Novel sep-1 mutants share osmotic sensitivity and cytokinesis defects but are competent for chromosome segregation. (A) A schematic representation of the C. elegans SEP-1 protein denoting the locations of the amino acid substitutions encoded by the mutations ax110, ax521 and e2406ts, as well as av103, the intragenic suppressor of sep-1(e2406ts). The regions with corresponding homology to the separase ortholog pfam group (peptidase C50) and the catalytic histidine and cysteine residues are indicated. The truncated and frame-shifted protein predicted to be expressed from the sep-1(ok1749) allele is also shown. The shaded box represents the out-of-frame residues that are encoded before the premature stop codon. (B,C) In a live sep-1(+) embryo (B) only the first polar body (indicated by a white arrow) was stained by DAPI (yellow), whereas in a live sep-1(ax110) embryo (C), the polyploid zygotic DNA stained with DAPI. The embryos expressed GFP::histone to reveal zygotic DNA (green). (D–F) Methanol-fixed embryos stained with anti-tubulin antibodies (green) and TOTO-3 (red). (D) A two-cell wild-type embryo, (E) a ‘four-cell’ polyploid ax110 embryo and (F) a ‘two-cell’ polyploid ax521 embryo. The arrowheads indicate successful chromosome separation at anaphase. Scale bars: 10 μm.

Novel sep-1 mutants have weak nondisjunction but severe cytokinesis defects during mitosis. Embryos of the indicated genotypes expressing GFP::H2B were imaged either in meiosis or mitosis (A–P) and scored for meiotic and mitotic chromosome segregation defects and completion of polar body extrusion during the meiotic divisions or cytokinesis during mitosis (Q–T). (A–D) A GFP::H2B-expressing wild-type embryo during the first mitotic division. Chromosome segregation and cytokinesis occurred normally in all embryos (n=5; S,T), resulting in a normal two-cell embryo (D). (E–H) sep-1(ax110) embryos showed weak chromosome nondisjunction during mitosis (n=6; F,G,S) but completed cytokinesis only 17% of the time (Q), resulting in polyploid one-cell embryos in the second mitotic division (n=6, H). sep-1(ax110) embryos did not have any nondisjunction during meiosis I despite failing polar body extrusion in every case we observed (n=7; S,T). (I–L) Partial depletion of sep-1 by RNAi caused severe nondisjunction in all embryos (n=4; J,K,S) but only disrupted cytokinesis 25% of the time, similar to our previous results (n=4; L,T) (Bembenek et al., 2010). Stronger sep-1(RNAi) treatments completely prevented chromosome segregation (data not shown). (M–P) Loss of pph-5 activity did not rescue the loss-of-function phenotypes of sep-1(RNAi) embryos (n=6; N,O,S) and cytokinesis failures were observed in 33% of the embryos, similar to the sep-1 RNAi phenotype in wild-type embryos (n=6; P,T). In sep-1(ax110);pph-5(av101) embryos, no chromosome segregation defects (n=7; S) or cytokinesis failures were observed (n=6; T) during either meiosis or mitosis. Scale bar: 10 μm.

Quantification of the CGE defect of sep-1 mutants. (A) Average total number of CGE events per embryo. (B) Average rate of CGE events per embryo per minute. (C) Average duration of CGE window (the average time elapsed between the first and last CGE event, in seconds). At least six embryos were imaged for each genotype, and the bars indicate standard deviations. The data points for sep-1(e2406ts) and sep-1(RNAi) are from Bembenek et al. (Bembenek et al., 2007). (D–I) Chromosome separation occurred in both av101 and ax110 embryos (midplane images D and F), however, ax110 had reduced numbers of CGE events (cortical time-projected images, E and G). CGE was restored in suppressed sep-1(ax110); av101 (I). White arrows indicate sites of CGE. Scale bars: 10 μm.

Subcellular localization of SEP-1 is defective in mutants. Wild-type (N2), sep-1(ax521), sep-1(ax110), sep-1(ax110); pph-5(av101) and pph-5(av101) embryos were stained with anti-SEP-1 antibodies (Red; all panels), either anti-α-tubulin to label the spindle (Green; A,C,D,E,G,H,I,K,L,M,O,P,Q,S,T) or FITC::WGA to label cortical granules (green; B,F,J,N,R), and DAPI (blue; all panels). Images in the metaphase I column show SEP-1 localization (Red only), and the insets are 2×magnified images of the spindle shown in all three colors. The upper right images in the anaphase I column are merged images of anti-SEP-1 and the FITC::WGA. Colocalization appears yellow. Images in the first mitosis column are taken from embryos observed during the first mitotic metaphase, and the embryos depicted in the MC mitosis column are of multicellular (or multinucleate) embryos. Scale bars: 10 μm.

Quantification of SEP-1 localization. The presence of SEP-1 at cortical filaments, the meiotic spindle, cortical granules, and the mitotic spindle matrix was scored in each strain and the embryonic cell cycle stage was determined independently by inspecting the chromosome configuration with DAPI staining. The number of positively stained cells and the total number of cells examined for a particular cell cycle stage are shown. *The cortical granules of sep-1(ax110); pph-5(av101) were labeled with SEP-1 antibodies, however, the overall intensity was reduced compared with wild-type embryos.

Schematic of the PPH-5 protein and PPH-5 expression data. (A) A schematic representation of the C. elegans PPH-5 protein illustrates the arrangement of four tandem TPR domains and the PP2A-like phosphatase domain. The amino acid substitution caused by av101 and the 55 amino acids removed by the tm2979 deletion are indicated. (B) Mutant PPH-5 proteins were expressed. An immunoblot showing PPH-5 expression in sep-1(+) and sep-1(ax110) animals bearing the pph-5(av101) (av) or pph-5(tm2979) (tm) suppressor mutations. Each lane was loaded with protein equivalent to 50 gravid hermaphrodites. Actin was used as a loading control, and the asterisk indicates a non-specific cross-reactive band. Full-length and internally deleted PPH-5 proteins are expected to migrate at 56 and 50 kDa, respectively.

Expression and localization pattern of pph-5 reporters. (A–C) A four-cell embryo expressing itIs37[pie-1p::mCherry::H2B::pie-1e] (A), avIs74[pie-1p::GFP(LAP)::PPH-5::pie-1e] (B), and the merged image (C). GFP::PPH-5 localized to the spindle matrix in mitotic metaphase cells. The white arrows in A–C indicate the metaphase plate. Scale bar in C: 10 μm. (D–I) pph-5 regulatory sequences were sufficient to drive expression of a mCherry::histone reporter in all somatic and germline tissues (D,G). A somatically expressed GFP reporter (avIs112[manf-1p::GFP::manf-1::manf-1e]) is shown in E and H, with merged images in F and I. The white dashed boxes in D–F are enlarged in G–I, respectively. The white arrowhead in G indicates the −1 oocyte, and the bracket denotes the spermatheca, which contains mCherry-positive spermatocytes. Scale bars in F and I: 100 μm.