Figure S2. CNEP-1 restricts nuclear... | Rockefeller University Press

Figure S2.

$CNEP-1 restricts nuclear membrane incorporation during NE sealing processes, related to Fig. 2 and Fig. 3. (A) Overview image from an electron tomogram in late anaphase I. Scale bar, 500 nm. To the right are two regions of the electron tomogram where the z-slices are shown. Number indicates spacing between the z-slices. On the far right are 3D models from the two regions where the nuclear and cytoplasmic membranes (magenta) and MTs (green) intersecting the nuclear holes are traced. Scale bars, 250 nm. (B) Schematics of the cnep-1 and lem-2 gene loci and the deletion alleles used in this study. (C) Example regions with stacked parallel nuclear membrane sheets (yellow arrowheads) that are in close proximity to cytoplasmic membranes that contact the NE in electron tomograms from meiosis I and II. Scale bar, 100 nm. To the right are the fraction of regions with parallel stacked nuclear membrane sheets. N = no. of regions with cytoplasmic-nuclear membrane contacts. (D) Confocal images from a time lapse series of oocytes expressing GFP::LEM-2 for the indicated conditions. Green arrowhead marks GFP::LEM-2 plaque, and yellow arrowheads mark internal membrane extension. Scale bar, 2.5 µm. Below is the percent of nuclei with GFP::LEM-2 marked internal nuclear membranes at the time of NE formation after meiosis II. (E) Confocal images of sperm-derived pronuclei from time lapse series of embryos expressing GFP::LEM-2 Scale bar, 2.5 µm. To the right are percentages of pronuclei that have interior nuclear membranes marked with GFP::LEM-2 for the indicated conditions. Yellow arrowheads mark GFP::LEM-2 plaque and internal membrane extensions. n = number of embryos. (F) Average ± SD time (seconds) of events relative to anaphase II onset for control and cnep-1Δ embryos scored from time lapse series of embryos expressing GFP::LEM-2 and mCherry::Histone2B, n = no. of embryos. (G) Plot of individual times (seconds) that the meiotic spindle marked with GFP::α-tubulin no longer is adjacent to the chromatin of the oocyte-derived pronucleus. Time is relative to anaphase II onset. (H) Confocal images of the oocyte-derived pronucleus from fixed embryos probed for CHMP-7 and also GFP::LEM-2 (not shown) for the indicated conditions. To the right are the percent of embryos with CHMP-7 or VPS-32 accumulation at the GFP::LEM-2 plaque for the indicated conditions, n = no. of embryos.$

CNEP-1 restricts nuclear membrane incorporation during NE sealing processes, related to Fig. 2 and Fig. 3. (A) Overview image from an electron tomogram in late anaphase I. Scale bar, 500 nm. To the right are two regions of the electron tomogram where the z-slices are shown. Number indicates spacing between the z-slices. On the far right are 3D models from the two regions where the nuclear and cytoplasmic membranes (magenta) and MTs (green) intersecting the nuclear holes are traced. Scale bars, 250 nm. (B) Schematics of the cnep-1 and lem-2 gene loci and the deletion alleles used in this study. (C) Example regions with stacked parallel nuclear membrane sheets (yellow arrowheads) that are in close proximity to cytoplasmic membranes that contact the NE in electron tomograms from meiosis I and II. Scale bar, 100 nm. To the right are the fraction of regions with parallel stacked nuclear membrane sheets. N = no. of regions with cytoplasmic-nuclear membrane contacts. (D) Confocal images from a time lapse series of oocytes expressing GFP::LEM-2 for the indicated conditions. Green arrowhead marks GFP::LEM-2 plaque, and yellow arrowheads mark internal membrane extension. Scale bar, 2.5 µm. Below is the percent of nuclei with GFP::LEM-2 marked internal nuclear membranes at the time of NE formation after meiosis II. (E) Confocal images of sperm-derived pronuclei from time lapse series of embryos expressing GFP::LEM-2 Scale bar, 2.5 µm. To the right are percentages of pronuclei that have interior nuclear membranes marked with GFP::LEM-2 for the indicated conditions. Yellow arrowheads mark GFP::LEM-2 plaque and internal membrane extensions. n = number of embryos. (F) Average ± SD time (seconds) of events relative to anaphase II onset for control and cnep-1Δ embryos scored from time lapse series of embryos expressing GFP::LEM-2 and mCherry::Histone2B, n = no. of embryos. (G) Plot of individual times (seconds) that the meiotic spindle marked with GFP::α-tubulin no longer is adjacent to the chromatin of the oocyte-derived pronucleus. Time is relative to anaphase II onset. (H) Confocal images of the oocyte-derived pronucleus from fixed embryos probed for CHMP-7 and also GFP::LEM-2 (not shown) for the indicated conditions. To the right are the percent of embryos with CHMP-7 or VPS-32 accumulation at the GFP::LEM-2 plaque for the indicated conditions, n = no. of embryos.

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