Fig. 6

Fig. 6

Suf/Spastizin controls cortical granule maturation.

(A) Electron micrographs of high-pressure frozen stage III oocytes. Lysosomes (white arrows) are fragmented in suf/spastizin mutants. Cortical granules (white arrowheads) show a dense-core (dashed circle), which is lost in mutant oocytes. Note, that we consistently generated wrinkles visible as black lines in mutant cortical granules, which indicate sectioning artifacts, but suggest a different composition. Scale bar: 10 µm. (B) Optical section of stage III oocytes showing the accumulation of cortical granules stained with MPA-lectin (red) in suf/spastizin mutant mothers. Scale bar: 50 μm. (C) Scheme visualizing the exocytosis of cortical granules (red arrowheads, left panel) after egg activation, which leads to chorion elevation (blue arrowheads, right panel) at the beginning of embryogenesis. (D) Cortical granules labeled with MPA-lectin (red) are released after activation in wt, but not in mutants. Scale bar: 50 µm. (E) Fertilized embryos 30 mpf (min post fertilization) from +/- (left panel) and -/- suf/spastizin mutants (right panel). Note the decreased elevation of the chorion in mutants. Scale bar: 100 μm. (F) Actin cortex of stage V eggs stained with phalloidin (green) at 0, 60 and 180 s after activation. Note the fusion of vesicles in wt eggs after 60 s, visible as black holes in the actin meshwork, and the formation of actin patches at 180 s after exocytosis completion. Scale bar: 50 μm. (G) VAMP4 (green) labels immature secretory granules and accumulates in suf/spastizin mutant oocytes. Scale bar 50 µm. (H) Stage III oocytes from +/- (wt) or -/- suf/spastizin mutants (mut) after 12–16 h incubation in L-15 medium. Scale bar: 50 μm (I) Stage III oocytes from -/- suf/spastizin mutants after injection of plasmid encoding wt or mutant Suf (p96re allele). Note that the mutant Suf^p96re injected oocytes also show chorion elevation similar to wt Suf injected oocytes.