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Files in this Data Supplement:
Fig. S1. Additional lectin staining. Concalavin A (A) and Pisum sativum agglutinin (B) labeled 1 μm vesicles (arrows) at the cortex in meiosis I embryos. Lens culinaris agglutinin (C) did not label these vesicles, and all three labeled a dense population of cytoplasmic vesicles. Soybean agglutinin (red, tubulin in green, DNA in blue) (D), Malcura pomifera agglutinin (MPA) (E) and Griffonia simplicifolia lectin I (GSL I) (F) stained the vitelline layer of oocytes which is raised at fertilization and remains the outermost layer of the eggshell after cortical granule exocytosis. (G-I) During prometaphase I, WGA-labeled cortical granules were clustered (G) and associated with reticulate ER labeled with SP12::GFP (H), merged in I, similar to Fig. 2B. (J-L) WGA-labeling (J) colocalized with CAV-1::GFP (K), merged in L, in an embryo during early anaphase I. Scale bar: 10 μm.
Fig. S2. Depletion of OID proteins affects cortical granules. wee-1.3(RNAi) oocytes had an abnormal population of aggregated UGTP-1::GFP vesicles (A, arrow). Cortical granules were largely obscured by these aggregates, but could occasionally be observed (A, arrowhead). wee-1.3(RNAi) oocytes had apparently normal CAV-1::GFP localization, except that sometimes the vesicles moved prematurely to the cortex (arrow, B); animals fed 48 hours. czw-1(RNAi) caused enlarged UGTP-1::GFP puncta (arrow, C), whereas CAV-1::GFP was dispersed into small vesicles (D); animals fed 48 hours. syn-4(RNAi) oocytes had abnormal populations of UGTP-1::GFP vesicles, sometimes accumulated at the plasma membrane (arrow, E). CAV-1::GFP vesicles were abnormally distributed (−4 oocyte, F) sometimes at the cortex (−3 oocyte, F) in syn-4(RNAi) oocytes; animals fed 48 hours. (G-L) Phenotypes were similar with either UGTP-1::GFP (not shown) or CAV-1::GFP (shown). cyk-3(RNAi) embryos retained large clusters of cortical granules (arrow, G), while cdk-1(RNAi) (arrow, H) and ify-1(RNAi) (arrow, I) retained cortical granules distributed throughout the cortex; animals fed 30 hours. (J-L) Normal trafficking of cortical granules occurred in cpg-1/cpg-2(RNAi) (J, image cropped to reduce gap between −1 oocyte and embryos), sqv-4(RNAi) (K), and cks-1(RNAi) (L). In each of these, vesicles observed in the oocyte (arrows, vesicles are smaller in J,K) have fused with the plasma membrane in the +1 embryo. CAV-1::GFP is subsequently endocytosed in the +2 embryo and was degraded, similar to WT embryos (Sato et al., 2006). Scale bar: 10 μm.
Movie 1. The wave of secretion during anaphase I. Embryo expressing histone::GFP and labeled with FM2-10 was imaged at five frames per second by SFC. As homologous chromosomes separate, vesicle fusion events can be seen in the cortex. 15 frames per second playback.
Movie 2. Correlative MPLSM imaging of exocytosis. Embryo labeled with FM2-10 (left) and simultaneous imaging of bright-field (right) demonstrated that vesicle fusion events visible in the fluorescent image occurred simultaneously with perturbations in the plasma membrane, visible in the bright-field image. Images taken at three frames per second; eight frames per second playback.
Movie 3. Correlative MPLSM imaging of exocytosis. Same as Movie 2 but using a fluorescent dextran instead of FM2-10.
Movie 4. UGTP-1::GFP vesicle exocytosis. Vesicle labeled with UGTP-1::GFP (left) and simultaneous imaging of bright-field (right) demonstrated that loss of UGTP-1::GFP-labeled vesicles (arrow) occurred simultaneously with perturbations to plasma membrane (arrow) visible in the bright-field image. Images taken at two frames per second; eight frames per second playback.
Movie 5. UGTP-1::GFP vesicle exocytosis. Fusion of UGTP-1::GFP-labeled vesicles allowed diffusion of an extracellular dextran into the vesicle cavity, which caused a dramatic increase in signal intensity. Images taken at three frames per second; eight frames per second playback.
Movie 6. CAV-1::GFP vesicle exocytosis. Exocytosis of CAV-1::GFP-labeled vesicles was imaged in a cortical plane using SFC in the presence of FM4-64 with simultaneous acquisition of green and red channels with the dual-view beam splitter. Images taken at 300 millisecond intervals; 15 frames per second playback.
Movie 7. GFP::SEP-1 dynamics. During oocyte maturation, GFP::SEP-1 accumulated on chromosomes and linear elements in the cortex, then later appeared on vesicles that disappeared during anaphase I. Images captured at six second time intervals, then 1.5 second intervals after metaphase I; eight frames per second playback.
Movie 8. GFP::SEP-1-labeled vesicle exocytosis. Vesicle labeled with GFP::SEP-1 (left) and simultaneous imaging of bright-field (right) demonstrated that loss of GFP::SEP-1-labeled vesicles (arrow, left) occurred simultaneously with perturbations to plasma membrane (arrow, right) visible in the bright-field image. Images taken every 610 milliseconds; eight frames per second playback.
Movie 9. GFP::SEP-1 vesicle exocytosis. Fusion of GFP::SEP-1-labeled vesicles allowed diffusion of an extracellular dextran into the vesicle cavity, which caused a dramatic increase in signal intensity. Images taken every 410 milliseconds; eight frames per second playback.
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