Loss of NPP-3 causes condensed chromosomes localizing to nuclear periphery.

(A) Representative confocal image (of the middle single layer) of GFP::H2B and mCherry::NPP-3 in the P1 cell (white square) in two-cell stage. The P1 cell is magnified and time-lapse images from prophase to anaphase are shown. The time point 0 s represents the nuclear envelope breakdown (NEBD). AO represents anaphase onset. Scale bar, 5 μm. (B) The nucleus in the center Z plane is divided into five concentric rings or circles with equal area, as region 1-5. Regions 4 and 5 are considered as nuclear peripheral regions. A GFP::H2B intensity threshold is set to represent the chromosomes. Pixels above the threshold are assigned to Regions 1 to 5 in the control and npp-3(RNAi) groups. The right panel shows a snapshot of the 3-dimensional chromosome distribution after Imaris surface simulation. (C) Bar graph showing the percentage of chromosome pixels in different regions in control and npp-3(RNAi) groups. The sample size is 12. Error bars represent mean ± SEM. Data were analyzed using Student’s t-test; ***p<0.001; ns: not significant. (D) Representative confocal images (maximum intensity projection) of chromosome dynamics marked by GFP::H2B from prophase to metaphase in the P1 cell. The time point 0 s represents the nuclear envelope breakdown (NEBD). Scale bar, 5 μm. (E) The dynamic changes of chromosome condensation parameter, in which 30% of pixels in the ROI analyzed is below the threshold scaled intensity (<77), in the control and npp-3(RNAi) embryos. Blue and purple areas indicate chromosome condensation phases in control group and npp-3(RNAi), respectively. The sample size is 5. Error bars show mean ± SEM.

Heterochromatin and telomere anchoring pathway are not involved into peripheral chromosomal localization.

(A) The volcano plot illustrates the distribution of genes based on their expression levels in the control and npp-3(RNAi) embryos. Up-regulated genes are shown in red, down-regulated genes in green, and genes with no significant deregulation in gray (no-DEGs). (B) Representative images of immunofluorescence staining for H3K9me3 and chromosomes (DAPI) in the P1 cell of control and npp-3(RNAi) embryos. Scale bar 5 μm. (C) Quantification of mean intensity of H3K9me3 in the P1 cells in (D). Each dot represents one sample. Error bars represent mean ± SEM. Data were analyzed using Student’s t-test; ***p<0.001. (D) Representative single-plane images of P1 cell with GFP:::H2B marked chromosomes in control, single and double RNAi. Scale bar 5 μm. (E) Representative images of P1 cell expressing GFP::H2B and SUN-1::mRuby in control and npp-3(RNAi) groups. Scale bar 5 μm. (F) Quantification of the mean intensity of SUN-1 in P1 cells of the control and npp-3(RNAi) embryo in the (B). Error bars represent mean ± SEM. Data were analyzed by Student’s t-test; **p<0.005. (G) Representative single plane images of P1 cell with GFP:::H2B marked chromosomes in control, single and double RNAi. Scale bar 5 μm. (H) Quantification of the chromosomal nuclear periphery localization in different conditions shown in the (D) and (G) by concentric assay shown in Figure 1B.

Nuclear rupture repair is required for chromosomes localizing to nuclear periphery.

(A) Representative confocal images in P1 cell of the control and npp-3(RNAi) embryos expressing NPP-1::GFP and mCherry::H2B.. White triangles indicate the sites of nuclear envelope rupture. Scale bar 5 μm. (B) Quantification of total gap size per nucleus in the (A). Each dot represents one nucleus. Error bars represent mean ± SEM. Data were analyzed using Student’s t-test; ***p<0.001. (C) and (E) Representative confocal images in P1 cell of the control and npp-3(RNAi) embryos expressing GFP::BAF-1 and mCherry::H2B (C); GFP::LEM-2 and mCherry::H2B (E). Scale bar 5 μm. (D) and (F) quantification of the mean intensity of BAF-1(C) and LEM-2 (E) in P1 cells of the control and npp-3(RNAi) embryos. Error bars represent mean ± SEM. Data were analyzed by Student’s t-test; **p<0.005. (G) Representative images of a single plane of mCherry::H2B marked chromosomes in control, single and double RNAi. Scale bar 5 μm. (H) Quantification of the chromosomal nuclear periphery phenotype in different conditions shown in the (I) by concentric assay shown in Figure 1B.

Spindle assembly checkpoint, centromere and kinetochore proteins are required for chromosomes localizing to nuclear periphery in NPP-3 disruption.

(A) The upper panel is the representative images of a single plane of P1 cell expressing GFP::H2B and mCherry::NPP-3 in control, different single RNAi. Scale bar 5 μm. The bottom panel is the quantification of the chromosomal nuclear periphery phenotype in different conditions shown in the upper panel by concentric assay shown in Figure 1B. (B) The upper panel is the representative images of a single plane of P1 cell expressing GFP::H2B and mCherry::NPP-3 in control, different double RNAi corresponding to (A). Scale bar 5μm. The bottom panel is the quantification of the chromosomal nuclear periphery phenotype in different conditions shown in the upper layer by concentric assay shown in Fig.1B. Data were analyzed by one-way ANOVA (and nonparametric or mixed); *p<0.05, ***p<0.001, ns: not significant when compared with control. Upper lane marked by red comparing with npp-3(RNAi) group. Bottom lane is comparing with control group.

Absence of NPP-3 causes extended prophase and metaphase through MDF-1 and MDF-2.

(A) Representative single-plane confocal image of P1 cell division from anaphase pf P0 cell to anaphase in control, npp-3(RNAi), mdf-1(RNAi), mdf-2(RNAi), npp-3(RNAi);mdf-1(RNAi) and npp-3(RNAi);mdf-2(RNAi) embryos BAF-1::GFP and mCherry::H2B. Triangles indicate the lagging chromosomes marked by lagging BAF-1. Scale bar 10 μm. (B) Quantification of the prophase duration of P1 cell from P0 cell nuclear envelope reassembly (NER) to P1 cell nuclear envelope breakdown(NEBD) in (A). Each dot represents one embryo. Error bars represent mean ± SEM. Data were analyzed by one-way ANOVA (and nonparametric or mixed); *p<0.05, ***p<0.001, ns: not significant. (C) Quantification of the duration of P1 cell from nuclear envelope breakdown (NEBD) to anaphase onset (AO) in (A). Each dot represents one embryo. Error bars represent mean ± SEM. Data were analyzed by one-way ANOVA (and nonparametric or mixed); *p<0.05, ***p<0.001, ns: not significant. (D) Percentage of lagging chromosomes marked by BAF-1 in different conditions shown in (A). Data were analyzed by Fisher’s test; *p<0.05, ***p<0.001, ns: not significant.

NPP-3 depletion disrupts early kinetochore assembly by reduction the import of HCP-1.

(A) Representative time-lapse image (maximum intensity projection) of GFP::MDF-1 and mCherry::H2B in the P1 of control and npp-3(RNAi) embryos. White triangles indicate MDF-1 accumulation on chromosomes. Scale bar 5 μm. (B) Quantification of MDF-1 integrated intensity at 30 s before NEBD and metaphase in (A). In the prolonged metaphase upon NPP-3 depletion, “metaphase” is defined as the timepoint in which the highest intensity of MDF-1 is observed. Error bars represent mean ± SEM. Data were analyzed using student’s t-test, *p<0.05. (C) Representative confocal images of (a single plane of) later embryos (>25 cells) expressing GFP::MDF-1 and mCherry::H2B in control and npp-3(RNAi) group. White arrow indicates the MDF-1 foci. Scale bar 5 μm. (D) Representative time-lapse image (maximum intensity projection) of GFP::MDF-2 and mCherry::H2B in the P1 of control and npp-3(RNAi) embryos. Scale bar 5 μm. (E) Quantification of MDF-2 integrated intensity at metaphase. Error bars represent mean ± SEM. Data were analyzed using student’s t-test, *p<0.05. (F) Representative time-lapse image (maximum intensity projection) of HCP-1::GFP and mCherry::H2B in the P1 of control and npp-3(RNAi) embryos. Scale bar 5 μm. (G) Quantification of HCP-1 integrated intensity at 30s before NEBD and metaphase. Error bars represent mean ± SEM. Data were analyzed using student’s t-test, *p<0.05.

Loss of MDF-1 in NPP-3 depletion leads to more nuclear fragmentation and DNA damage.

(A) Representative single-plane confocal images of nuclei from late-stage embryos (about 20-30 cell stage). expressing GFP::BAF-1 and mCherry::H2B in control, mdf-1(RNAi), npp-3(RNAi), and npp-3(RNAi); mdf-1(RNAi) conditions. Scale bar 2 μm. (B) Representative images of HUS-1 and chromosome localization in the embryonic stage (about 20-30 cell stage). Arrowhead indicates the colocalization of HUS-1 foci and chromosome. Scale bar 2 μm. (C) Quantification of HUS-1 foci per nucleus including all nuclei in the embryos in different conditions in (B). Error bars represent mean ± SEM. Data were analyzed by one-way ANOVA (and nonparametric or mixed); *p<0.05, ***p<0.001, ns: not significant. (D) During mid-prophase, NPP-3 depletion causes chromosomes to localize to the nuclear periphery, depending on some spindle assembly checkpoint (SAC) components (MDF-1, MDF-2), inner kinetochore proteins, BAF-1, and LEM-2. This response prevents lagging chromosomes, DNA damage and micronuclei formation. SAC components MDF-1 and MDF-2 are also required for the prophase extension observed in NPP-3 depletion. During prometaphase, import of kinetochore proteins KNL-1, BUB-1 and HCP-1 is reduced and more MDF-1, MDF-2 and BUB-1 accumulate on chromosomes. The SAC is activated, resulting in an extended interval from nuclear envelope breakdown (NEBD) to anaphase onset when NPP-3 is depleted.

NPP-3 is essential for embryonic development.

(A) Representative confocal images in P1 cell of the control, npp-2(RNAi), npp-3(RNAi), npp-4(RNAi), npp-7(RNAi), and npp-13(RNAi) embryos expressing NPP-1::GFP and mCherry::H2B. Scale bar 5 μm. (B) Representative confocal image (single layer) of GFP::H2B and mCherry::NPP-3 in the 15-cell stage from prophase to anaphase. White area in the top plane is the time-lapse region for the bottom plane, Scale bar,10 μm for top plane and 5 μm for the bottom one. (C) Representative confocal image (single layer) of GFP::H2B and mCherry::NPP-3 in the 15-cell stage in control and npp-3(RNAi) embryos. Scale bar,10 μm. (D) Quantification of mCherry::NPP-3 intensity in the (C). (E) (G) Selected representative confocal images of all chromosomes localizing at the nuclear periphery in the control and air-1(RNAi) embryos expressing H2B::GFP and mCherry::NPP-3 (E) and GFP::NPP-7 and mcherry::H2B (G). The upper right image is the zoom-in view of the nucleus. Scale bar 10 μm. (F) (H) The intensity of GFP and mCherry is normalised to the average intensity along the nuclear envelope and plotted in the control and air-1(RNAi) nucleus from (E) and (G). (I) Representative confocal image of embryos expressing GFP::H2B and mCherry::PH after 15 hours of imaging from P0 cell in the control and npp-3(RNAi) groups. Scale bar 10 μm. (J) Quantification of embryonic viability in the control and npp-3(RNAi) embryos.

Chromosome localizing to nuclear periphery caused by NPP-3 depletion are transcriptionally repressed.

(A) and (B) GO enrichment of up-regulated genes (A) and down-regulated genes (B) in biological process. (C) and (D) Alignment of up-and down regulated genes to chromosome location in different fold- change threshold.

NPP-3 depletion disrupts nuclear envelope integrity and induces DNA damage.

(A) Representative maximum projection time-lapse images of LacI::GFP at different time points following anaphase onset in the P1 cell. Scale bar 5 μm. (B) Quantification of LacI intensity during the nuclear envelope reassembly process in the P1 cell. Mean with SEM. (C) Representative single-plane confocal images of P1 cells expressing NPP-1::GFP in control (left panel) and npp-3(RNAi) groups (right panel). The raw confocal image was set to 8 bits and subjected to thresholding. This process simplified the intensity values, setting the minimum to 0 and the maximum to 255. The gap size was defined as the distance between the two neighboring peaks at 255. (D) Quantification of total gap size per nucleus in the different cell stages. Each dot represents one nucleus. Error bars represent mean ± SEM. Data were analyzed using Student’s t-test; *p<0.05, **p<0.005, ***p<0.001. (E) Representative maximum projection images of P1 cell expressing HUS-1::GFP and mCherry::H2B in control and npp-3(RNAi) groups, Scale bar 5 μm. (F) Representative single-plane images of 20-30 cell stage embryos expressing HUS-1::GFP and mCherry::H2B in control and npp-3(RNAi) groups. The upper right image is the zoom-in view of the nucleus. Scale bar 5 μm. (G) Representative single plane images of P1 cell with GFP:::H2B marked chromosomes in control, npp-3(RNAi),chk-1(RNAi) and npp-3(RNAi);chk-1(RNAi). Scale bar 5 μm. (H) Quantification of the chromosomal nuclear periphery localization in different conditions shown in the (G) by concentric assay shown in Figure 1B.

MDF-1, MDF-2 and KNL-1 are required for SAC activation at prometaphase upon NPP-3 depletion.

(A) Representative confocal image (single layer) of GFP::H2B and mCherry::NPP-3 in the P1 cell in control, npp-3(RNAi); mdf-1(RNAi) and npp-3(RNAi); mdf-1(RNAi) groups. Scale bar 5 μm. (B) Quantification of NPP-3 intensity at the nuclear envelope in the (A). Error bars represent mean ± SEM. Data were analyzed using Student’s t-test;***p<0.001, ns: not significant. (C) Representative time-lapse image of GFP::H2B in the P1 cell from prophase to anaphase in different conditions (Figure 3I and 3G). Scale bar 5μm. (D) Quantification of the duration of P1 cell from nuclear envelope breakdown (NEBD) to anaphase onset (AO) in (C). Each dot represents one embryo. Error bars represent mean ± SEM. Data were analyzed by one-way ANOVA (and nonparametric or mixed); *p<0.05, ***p<0.001, ns: not significant. Groups showing a significant difference compared to control are marked in red, while groups with significant differences compared to npp-3(RNAi) are marked in green.

NPP-3 depletion reduces the import of KNL-1, BUB-1 and HCP-1 during prophase in P1 cell.

(A) Representative time-lapse image (maximum intensity projection) of GFP::MDF-1 and mCherry::H2B in the EMS cell of control and npp-3(RNAi) embryos. Arrows indicate MDF-1 accumulation on chromosomes. Scale bar 5 μm. (B) Quantification of MDF-1 intensity at 30s before NEBD and metaphase in the (A). Error bars represent mean ± SEM. Data were analyzed using student’s t-test, **p<0.005, ***p<0.001. (C) Representative time-lapse image (maximum intensity projection) of GFP::MDF-2 and mCherry::H2B in the EMS cell of control and npp-3(RNAi) embryos. Scale bar 5 μm. (D) Quantification of MDF-2 intensity at 30s before NEBD and metaphase in the (C). Error bars represent mean ± SEM. Data were analyzed using student’s t-test, ***p<0.001. (E) (G) (I) (K) Representative time-lapse image (maximum intensity projection) of GFP::SAN-1(E), GFP::HCP-3 (G), KNL-1::GFP (I) and BUB-1::GFP (K) with mCherry::H2B in the P1 of control and npp-3(RNAi) embryos. Scale bar 5 μm. (F) (H) (J) (L) Quantification of SAN-1(F), HCP-3 (H), KNL-1 (J) and BUB-1 (L) intensity at 30s before NEBD and metaphase in the (E), (G) (I) (K). Error bars represent mean ± SEM. Data were analyzed using student’s t-test. *p<0.05, **p<0.005, ***p<0.001.

Loss of MDF-1 in NPP-3 depletion leads to more nuclear fragmentation, DNA damage, and higher lethality.

(A) Representative single-plane confocal images of 20-30 cell-stage embryos expressing GFP::BAF-1 and mCherry::H2B in control, mdf-1(RNAi), npp-3(RNAi), and npp-3(RNAi); mdf-1(RNAi) conditions. White area indicates the micronuclei shown in the Figure 7A. Scale bar 10 μm. (B) Representative images of HUS-1 and chromosome localization in the embryonic stage (about 20-30 cell stage). The white area is the region of magnified confocal images of in the Figure 7B. Scale bar 10 μm. (C) The average embryonic viability in the control, mdf-1(RNAi), npp-3(RNAi), and npp-3(RNAi); mdf-1(RNAi) conditions. The data were collected from three plates in each group, with three worms analyzed per plate. Experiments were performed twice.

Nuclear peripheral chromosome localization is not solely depending on the nuclear envelope rupture.

Normalized intensity ratio LacI::GFP (nuclear versus cytoplasmic) changes over time following anaphase onset in P0 cells of control, npp-2(RNAi), npp-3(RNAi), npp-4(RNAi), npp-7(RNAi), and npp-13(RNAi) groups. Error bars show mean ± SEM. Sample size is 6.

Worm strains

Primer List