Cortical granule exocytosis in C. elegans is regulated by cell cycle components including separase

Abstract

In many organisms, cortical granules undergo exocytosis following fertilization, releasing cargo proteins that modify the extracellular covering of the zygote. We identified cortical granules in Caenorhabditis elegans and have found that degranulation occurs in a wave that initiates in the vicinity of the meiotic spindle during anaphase I. Previous studies identified genes that confer an embryonic osmotic sensitivity phenotype, thought to result from abnormal eggshell formation. Many of these genes are components of the cell cycle machinery. When we suppressed expression of several of these genes by RNAi, we observed that cortical granule trafficking was disrupted and the eggshell did not form properly. We conclude that osmotic sensitivity phenotypes occur because of defects in trafficking of cortical granules and the subsequent formation of an impermeable eggshell. We identified separase as a key cell cycle component that is required for degranulation. Separase localized to cortically located filamentous structures in prometaphase I upon oocyte maturation. After fertilization, separase disappeared from these structures and appeared on cortical granules by anaphase I. RNAi of sep-1 inhibited degranulation in addition to causing extensive chromosomal segregation failures. Although the temperature-sensitive sep-1(e2406) allele exhibited similar inhibition of degranulation, it had minimal effects on chromosome segregation. These observations lead us to speculate that SEP-1 has two separable yet coordinated functions: to regulate cortical granule exocytosis and to mediate chromosome separation.

Fig. 1. Identification of cortical granules

(A–C) Succynilated WGA-labeled cortical granules in C. elegans oocytes (A), and prometaphase I embryos (B), but not in embryos after meiosis I (C) (DNA in blue). (D–F) WGA-labeled cortical granules (D) are associated with SP12::GFP-labeled reticulate ER (E) at the cortex of a metaphase I embryo; merge in F. (G–I) UGTP-1::GFP-labeled 1 µm vesicles were clustered in prometaphase I (arrows, G) and redistributed across the cortex by metaphase I (arrows, H). Smaller cytoplasmic puncta remain after loss of the 1 µm vesicles (arrows, I). (J–L) WGA staining (J) co-localized with UGTP-1::GFP (K) in cortical granules in the cortex of a metaphase I embryo; merge in L. Scale bar: 10 µm.

Fig. 2. TEM of cortical granules

(A) A mature C. elegans oocyte contains cortical granules (arrows); inset shows higher magnification of vesicles near the oocyte chromosomes (asterisks). (B) An embryo within the spermatheca contains a cortical granule near the plasma membrane (upper right arrow), and a cluster of heterogeneous vesicles (lower arrows). (C) A metaphase I embryo contains cortical granules (arrows) distributed across the cortex (asterisk denotes chromosome in spindle). The cortical granules are found in close association with reticulate ER (A–C). (D) Embryo at metaphase II (chromosome in spindle indicated by white asterisk) lacks cortical granules, and the polar body (black asterisk) is trapped between eggshell layers (arrows). Scale bar: 2 µm.

Fig. 3. A wave of exocytosis during anaphase I

C. elegans embryos expressing histone::GFP and labeled with the plasma membrane dye FM2–10 were imaged using SFC every 200 milliseconds. After the chromosome separation initiated (A), exocytic events (arrows) occurred near the spindle (B) and spread across the cortex (C,D) before the completion of anaphase I. Images in C and D are maximum projection summations of several frames in which a vesicle fusion event occurs. A gap is formed between the plasma membrane and the vitelline layer near the polar body (E, bracket). (F) Images of the plasma membrane before and after exocytosis, labeled with FM2–10 or a fluorescent dextran. Scale bar: 10 µm.

Fig. 4. RNAi of OID genes affects cortical granules

(A) Organization of the C. elegans gonad, showing approximate cell cycle stages based upon the position of oocytes and embryos in wild-type animals. (B–D) Cortical granules labeled with UGTP-1::GFP (indicated by arrows; insets show higher magnification of vesicles). In wild-type animals, embryo +1 was undergoing meiosis I and contains cortical granules, whereas embryo +2 was in the first mitotic division and lacks cortical granules. (C) chs-1(RNAi) oocytes and embryos showed the same UGTP-1::GFP pattern as the wild type. (D) apc-2(RNAi) caused retention of clustered UGTP-1::GFP-labeled cortical granules. (E) sep-1(RNAi) caused retention of UGTP-1::GFP-labeled cortical granules in the first three embryos. Scale bar: 10 µm.

Fig. 5. TEM of APC/C mutant and sep-1(RNAi) embryos

(A) The +2 embryo in a mat-1(ye121) C. elegans did not exit meiosis (asterisk indicates chromosomes in meiotic spindle) and retained cortical granules (arrows). (B) sep-1(RNAi) embryos were not arrested, but retained cortical granules (arrows). (C,D) Comparison of eggshell structures. In wild-type meiosis I embryos that contain cortical granules, a single vitelline layer was present (C). In mitotic embryos, cortical granules were lost and a three-layer eggshell (arrows) structure was observed (D). The +2 embryo in APC/C mutant (E) and sep-1(RNAi) (F) animals contained cortical granules and had a single vitelline layer (arrows), similar to immature wild-type eggshells. Scale bars: 3 µm in A,B; 0.5 µm in C–F.

Fig. 6. The dynamics of the exocytic wave during anaphase I

C. elegans embryos labeled with histone::GFP and FM2–10 were imaged every 333 milliseconds using MPLSM. (A) Total exocytic events observed in wild-type, chs-1(RNAi) and cks-1(RNAi) embryos were nearly twice that of sep-1(e2406) and sep-1(RNAi) embryos. (B) Kinetic profiles of chromosome separation (plotted as a line) and exocytic events (columns) from single embryo recordings representative of average dynamics. Images from the movies are shown in C–K; brackets indicate distance measured between chromosomes, arrows indicate quantitated exocytic events. In wild type (green), degranulation initiated after chromosomes separated by 1.5 µm, and completed before polar body extrusion. In sep-1(RNAi) (red), chromosome separation was severely reduced, the wave of exocytosis was reduced and took much longer. By contrast, in sep-1(e2406) embryos (blue) chromosomes initially separated normally, but still had a reduced level of exocytosis that began after chromosomes separated by 2.5 µm. Scale bar: 10 µm.

Fig. 7. Separase localizes to cortical granules in C. elegans

(A) Before ovulation, cytoplasmic GFP::SEP-1 rapidly accumulated in the nucleus, on chromosomes (asterisks) and cortical filaments (arrows). By metaphase I, GFP::SEP-1 was lost from filaments and appeared on cortical granules (B,C, arrows). GFP::SEP-1 disappeared during the exocytic wave in anaphase I and accumulated on the cortex near the polar body (D, arrow). (E,F) During mitosis, SEP-1::GFP localized to centrosomes (arrows), chromosomes (asterisks) and a diffuse cloud around the spindle. (G–I) Immunofluorescence with α-SEP-1 (green) and DNA (blue). (G) Separase-labeled vesicles (arrows) were absent from the vicinity of the spindle in an embryo that had partly completed the exocytic wave during anaphase I (maximum projection image). Separase localizes to six central spindle elements (arrowhead) between homologous chromosomes and accumulates on the cortex near the polar body (asterisk indicates sperm pronucleus). (H) During prometaphase I, separase localized to filaments in the cortex (arrows) and in the spindle (asterisk). (I) Separase appeared on centrosomes (arrow) and chromosomes (asterisk) during mitosis. Scale bar: 10 µm.

Fig. 8. Separase co-localization during meiosis I in C. elegans

During prometaphase I, the outer kinetochore protein, HIM-10 (A, red), co-localized in cortical filaments with separase (B, green); merge in (C). During anaphase I, cortical granules labeled with WGA (D, red) are also labeled with separase (E, green); merge in F, maximum projection image of four cortical planes. A sep-1(e2406) mutant embryo in anaphase I had cortical granules labeled with WGA (G, red) that were not labeled with separase antibody (H, green), although separase was still present but reduced on the anaphase spindle (arrow); merge in I. Scale bar: 10 µm.

Fig. 9. Regulation of cortical granule trafficking in C. elegans

Depiction of several steps in cortical granule trafficking and eggshell formation, along with the OID genes involved. Separase localization is indicated. Immature oocytes form cortical granules (red) as they grow while separase remains in the cytoplasm. Just before ovulation, separase accumulates in the nucleus, on oocyte chromosomes (blue) and cytoplasmic filaments. The vitelline layer (brown) separates from the plasma membrane (green) around the time of fertilization (sperm pronucleus in black), and cortical granules cluster near the cortex. Separase is lost from filaments and accumulates on cortical granules as they redistribute in the cortex. Separase is lost during the wave of cortical granule exocytosis in anaphase I. Subsequently, the cargo of cortical granules assembles into the chitin (pink) and lipid (red) layers, which are permeable until late meiosis II.