Intertwined Functions of Separase and Caspase in Cell Division and Programmed Cell Death

Abstract

Timely sister chromatid separation, promoted by separase, is essential for faithful chromosome segregation. Separase is a member of the CD clan of cysteine proteases, which also includes the pro-apoptotic enzymes known as caspases. We report a role for the C. elegans separase SEP-1, primarily known for its essential activity in cell division and cortical granule exocytosis, in developmentally programmed cell death when the predominant pro-apoptotic caspase CED-3 is compromised. Loss of SEP-1 results in extra surviving cells in a weak ced-3(-) mutant, and suppresses the embryonic lethality of a mutant defective for the apoptotic suppressor ced-9/Bcl-2 implicating SEP-1 in execution of apoptosis. We also report apparent non-apoptotic roles for CED-3 in promoting germ cell proliferation, meiotic chromosome disjunction, egg shell formation, and the normal rate of embryonic development. Moreover, loss of the soma-specific (CSP-3) and germline-specific (CSP-2) caspase inhibitors result in CED-3-dependent suppression of embryonic lethality and meiotic chromosome non-disjunction respectively, when separase function is compromised. Thus, while caspases and separases have evolved different substrate specificities associated with their specialized functions in apoptosis and cell division respectively, they appear to have retained the residual ability to participate in both processes, supporting the view that co-option of components in cell division may have led to the innovation of programmed cell suicide early in metazoan evolution.

Figure 1

Pro-apoptotic action of separase in embryos and the germline. (A) Phylogenetic tree showing relationships of metazoan caspases and separases. Dm, Drosophila melanogaster; Hs, Homo sapiens; Mm, Mus musculus. Ce, C. elegans. (B) and (C) Subnormal numbers of cell corpses in sep-1(ts) embryos. In (B), cell corpses were scored at the “comma” stage at the indicated temperature. Total number of embryos scored is shown in brackets. (C) average number of cell corpses in N2 and sep-1(e2406) at the indicated stage and temperature. Error bars are ±SD. At least 16 embryos were scored for each stage. (D) Extra surviving cells in the anterior pharynx of sep-1(e2406ts) and ced-3(n2443), a weak allele of ced-3, L1 larvae. Mid-stage embryos were raised to 21 °C after completion of early divisions at the permissive temperature (15 °C) to prevent developmental arrest. (E) Decreased germ cell apoptosis in sep-1(e2406) mutants. Germ cell corpses were scored in both gonad arms of young adult N2 and sep-1(e2406) animals (50 worms were scored for each genotype) at 15 °C. (F) sep-1(e2406) suppresses ced-9(If) lethality. The hatching rate of embryos produced by sep-1(e2406); ced-9(n1950 n2161) animals and ced-9(n1950 n2161) animals are shown. Total number of embryos scored is shown above each bar. Values are expressed as mean ± SD.

Figure 2

Delayed embryonic development in ced-3 mutants. (A) Slowed embryonic development in ced-3 mutants. Embryos of the indicated genotype were allowed to develop at 22.5 ± 0.5 °C and the time from 2-cell to hatching was measured. Stippled bars: ced-3 alleles that show a moderate defect in PCD (33). Striped bars: ced-3 alleles that show a strong defect in PCD. (B) Embryonic survival. Embryos of the indicated genotype and temperature were scored for hatching as the measure of embryonic viability. Each data point was obtained from >154 embryos. (C) Partial suppression of sep-1(-) embryonic lethality by csp-3(-). Embryos of the indicated genotype were scored as in A. Each data point was obtained from>1000 embryos.

Figure 3

CED-3 enhances early cell division defects in separase mutants. (A) Progression of GFP-labeled mitotic chromosomes during zygotic cleavage. One-cell embryos of the indicated genotype, carrying the HIS-2B::GFP marker, are shown at pronuclear meeting (defined as t = 0; left panels), maximum metaphase alignment (middle panels), and immediately following separation of chromosomes (right panels, with the exception of the sep-1(e2406); ced-3(n717) embryo, which failed in chromosome separation). Embryos were dissected from young adults that had been pre-incubated at 21 °C for 90 mins and allowed to undergo cleavage at the same temperature. The time following pronuclear meeting is indicated in seconds, with the standard deviation reported for all embryos (see B) that underwent chromosome segregation with the exception of the last sep-1(-); ced-3(-) panel, in which time in indicated in minutes. Scale bar, 10 µm. The arrow and arrowhead indicate pseudocleavage and cleavage furrows respectively which are absent in the double mutant embryos. The asterisks show the chromatin condensation and separation defects in the last two panels of the double mutant embryos. (B) Average time for chromosome segregation in all embryos of the indicated genotype (n = 20 per genotype) in which separation of chromosomes was successful. The time from metaphase alignment to the start of chromosome segregation, with standard deviation, is indicated. For all experiments, embryos were imaged every 30 sec with a 500 ms exposure time.

Figure 4

Synergy of CED-3 and SEP-1 in gonadal cell proliferation. (A) % sterility of adults grown at 15 °C. (B) Quantitation of gonadal (both somatic and germline) nuclei in larvae of the indicated genotype. Synchronized L1 worms (n = 50 for each data point) were grown at 25°C, and samples collected every 4 hrs. Larvae were fixed in Carnoy’s solution, stained with DAPI, and gonadal cells counted. (C), (E) and (G), DIC images of N2, sep-1(e2406) and sep-1(e2406); ced-3(n717), respectively, 24 hours after initiation of feeding of synchronized L1 larvae grown at 25^oC. (D), (F) and (H), corresponding DAPI images of the same animals.