Table 1.
Strains used in this study.
Figure 1.
SEP-1PD::GFP transgenic worm lines.
A. Microparticle bombardment of homozygous unc-119(ed3); sep-1(+) worms with plasmid DNA bound to gold beads. The plasmid (enlarged panel) contains the sep-1 coding sequence with mutation in the protease domain (C1040S) fused to GFP under control of the pie-1 promoter and an unc-119(+) rescue sequence, allowing for identification of transformed worm lines. The designated alleles and transgene are homozygous in the resulting transgenic worm line. B-M: SEP-1PD::GFP (top row) and SEP-1WT::GFP (bottom row) localization in newly fertilized embryos with H2B::mCherry. Embryos were imaged after 5 generations removed from gfp RNAi feeding (see text and Figure 2). During meiosis I, SEP-1PD::GFP and SEP-1WT::GFP localize to chromosomes and the meiotic spindle (asterisk, B,C, E, F). During prometaphase, separase appears on cortical filaments that appear as puncta depending on whether they are oriented parallel to the focal plane (arrowheads, B and E, insets show examples of filaments oriented properly). Separase is localized to cortical granules by the onset of anaphase (arrows, C and F). During polar body extrusion, SEP-1PD::GFP and SEP-1WT::GFP accumulate at the base of the polar body (base of the polar body designated by arrows D and G, respectively) between the separating anaphase chromosomes (chromosomes designated by arrowheads, D and G). SEP-1PD::GFP also accumulates strongly on the plasma membrane of the embryo after cortical granule exocytosis (D). During the indicated stages of mitosis (H-M), SEP-1WT::GFP and SEP-1PD::GFP localize to chromosomes (asterisk) and centrosomes (arrowhead). During cytokinesis, SEP-1PD::GFP accumulates at the cleavage furrow (arrow, J).
Figure 2.
SEP-1PD::GFP worm lines can be maintained on gfp RNAi.
A. Embryonic lethality of SEP-1PD::GFP line on gfp RNAi and after removal onto OP50 plates at 20°C (left) or 25°C (right). Each data point with error bars represents the average of embryonic lethality from 10 singled worms +/− SEM. B. Average generation +/− SEM that could be propagated for the SEP-1PD::GFP line after removal from gfp RNAi when the indicated number of worms are picked at each generation and kept at 20°C. C. Percentage of sterile animals in the SEP-1PD::GFP line after removal from gfp RNAi.
Figure 3.
Propagation of the SEP-1PD::GFP transgene by backcrossing.
A. The diagram represents the strategy used to propagate the SEP-1PD::GFP transgene using males. Transgenic unc-119(ed3)/unc-119(ed3); sep-1(+)/sep-1(+); SEP-1PD::GFP/- males heterozygous for the transgene are continually crossed to unc-119(ed3) hermaphrodites to generate heterozygous sep-1(+)/sep-1(+); SEP-1PD::GFP/- males and hermaphrodites in the unc-119(ed3) background. The resulting progeny (male and hermaphrodite) that carry the SEP-1PD::GFP transgene are readily identified by mobility because they are Unc rescued due to presence of the transgene. B. Embryonic lethality in F2 broods from singled F1 sep-1(+)/sep-1(+); SEP-1WT::GFP/- or sep-1(+)/sep-1(+); SEP-1PD::GFP/- hermaphrodites at the indicated temperature. C. Embryonic lethality in the F2 after the indicated number of backcrosses of heterozygous sep-1(+)/sep-1(+); SEP-1WT::GFP/- or sep-1(+)/sep-1(+); SEP-1PD::GFP/- transgenic males to unc-119 hermaphrodites. Data points represent the average of a group of 10 singled worms +/− SEM.
Figure 4.
Wild-type SEP-1, but not protease-dead SEP-1, rescues sep-1 mutants.
A. Lines with SEP-1PD::GFP transgene were maintained on gfp RNAi at 20°C and removed for several generations to allow transgene expression. Balanced worms were then singled and progeny were analyzed. B. Percentage of progeny that are balanced mutant or homozygous mutant from singled sep-1(e2406)/hT2g hermaphrodites homozygous for the indicated transgene. C. Percentage of progeny that are balanced mutant or homozygous mutant from singled sep-1(ok1749)/hT2g (referred to as Δ) hermaphrodites with indicated transgene at 20°C.
Figure 5.
Genetic interactions of wild-type or protease-dead SEP-1 with sep-1(e2406).
A. Crossing scheme of heterozygous SEP-1WT::GFP or SEP-1PD::GFP transgenic males to sep-1(e2406) homozygous hermaphrodites. GFP transgene expression in the F1 was determined by microscopy after the shift to 20°C. Only progeny from animals expressing SEP-1WT::GFP or SEP-1PD::GFP were analyzed. B. The table shows embryonic lethality in the F1 and F2 progeny when males carrying the indicated transgene were used in the initial cross.