Figure 4. GNU-mKATE2 co-localizes with TRAL-GFP and ME31B-GFP granules in mature oocytes.

Mature oocytes were isolated from gnu^wt-mkate2;tral-gfp or me31b-gfp;gnu^wt-mkate2 females, fixed, and the vitelline membrane removed manually. Oocytes were stained with the anti-GFP booster and imaged by confocal microscopy for fluorescence at 488 nm to detect GFP and 568 nm to detect mKATE2. mKATE2 signal was detected without the use of a booster. Co-localization was measured by quantification of overlap between GFP+ granules and mKATE2+ granules using the surface-surface co-localization algorithm in Imaris (Bitplane). (A) Representative image of gnu^wt-mkate2;tral-gfp oocytes. Co-localizing GNU-mKATE2 (magenta) and TRAL-GFP (green) granules are colored in white. The images shown are single slices of confocal z-stacks from one oocyte. Bottom images show the insets of each panel (dashed yellow box). Scale bar represents 20 μm. (A’) Venn diagram of quantified co-localization between GNU and TRAL granules. GNU and TRAL co-localize in 33.6±5.2% of all granules quantified. GNU-containing TRAL granules represent approximately a third of TRAL granules scored. Values are averaged across eight oocytes. (B) Representative image of me31b-gfp;gnu^wt-mkate2 oocytes. Co-localizing GNU-mKATE2 (magenta) and ME31B-GFP (green) granules are colored in white. The images shown are single slices of confocal z-stack from one oocyte. Bottom images show the insets of each panel (dashed yellow box). Scale bar represents 20 μm. (B’) Venn diagram of quantified co-localization between GNU and ME31B granules. GNU and ME31B co-localize in 57.8±4.6% of all granules quantified. GNU-containing ME31B granules represent approximately half of ME31B granules scored. Values are averaged across eight oocytes.

Figure 4—figure supplement 1. GNU-mKATE recapitulates mature oocyte localization of GNU-GFP.

Mature oocytes were isolated from gnu^wt-mkate2;gnu^wt-gfp or gnu^wt-mkate2;pdi-gfp females, fixed, and the vitelline membrane removed manually. Oocytes were stained and imaged as in Figure 4. (A) Representative image of gnu^wt-mkate2;gnu^wt-gfp oocytes. Co-localizing GNU-mKATE2 (magenta) and GNU-GFP (green) granules are colored in white. (A’) Venn diagram of quantified co-localization between GNU-mKATE2 and GNU-GFP granules. GNU-mKATE2 and GNU-GFP co-localize in 69.9±4.1% of all granules quantified. (B) Representative image of gnu^wt-mkate2;pdi-gfp oocyte. Co-localizing GNU-mKATE2 (magenta) and ER (based on the ER-resident protein PDI-GFP in green) are colored in white. (B’) Venn diagram of quantified co-localization between GNU and ER. GNU and the ER marker (PDI-GFP) co-localize in 6.9±3.2% of all granules quantified. In (A) and (B), the images shown are maximum intensity projections of a z-series from one oocyte. Scale bar represents 20 μm. In (A’) and (B’), values are averaged across eight oocytes.