 oocyte. QuickTime (4.4 MB) movie of a reconstruction of MSP (red) RME-2 (green) staining in a mated female, focusing on the �1 oocyte. The movie is a reconstruction of the data stack represented in Fig. 3A. The oocyte has been cut both parallel and orthogonal to the proximodistal axis. Three individual views of the movie are shown in Fig. 3B. Move the cursor on the movie to play, and rotate through 360� to visualize MSP at the surface and within the �1 oocyte.
  • Movie 5 - Movie 5. Apparent budding of MSP puncta from spermatozoa. QuickTime (4.6 MB) projection of a confocal 3D data stack of MSP (red) and MSD (green) proteins localized in a whole-mount preparation of a mated female. The DIC channel is shown in blue. The yellow elliptical structures are spermatozoa in extracellular spaces surrounding embryos in the uterus. The red staining at a distance from the spermatozoa corresponds to extracellular MSP puncta. Note the red blebs at the margins of the spermatozoa. The MSD proteins are excluded from these apparent budding structures.
  • Movie 6 - Movie 6. 3D reconstruction of MSP-containing buds from spermatozoa. QuickTime (3.1 MB) movie of a 3D reconstruction of MSP budding structures from a spermatozoa. MSP (red) and MSD (green) were visualized in whole-mount preparations of mated female. The movie shows a cylindrical slice of a spermatozoan in the uterus. MSP and MSD proteins co-localize in spermatozoa, except in the MSP projections. Note the free MSP puncta. Put cursor on the movie to rotate through 360�.
  • Movie 7 - Movie 7. Spermatids generate extracellular MSP in the punctate form in the gonad arm. QuickTime (208 KB) projection of a confocal 3D data stack of MSP (red) in a dissected gonad from a spe-8(hc50) hermaphrodite (data from Fig. 6B). A single spermatid (round, red object) is seen in the gonad arm. Note the wide distribution of MSP puncta in the gonad arm. The spermatid is approximately 5 mm.