Introduction

The nuclear envelope (NE) consist of two phospholipid bilayer—the inner nuclear membrane (INM) and the outer nuclear membrane (ONM), along with associated components such as nuclear membrane proteins, the nuclear lamina, and nuclear pore complexes (NPCs) (1). The ONM is continuous with the endoplasmic reticulum (ER), and during telophase, nuclear membrane proteins and ER-derived lipid reform around chromosomes to reestablish the NE (2, 3). The INM is linked to the nuclear lamina, a meshwork of intermediate filaments primarily composed of lamin proteins that provides structural support and serves as an attachment platform for chromatin (4). Mutations in lamins lead to a spectrum of inherited disorders known as laminopathies (5, 6). The lamina is indirectly connected to the cytoskeleton through LINC (Linker of nuleoskeleton and cytoskeleton) complexes to facilitate the positioning and movement of chromosomes (7).

The nuclear envelope serves as a selectively permeable barrier that regulates molecular exchange between the nucleus and cytoplasm through specialized transport channels, most prominently the nuclear pore complexes (NPCs) (8). The NPC is positioned at the junction of the INM and ONM and can be subdivided into seven structural subcomplexes: the cytoplasmic ring, nucleoplasmic ring, cytoplasmic filament, transmembrane region, inner ring, central channel, nuclear basket and linker (9). Beyond mediating transport, the NE also participates in key cellular processes such as chromatin organization, epigenetic regulation, gene expression, DNA repair and replication, and cell cycle control (9–11).

Heterochromatin predominantly localizes to the nuclear periphery and consist of densely packed, transcriptionally silent regions enriched with modifications such as H3K9me3 and H3K27me3 (12). Its peripheral positioning contributes to gene regulation and genome stability during differentiation (13–15). The nuclear lamina and nuclear pore complexes (NPCs) are key mediators of heterochromatin anchoring, particularly inner ring and nuclear basket nucleoporins. Certain nucleoporins, known as adaptor nucleoporins, acting as bridges that connect chromatin to the NPC or serving as platforms for nuclear transport receptors and regulatory factors (16–18). For instance, the nuclear basket protein NUP153 binds across mammalian and fly genomes, as shown in multiple ChIP-seq studies, targeting silent genes, active promoters and enhancers, and CTCF-binding sites (19). Additionally, the linker component NUP93 and inner ring components NUP188 and NUP205 predominantly associate with facultative heterochromatin regions, such as promoter regions of the HOXA gene cluster, anchoring them to the nuclear periphery to promote transcriptional repression in diploid colorectal cancer (DLD1) cells (20). In C. elegans, depletion of the linker component NPP-13/NUP93 or inner ring component NPP-3/NUP205 disrupts transport of molecules smaller than 70 kDa, impairs cell cycle progression, and triggers abnormal chromosome condensation at the nuclear periphery (21, 22). However, the precise mechanisms by which NPP-3/NUP205 regulates chromosome localization remain unclear, and whether it specifically targets particular chromosome domains or acts globally requires further investigation of its interaction networks and functional pathways.

In C. elegans, which possess holocentric chromosomes lacking defined peri-centromeric regions, heterochromatin markers such as H3K9me3 are enriched along chromosome arms (23) and associate with lamina-associated domains (LADs) to promote gene repression and genome organization during differentiation (24). In the early- to mid-stage embryos, the chromodomain protein CEC-4 binds directly to H3K9me3 to anchor heterochromatin to the nuclear periphery, without affecting transcription repression (24, 25). Following differentiation, a second heterochromatin-sequestering pathway is activated. For example, MRG-1 functions as a redundant chromatin anchor in intestinal cells upon CEC-4 depletion (26).

Telomeres at chromosome ends cluster at the nuclear envelope during various cellular processes across species, including interphase in budding yeast (27), meiotic prophase in the C. elegans gonad, embryonic prophase (28, 29) and post-mitotic nuclear assembly in human cells (30). This clustering is mediated by the nuclear envelope protein SUN1 in humans/yeast (SUN-1 in C. elegans) and the telomere shelterin component RAP1 in yeast and human cells (27, 30) or telomere-binding protein POT-1 in C. elegans early embryos (29). During meiosis I prophase in C. elegans, SUN-1 localizes to the inner nuclear membrane where it interacts with pairing center proteins such as HIM-8, while the KASH domain protein ZYG-12 localizes in the outer nuclear membrane and interacts with cytoplasmic dynein (31). The SUN-1-ZYG-12 interaction transmits force generated by the cytoskeleton across the nuclear envelope to chromosomes, facilitating chromosome movement and homologous pairing (28, 32).

Structural alterations of the nuclear envelope caused by external stimuli, mechanical stress, or mutations in nuclear envelope components can trigger nuclear envelope rupture, activates repair processes, and reorganize chromatin through proteins such as Barrier-to- Autointegration Factor 1 (BAF-1) and LEM-2 (33, 34). These repair mechanisms resemble the active preassembly of the NE during mitosis (35). Similarly, in LMN-1/laminB-deficient C. elegans embryos, condensed chromosomes localize to the nuclear periphery, with LEM-2 accumulating at rupture sites (36).

Under respiratory stress conditions such as anoxia (oxygen deprivation) or treatment with respiratory chain inhibitors like sodium azide, nearly all chromosomes in C. elegans embryos relocalize to the nuclear periphery during prophase (37). Similar chromosome peripheral clustering occurs in Drosophila embryos (38). Notably, both organisms can fully recover from anoxia without lasting damage (37, 38), suggesting that peripheral chromosome positioning may serve as a transient protective response to respiratory stress. In C. elegans embryos, NPP-16 supports survival through anoxia and is associated with prophase arrest, while CDK-1 also contributes to this arrest (37). However, the mechanisms and functional significance of this chromosomal relocalization remain largely unexplored. On the other hand, the Roth lab demonstrated that the spindle assembly checkpoint is essential for anoxia-induced metaphase arrest and survival (39), though whether prophase and metaphase arrests are mechanistically linked remains unclear.

In this study, we investigated the phenotypic consequences of disrupting the inner ring component NPP-3/NUP205 in C. elegans embryos. NPP-3 depletion extends the duration of prophase and prometaphase. NPP-3 depletion. Notably, chromosomes in NPP-3-depleted embryos localize to the nuclear periphery, with occasional chromosome segregation defects associated with nuclear rupture. Chromosome peripheral localization in NPP-3-depleted embryos does not depend on heterochromatin or telomere anchoring pathways but instead involves centromere-kinetochore components, the spindle assembly checkpoint components, and nuclear rupture repair machinery. Spindle assembly checkpoint is necessary for the extended prophase and prometaphase observed in early embryos following NPP-3 depletion. Additionally, DNA damage checkpoints is activated in late-stage embryos after NPP-3 depletion, with increased DNA damage upon simultaneous depletion of NPP-3 and MDF-1. These results suggest that chromosome positioning at the nuclear periphery may serve a protective role, mitigating DNA damage and ensuring accurate chromosome segregation. Our findings provide new insights into the connection between nuclear envelope integrity and chromosome positioning during cell division, with implications on nuclear organization under stress conditions.

Results

NPP-3 depletion causes chromosomes to cluster at the nuclear periphery

To investigate the effect of nucleoporins on chromosome behavior, we depleted several representative nucleoporins from different subcomplexes: NPP-2 (cytoplasmic and nucleoplasmic ring), NPP-4 (central channel), NPP-3 and NPP-13 (inner ring), and NPP-7 (nuclear basket). Depletion of these NUPs disrupted nuclear envelope integrity, causing small but numerous ruptures along the nuclear envelope (Figure S1A). Notably, in P1prophase nuclei of two-cell stage embryos, condensed chromosomes localized closed to the nuclear periphery in npp-3(RNAi), npp-7(RNAi) or npp-13(RNAi) (Figure S1A). This peripheral chromosome redistribution aligned with prior observations for depletion of the NPC inner ring components (21). In contrast, no such chromosomal localization occurred in npp-2(RNAi) or npp-4(RNAi) embryos.

To gain deeper insights into the role of inner ring subcomplex in chromosome organization, we examined the subcellular distribution of NPP-3 throughout the cell cycle. We generated a CRISPR/Cas9-engineered strain expressing mCherry-tagged NPP-3 at the endogenous locus and co-expressing GFP::H2B in live-cell imaging. In both transcriptionally quiescent P1 cells and transcriptionally active 15-cell-stage embryos, mCherry::NPP-3 predominantly localized to the nuclear envelope during interphase to prophase (Figure 1A and S1B). Upon nuclear envelope breakdown (NEBD), it adopted a characteristic two-half-circle pattern, absent in the vicinity of the centrosomes (Figure 1A and S1B)), consistent with previous observations (22). Depletion of NPP-3 by feeding RNA interference substantially reduced the mCherry::NPP-3 signal at the nuclear envelope, confirming that this CRISPR/Cas9-engineered mCherry::NPP-3 reporter faithfully reflected endogenous NPP-3 localization (Figure S1C and S1D).

Figure 1.

As chromosomes puncta localized to nuclear periphery after NPP-3 depletion, we hypothesize whether there is a negative regulation of NPP-3 on chromosome nuclear peripheral localization. The centrosomal kinase AIR-1/Aurora A and centrosomes regulate the loss of NPP-3 from the nuclear envelope during mitotic entry (22). Thus, NPP-3 form two-half-circles localization in prophase in wildtype. Depletion of AIR-1/Aurora A in C. elegans embryos results in the presence of multiple centrosomes (22, 40). The relationship between AIR-1, centrosomes and NPP-3 provides an opportunity to investigate the potential effect of AIR-1 depletion on localization of NPP-3 at the nuclear envelope. To explore whether NPP-3 localization regulates chromosomes positioning along the nuclear envelope, we depleted AIR-1/Aurora A to induce the formation of multiple centrosomes - a process associated with the disappearance of NPP-3 near the multiple centrosomes. Upon AIR-1 depletion, mCherry::NPP-3 showed fragmented localization rather than a whole circle on the nuclear envelope in the prophase of 20-30-cell stage embryos (Figure S1E). Additionally, chromosomes labeled with H2B::GFP were positioned in proximity to the nuclear envelopes, but specifically did not directly associate with the sites containing mCherry::NPP-3 foci (Figure S1E). The peaks of mCherry::NPP-3 and H2B::GFP signals exhibited an inverse relationship in air-1(RNAi) embryos (Figure S1F), suggesting that NPP-3 negatively regulates chromosome nuclear periphery localization. As depletion of NPP-7 also showed chromosomes localizing to nuclear periphery (Figure S1A), the relationship between NPP-7 and chromosome was also tested. Interestingly, similar to mCherry::NPP-3, chromosomes also localized at sites with low intensity of GFP::NPP-7 in air-1(RNAi) embryos (Figure S1G and S1H). These observations suggest that condensed chromosomes tend to localize at nuclear envelope sites without NPP-3 or NPP-7.

NPP-3 depletion leads to earlier chromosome condensation

To uncover the underlying the aberrant chromosomal organization observed upon NPP-3 depletion, we take advantage the well-characterized mitotic dynamics of the P1 cell in C. elegans embryos. Live-cell imaging of GFP::H2B-labeled nuclei in npp-3(RNAi) embryos revealed a pronounced redistribution of all chromosomes toward the nuclear periphery (Figure 1B). This redistribution was evident in both two- and three-dimensional reconstructions generated through Imaris surface rendering (Figure 1B and 1C). To assess the functional implications of this altered chromosomal positioning induced by NPP-3 depletion, we quantified chromatin condensation dynamics in the P1 cell using the approach described by Maddox et al. (41). During prophase, a nucleus-confined square region was rescaled at each time point to normalize intensity distributions (0–255), minimizing artifacts due to changes in nuclear size and photobleaching. In control embryos, chromatin condensation began approximately 420 seconds prior to nuclear envelope breakdown (NEBD) and progressed to rod-shaped chromosomes by 120 seconds before NEBD (Figure 1D and 1E). In contrast, npp-3(RNAi) embryos exhibit an earlier onset of condensation, detectable as early as 630 seconds prior to NEBD, while reaching a comparable level of chromatin compaction at NEBD (Figure 1D and 1E). In terms of development, control embryos advanced to the three-fold stage within ∼15 hr after P1 division, whereas NPP-3-depleted embryos arrested during early gastrulation, with chromosomes persistently localized at the nuclear periphery (Figure S1I). This developmental arrest correlates with the embryonic lethality observed in viability assays, confirming the essential role of NPP-3 in embryogenesis (Figure S1J). Together, our findings demonstrate that NPP-3 depletion advances the timing of chromatin condensation, drives peripheral chromosome positioning, and impairs normal embryonic development.

NPP-3 depletion results in increased heterochromatin and transcription silencing

Heterochromatin and transcriptionally repressed genes are typically positioned at the nuclear periphery. This organization is mediated by interactions between specific histone modifications and their associated binding proteins, and is characterized by the enrichment of key heterochromatic marks such as H3K9me2, H3K9me3, and H3K27me3 at the nuclear lamina (42). Given that NPP-3 depletion causes chromosome to relocalize to the nuclear periphery and triggers earlier chromatin condensation, we hypothesized that this peripheral repositioning might be associated with transcriptional repression.

To test this, we performed RNA sequencing to compare the global gene expression profiles between control and npp-3(RNAi) embryos. Compared to the control embryos, 30% of genes were downregulated, 2% were upregulated, and 68% remained unchanged upon NPP-3 depletion (Figure 2A), indicating large-scale transcriptional repression. Gene Ontology analysis revealed that up-regulated genes were primarily associated with mitotic cell cycle phase transitions and cyclin-dependent protein serine/threonine kinase activity (Figure S2A), whereas down-regulated genes were enriched for functions related to RNA polymerase II and larval development (Figure S2B). Notably, chromosomal mapping of both up- and down-regulated genes showed no positional bias (Figure S2C and S2D).

Figure 2.

To further investigate the chromatin state at the nuclear periphery, we conducted immunofluorescence staining with an antibody against the heterochromatin marker H3K9me3 in fixed embryos. In control embryos, H3K9me3 foci were primarily observed on subsets of chromosomes arms/ends near the nuclear periphery. In contrast, npp-3(RNAi) embryos displayed a greater number of H3K9me3 foci and higher mean fluorescence intensity across most chromosomes at the nuclear periphery (Figure 2B and 2C). These findings suggest that chromosomes relocated to the nuclear periphery in NPP-3-depleted embryos adopt a more heterochromatic, transcriptionally repressed state.

Heterochromatin and telomere anchoring pathways are not involved in peripheral chromosomal localization

In C. elegans embryos, the inner nuclear membrane protein CEC-4 is known to anchor H3K9-methylated heterochromatin at the nuclear periphery (43). To investigate whether CEC-4 is required for the peripheral chromosome accumulation caused by NPP-3 depletion, we examined chromosome localization following CEC-4 depletion alone and in combination with NPP-3 depletion. Depletion of CEC-4 alone did not alter chromosome positioning (Figure 2D and 2H). However, co-depletion of NPP-3 and CEC-4 resulted in a peripheral localization phenotype indistinguishable from npp-3(RNAi) alone (Figure 2D and 2H), demonstrating that CEC-4 is dispensable for this process.

An alternative mechanism for chromosome anchoring at the nuclear periphery involves telomere clustering mediated by the SUN-1/POT-1 pathway in C. elegans, which is essential for meiotic progression (29). To investigate whether the peripheral chromosome localization observed upon NPP-3 depletion depends on the SUN-1-POT-1 pathway, we analyzed SUN-1 distribution and performed genetic epistasis experiments. Our results showed that npp-3(RNAi) did not alter SUN-1 localization or its fluorescence intensity at the nuclear envelope (Figure 2E and 2F). Moreover, depletion of SUN-1 or POT-1 individually did not affect chromosome positioning, and their co-depletion with NPP-3 failed to alter the peripheral accumulation phenotype (Figure 2G and 2H). Collectively, these findings indicate that the peripheral chromosome localization induced by NPP-3 depletion is independent of the SUN-1/POT-1-mediated telomere anchoring pathway.

Nuclear rupture repair is required for chromosomes localizing to nuclear periphery in NPP-3 disruption

Given that NPP-3 localizes to the nuclear envelope, we hypothesized that NPP-3 depletion might compromise nuclear envelope integrity and consequently alter chromosome status. This hypothesis is based on previous findings that nuclear envelope disruption can trigger chromosome condensation at rupture sites (33, 44) and induce DNA damage (45). To test this, we used a strain co-expressing NUP channel component NPP-1/NUP43::GFP and mCherry::H2B to visualize nuclear envelope structure and chromatin organization. Nuclear envelope permeability was further assessed using a LacI-tagged GFP reporter (46). In control embryos, the NPP-1 signal appeared continuous, indicating an intact nuclear envelope. In contrast, in npp-3(RNAi) embryos, the NPP-1 signal displayed numerous gaps and discontinuities (Figure 3A and 3B), signifying structural compromise of nuclear envelope. To quantify the extent of nuclear envelope damage, we performed line-scan analysis along the nuclear envelope at different embryonic stages. This analyses revealed a progressive increase in both the size and number of rupture sites from the one-cell to the multicellular stage in npp-3(RNAi) embryos (Figure S3C and S3D). This structural compromise was accompanied by impaired nuclear envelope barrier function, as indicated by a significant reduction in the nuclear import of the LacI-GFP reporter (Figure S3A and S3B), consistent with previous findings that NPP-3 regulates nuclear permeability (22).

Figure 3.

Nuclear envelop rupture triggers a repair response involving recruitment of Barrier-to-Autointegration Factor (BAF-1) and LEM-domain protein LEM-2, both of which can promote chromatin condensation and repositioning (36, 47–49). To determine whether this repair pathway contributes to peripheral chromosome accumulation upon NPP-3 depletion, we examined the roles of BAF-1 and LEM-2. Consistent with nuclear envelope ruptures, npp-3(RNAi) embryos exhibited increased accumulation of both BAF-1 and LEM-2 at the nuclear periphery (Figure 3C to 3F). While baf-1 (RNAi) alone and npp3(RNAi);baf-1 (RNAi) double depletion produced severe chromosomes clustering that precluded epistatic analysis of chromosome positioning, we examined LEM-2 single and double depletion with NPP-3. lem-2(RNAi) alone did not alter chromosome positioning, whereas co-depletion of NPP-3 and LEM-2 partially suppressed the peripheral chromosome accumulation phenotype (Figure 3G and 3H). These findings are consistent with a model where NPP-3 depletion causes nuclear envelope rupture and BAF-1-dependent recruitment of LEM-2 to the affected regions (35), which may promote chromosome repositioning to the periphery.

Additionally, during embryonic development, nuclear rupture size increases (Figure S2D), correlating with compromised nuclear integrity and potential accumulation of DNA damage from cytoplasmic nucleases over time (50). Notably, the DNA damage sensor HUS-1 (a 9-1-1 complex component) colocalized with chromosomes in npp3(RNAi) in 20 to 30-cell-stage embryos but not in P1 cell (Figure S3E and S3F). These HUS-1 foci indicated activation of the DNA damage checkpoint during 20-30 cell stage embryogenesis upon NPP-3 depletion. Additionally, double depletion of NPP-3 and CHK-1 did not revert NPP-3-induced peripheral chromosome localization in P1 cells (Figures S3G and S3H), confirming that this relocalization is independent of the DNA damage checkpoint pathway.

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

Under respiratory stress conditions such as anoxia or sodium azide treatment, C. elegans embryos exhibit a distinctive prophase phenotype in which nearly all chromosomes localize near the nuclear periphery (37). Loss of nucleoporin NPP-16/NUP50 suppresses prophase arrest under anoxia, suggesting a role for nucleoporins in this process. The nuclear envelope localization of the spindle assembly checkpoint (SAC) component MDF-1/MAD1 depends on interactions with nuclear pore complex NUP153 in human cells (51) and NPP-5/NUP107 in C. elegans (52). Such nuclear envelope localization of MAD1 has been shown to contribute to SAC activation before nuclear envelope breakdown (53, 54).

On the other hand, the SAC plays a crucial role in anoxia-induced metaphase arrest and survival of C. elegans embryos (39). Oxygen deprivation also induces chromosome relocalization toward the nuclear periphery (38, 55). Moreover, in Drosophila embryos, SAC components such as MPS1 and BUB3 accumulate on chromosomes at the spindle midzone during metaphase and subsequently relocalize to the centrosomes during interphase under anoxic conditions (56). Although the SAC does not appear to be required for anoxia-induced prophase arrest (39), it remains unclear whether the SAC can function during interphase and prophase, and if it is involved in controlling chromosome positioning following anoxia treatment.

To investigate whether SAC components and centromere-kinetochore contribute to the peripheral chromosome localization observed upon NPP-3 depletion, we performed epistatic analysis of chromosome localization. Individual depletions were carried out for SAC proteins MDF-1/MAD1, MDF-2/MAD2, SAN-1/MAD3, and for inner kinetochore proteins HCP-3/CENP-A, HCP-4/CENP-C, and KNL-1, as well as outer kinetochore proteins NDC-80, BUB-1, BUB-3, and HCP-1/CENP-F, both alone and in combination with npp-3(RNAi). Chromosomal positioning during prophase in P1 cells was assessed. Efficient depletion of npp-3 RNA was verified by NPP-3::mCherry signal (Figure S4A and S4B). We found that depletion of MDF-1 or MDF-2 alone did not affect chromosomal positioning; however, co-depletion with NPP-3 abolished peripheral chromosome localization, restoring central nuclear localization comparable to controls (Figures 4A and 4B). On the other hand, depletion of inner kinetochore components (HCP-3, HCP-4, KNL-1) did not alter prophase chromosome positioning, although it disrupted chromosome segregation (Figures 4A and S4C). Notably, co-depletion of HCP-3 or HCP-4 with NPP-3 restored central chromosome positioning, whereas co-depletion of NPP-3 and KNL-1 retained partial peripheral chromosomal localization, suggesting distinct contributions of these kinetochore components to chromosome–nuclear envelope interactions..

Figure 4.

Conversely, depletion of outer kinetochore proteins (NDC-80, BUB-1, BUB-3, HCP-1) did not alter prophase chromosome positioning in P1 cells, although loss of NDC-80 or BUB-1 caused embryonic lethality at later developmental stages (57, 58). Importantly, co-depletion of these outer kinetochore proteins with NPP-3 did not alter the peripheral chromosome localization phenotype. Collectively, these findings reveal that SAC proteins MDF-1, MDF-2 and SAN-1, together with inner kinetochore components, play essential roles in regulating chromosome positioning at the nuclear periphery during prophase.

Absence of NPP-3 Activates the Spindle Assembly Checkpoint to extend both prophase and prometaphase

As SAC and kinetochore proteins are required for the peripheral chromosomal localization upon NPP-3 depletion, we wonder whether SAC is activated in prophase and prometaphase. Firstly, we monitored nuclear envelope and chromatin dynamics in the P1 cell by using a strain expressing BAF-1::GFP; mCherry::H2B. BAF-1 marks the nuclear membrane and serves as an indicator of NE remodeling timing (59), while also highlighting anaphase-bridged chromatin in lmn-1(RNAi) embryos (60). We found that NPP-3 depletion causes a significant delay in the cell cycle. Specifically, the duration of prophase—measured from nuclear envelope reassembly (NER) in the P0 cell to nuclear envelope breakdown (NEBD) in P1 cell—was prolonged to 950.0 ± 112.1 seconds, compared with 782.4 ± 75.1 seconds in control embryos (Figure 6A and 6B). Additionally, the interval between NEBD and anaphase onset (AO) was 156.0 ± 25.0 seconds in npp-3(RNAi) embryos, more than doubled of the duration in controls (79.5 ± 14.7 seconds) (Figure 5A and 5C).

Figure 5.

Figure 6.

The prolonged NEBD-to-AO interval is a characteristic hallmark of spindle assembly checkpoint (SAC) activation (61). To determine whether this delay is SAC-dependent, we co-depleted NPP-3 with a key SAC component, MDF-1/MAD1 or MDF-2/MAD2. Single depletion of MDF-1 (79.4 ± 23.6 s) or MDF-2 (72.0 ± 15.4 s) alone did not affect the NEBD-to-AO timing compared to controls (79.5 ± 14.7 s) (Figure 5A and 5C). However, co-depletion of NPP-3, alongside MDF-1 or MDF-2, significantly reversed the anaphase onset delay, reducing the interval to 91.5 ± 24.8 seconds for npp-3;mdf-1(RNAi) and 97.5 ± 23.6 seconds for npp-3;mdf-2(RNAi) (Figure 5A and 5C). Similarly, double depletion of NPP-3 with KNL-1 also abolished the delay, whereas depletion of other kinetochore or spindle assembly proteins, including SAN-1, HCP-3, HCP-4, NDC-80, BUB-1, BUB-3, and HCP-1, did not (Figure S4D). These findings indicate that the late mitotic delay caused by NPP-3 loss is mediated through SAC activation involving MDF-1, MDF-2, and KNL-1. Notably, although bypassing the SAC restores cell cycle timing, it exacerbates genomic instability. Co-depletion of NPP-3 with either MDF-1 or MDF-2 significantly increased the frequency of lagging chromosomes, as indicated by lagging BAF-1, compared to depletion of NPP-3 alone (Figure 5A and 5D).

Interestingly, co-depletion of NPP-3 and MDF-1 also abolished the prolonged prophase duration (793.8 ± 64.4 s) caused NPP-3 depletion (950.0 ± 112.1s) to levels comparable with controls (782.4 ± 75.1s), whereas co-depletion with MDF-2 resulted in only partial restoration (832.5 ± 66.5 s) (Figure 5C). Here, we show that the SAC components MDF-1 and MDF-2 have a role in prolonging prophase in NPP-3 depletion, which is a novel role beyond their known functions in prometaphase extension.

NPP-3 depletion causes the redistribution of SAC and kinetochore proteins during prophase

To investigate whether NPP-3 depletion activates SAC by promoting the recruitment of MDF-1/MAD1 and MDF-2/MAD2 to chromosomes from prometaphase to metaphase (58, 62, 63), We examined the dynamic localization of GFP-tagged MDF-1 and MDF-2 in control and npp-3(RNAi) embryos expressing mCherry::H2B. In control embryos, MDF-1 localized primarily to the nucleoplasm with mild nuclear envelope association during prophase; its signal diminished after NEBD and became undetectable on chromosomes by metaphase (Figure 6A). In contrast, NPP-3 depletion abolished MDF-1 nuclear envelope localization during prophase and caused moderate chromosome accumulation between NEBD and metaphase. Nuclear MDF-1 intensity in npp-3(RNAi) P1 cells showed a modest increase at ∼30 s before NEBD and at metaphase, when normalized to controls as described previously (64) (Figure 6A and 6B).

Compared to previous findings where MDF-1 aligns on chromosomes during monopolar spindles in zyg-1(RNAi) P1 cells, defective in centrosome duplication (62), we observed only a slight increase in MDF-1 intensity and no clear accumulation at metaphase in npp-3(RNAi) P1 cells. Potentially, SAC activity is weaker in the P1 cell upon NPP-3 depletion, as compared to ZYG-1 depletion with completely unattached kinetochores. Since SAC activity increases during embryogenesis with rising nuclear-to-cytoplasm ratio (65), we investigated whether MDF-1 exhibited any notable changes during embryonic development. MDF-1 intensity was also markedly elevated before NEBD and at metaphase in the EMS cell after NPP-3 depletion compared to controls, with bright MDF-1 foci co-localizing with chromosomes during prometaphase (Figure S5A and S5B). Furthermore, in the prophase of 25-cell stage, MDF-1 showed clear NE localization with diffuse signal inside the nucleus in the control embryos. Notably, bright MDF-1 foci appeared on chromosomes during prophase in embryos beyond the 25-cell stage upon NPP-3 depletion, contrasting to the nuclear envelope localization in controls (Figure 6C). These results demonstrate that NPP-3 depletion disrupts MDF-1 localization dynamics from prophase through metaphase, suggesting a potential additional role for MDF-1 during prophase.

MDF-2, another key component of spindle assembly checkpoint, exhibited a weak signal at NEBD followed by progressive chromosomal accumulation that peaked at metaphase in the P1 cell of control embryos. While there is no difference in MDF-2 signal in npp-3(RNAi) and control embryos at 30 seconds before NEBD in P1 cells, MDF-2 signal was slightly enhanced in the npp-3(RNAi) embryos compared to controls at metaphase (Figure 6D and 6E). Similar patterns were observed in EMS cells in npp-3(RNAi), which showed more pronounced metaphase chromosomal accumulation (Figure S5C and S5D).

Additionally, the localization pattern of the spindle assembly checkpoint protein SAN-1 is also altered. In control P1 cells, SAN-1 entered the nucleus after NEBD, whereas upon NPP-3 depletion, SAN-1 translocated into the nucleus prior to NEBD (Figures S5E and S5F). Importantly, SAN-1 did not accumulate on chromosomes in either control or npp-3(RNAi) groups, consistent with previous findings that SAN-1 is not recruited to chromosomes even upon SAC activation (58).

Collectively, these data suggest that NPP-3 depletion promotes the recruitment of MDF-1 and MDF-2 to chromosomes, potentially leading to spindle assembly checkpoint activation.

To investigate whether NPP-3 depletion impacts kinetochore assembly in C. elegans, we examined the dynamics of inner and outer kinetochore proteins—HCP-3, KNL-1, BUB-1, and HCP-1—from prophase through anaphase. HCP-3 fluorescence intensity at 30 seconds prior to NEBD and during metaphase was comparable between control and npp-3(RNAi) embryos (Figure S5G and S5H). In contrast, levels of KNL-1, BUB-1, and HCP-1 were significantly reduced at 30 seconds before NEBD in NPP-3-depleted embryos (Figures S5I to S5L and 6G). At metaphase, KNL-1 accumulated on chromosomes to similar intensities in both conditions (Figure S5J), whereas BUB-1 showed increased chromosomal association in npp-3(RNAi) embryos compared to control (Figure S5L), consistent with SAC activation via BUB-1 recruitment (66). HCP-1 levels, however, remained diminished at metaphase under NPP-3 depletion compared to control (Figure 6F and 6G), suggesting incomplete kinetochore assembly. Collectively, these results suggest that NPP-3 may be critical for the import of kinetochore components during prophase, and its depletion hampers kinetochore assembly but activates SAC during prophase and prometaphase.

Chromosome Anchoring at the Nuclear Periphery and Spindle assembly checkpoint MDF-1 are crucial for maintaining chromosome stability in NPP-3 depletion

Loss of the SAC abrogates the peripheral chromosomal localization induced by NPP-3 depletion during prophase (Figure 4A and 4B) and the prophase and prometaphase extension (Figure 5A and 5C). However, loss of the SAC also exacerbates genomic instability in NPP-3 depletion. In npp-3 RNAi, even with the SAC activation, lagging chromatin was observed in the AB (58.33%) and P1 (16.67%) cells treated compared to 0% in control group (Figure 5D). Co-depletion of NPP-3 with either MDF-1 or MDF-2 increased the frequency of lagging chromosomes compared to NPP-3 depletion alone in P1 cells, increasing to 45.45% for npp-3;mdf-1(RNAi) and 87.50% for npp-3;mdf-2(RNAi) (Figure 5A and 5D). Taken together, these findings indicate that SAC activation in response to defects caused by NPP-3 loss serves to prevent premature anaphase onset, with SAC components playing specific roles in regulating mitotic phases.

To investigate whether this peripheral chromosome positioning and extended cell cycle in NPP-3 depletion serves a protective function, we compared the effects of NPP-3 depletion alone versus the double depletion NPP-3 and MDF-1 on the chromosomes. We quantified the micronuclei, marked by GFP::BAF-1, and DNA damage, labelled by HUS-1::GFP (67, 68) in 20-30 cell-stage embryos. Double depletion of NPP-3 and MDF-1 resulted in micronuclei formation in 50% of embryos, a phenotype that was absent in single depletion conditions (Figures S6A and 7A). Following NPP-3 depletion, an average of 2.40 ± 1.62 HUS-1::GFP foci co-localized with chromatin at the 20–30 cell stage, compared with 0.045 ± 0.21 and 0.075 ± 0.27 in control and mdf-1(RNAi) groups, respectively (Figures S6B, 7B and 7C). Double depletion of NPP-3 and MDF-1 further increased HUS-1 foci to 3.34 ± 2.39 (Figures 7B and 7C).

Figure 7.

Consistent with these observations, embryonic viability was markedly reduced in the npp-3(RNAi) group (5.71% ± 2.48) relative to control (99.55% ± 0.78), whereas MDF-1 depletion alone had minimal effect (98.60% ± 1.95) (Figure S6C). Importantly, combined depletion of NPP-3 and MDF-1 produced a further significant decline in viability (1.38% ± 0.75), supporting the conclusion that loss of peripheral chromosome anchoring exacerbates genomic instability and severely impairs embryonic survival (Figure 7D).

Discussion

Here, we describe the phenotype of global chromosome localization to the nuclear periphery caused by NPP-3 depletion in C. elegans embryos. Our candidate gene screening results indicate that this localization depends on the spindle assembly checkpoint proteins, centromere and kinetochore proteins, and nuclear rupture repair factors, but not the heterochromatin or telomere anchoring pathways. Additionally, NPP-3 depletion impairs kinetochore assembly in prophase, extends prophase and prometaphase depending on the spindle assembly checkpoint (SAC) in embryos, and triggers a DNA damage response in late-stage embryos. Notably, we observed MDF-1 and HUS-1 foci on some chromosomes during prophase. In anoxia, chromosomes also localize to the nuclear periphery, and this arrest is reversible (37). Such chromosome relocalization response in anoxia could potentially reduce transcriptional activity, as in NPP-3 depletion, and lower energy consumption. We propose that the peripheral chromosome localization in NPP-3 depletion is not a mere defect but a protective mechanism to maintain genome stability (Figure 7D).

Not all nuclear rupture induces chromosomes localizing to the nuclear periphery

The nuclear envelope (NE) comprises several essential components, including the nuclear pore complex (NPC), nuclear lamina, and associated membrane proteins. Mutations in nucleoporins or lamins have been linked to nuclear rupture phenotypes that contribute to developmental abnormalities and a range of diseases. For example, Emery-Dreyfuss muscular dystrophy (EDMD) is associated with nucleoporin mutations (69), while laminopathies are resulted from lamin gene mutations (70). Additionally, disruptions in NE components have been implicated in neurodegenerative disorders such as Huntington’s disease and Alzheimer’s disease (18), highlighting the critical role of nuclear envelope integrity in cellular function and organismal development nuclear integrity.

In C. elegans embryos, lamin LMN-1 deficiency leads to prominent nuclear ruptures, accumulation of the inner nuclear membrane protein LEM-2 at rupture sites, and subsequent chromosome condensation at rupture sites, which results in the redistribution of all chromosomes to the nuclear periphery (36), similar to what we observed in NPP-3 depletion.

The depletion of the selected NUPs led to disruptions in the nuclear envelope integrity, as we observed small yet numerous nuclear rupture sites along the nuclear envelope (Figure S1A). Surprisingly, we further found that, at prophase, the condensed chromosomes were localized closed to the nuclear periphery in npp-3(RNAi), npp-13(RNAi) or npp-7(RNAi) in two-cell embryos (Figure S1A). However, such chromosomal localization was not observed in npp-2(RNAi) or npp-4(RNAi). As npp-3(RNAi), npp-4(RNAi) or npp-13(RNAi) embryos share similar permeability defects according to the dynamic of LacI (Figure S7), these results reveal an intriguing observation that chromosomal localization at the nuclear periphery is not directly correlated with the extent of permeability defect caused by NUPs depletions, but more specifically related to disruption of certain NPC subcomplexes in the inner ring and nuclear basket.

Potential relationship between BAF-1 and centromere-kinetochore proteins

According to the screening, nuclear rupture repair (BAF-1, LEM-2) and centromere-kinetochore proteins (HCP-3/CENP-A, HCP-4/CENP-C, KNL-1) are involved in nuclear peripheral chromosome localization caused by NPP-3 depletion. we propose that BAF-1 may serve as a crucial molecular link between the nuclear envelope and centromere-kinetochore complexes during chromosome localization to nuclear periphery, particularly under the condition of NPP-3 depletion. Barrier-to-Autointegration Factor (BAF) is a conserved nuclear envelope (NE) component that binds chromatin and helps chromosome anchoring to the NE (47). Additionally, BAF-1 was reported to interact with centromeric protein CENP-C, for proper kinetochore assembly in metaphase which in turn regulate the chromosome segregation accuracy in Drosophila (44, 71). The observed decrease in CENP-C and CENP-A levels upon BAF-1 depletion supports this notion, indicating that BAF-1 might directly or indirectly influence the recruitment or maintenance of these centromeric proteins during in metaphase (71). Therefore, the potential relationship between BAF-1 and centromere-kinetochore proteins may be integral to maintaining chromosomal stability, especially when nuclear envelope integrity is compromised. Further experimental validation is warranted to elucidate the mechanistic details of this interaction during cell division.

Spindle assembly checkpoint and DNA damage checkpoint are activated upon NPP-3 depletion

Based on Figure 5A and Figure 6A, NPP-3 depletion not only activates the spindle assembly checkpoint (SAC) during metaphase but also induces a redistribution of MDF-1 in prophase. The release of MDF-1 from the NE has been shown to facilitate earlier SAC activation by recruiting MDF-1/MDF-2 to assembled kinetochore prior to nuclear envelope breakdown in human cells (53). In C. elegans embryos, similar observations is noted upon of NPP-5 (an cytoplasmic ring Nup), where MDF-1 loses its nuclear envelope (NE) localization in prophase, and accumulates around chromosomes during metaphase, mirroring the effects seen with NPP-3 depletion (52). However, unlike NPP-3 depletion, chromosomes do not localize to nuclear periphery upon NPP-5 depletion (52), and it remains unclear whether the SAC is activated after NPP-5 depletion. Therefore, the loss of MDF-1 localization at the NE is not directly causing peripheral chromosome positioning.

However, with the extension of prophase in NPP-3 depletion dependent on MDF-1 and MDF-2, we suspect that SAC may be activated in prophase, potentially also through the mitotic checkpoint complex (MCC) (72), and this SAC activation could be required for the localization of condensed chromosomes at the nuclear periphery. We hypothesize that early condensation of chromosomes in prophase in NPP-3 depletion may mimic a prometaphase environment, but the delayed outer kinetochore protein assembly could create a microenvironment resembling prometaphase chromosomes with unattached kinetochores as large MDF-1 foci was found to localize around chromosomes in prophase (Figure 5C), potentially resulting in the detection of these chromosomes as “unattached”.

Additionally, NPP-3 depletion activates the DNA damage checkpoint (Figure S3F), likely due to DNA damage caused by compromised nuclear envelope integrity. Since both SAC and DNA damage response components such as MDF-1 and HUS-1 form foci at the nuclear periphery (Figures S3F and 5C), there may be a potential interplay between SAC activation and the DNA damage response following nuclear rupture. For example, MAD1/MAD2 have been implicated in DNA damage responses in both C. elegans and human cells during prophase, with RAD-51 foci associated with MDF-2/MAD2 in the proliferative zone of worm germlines (73). However, without MDF-1 in NPP-3 depletion, there are even more HUS-1 foci (Figure S7), suggesting that more DNA remains unrepaired without SAC activation.

The survival of C. elegans embryos through anoxia rely on the SAC components SAN-1/MAD-3, MDF-1 and MDF-2 (39). However, whether the prophase chromosome localization to the nuclear periphery is dependent on the SAC has not been analyzed. Investigating the functions of SAC components under various stresses, such as nuclear envelope rupture and DNA damage, beyond the regular detection of unattached kinetochores for prometaphase extension, will be a valuable avenue for future research to explore additional functions of SAC.

Materials and Methods

Experimental model and study participant details

C. elegans strains

All C. elegans strains were grown at 22°C on Nematode Growth Medium (NGM) plates seeded with Escherichia coli strain OP50. All strains used in this study are listed in Table S1.

Transgenic strain construction

The CRISPR-cas9 transgenic strains of endogenous NPP-3::mCherry was generated by SunyBiotech Company [strain name and genotype: PHX913 npp-3(syb913)]. The sequencing primers are listed in Table S2

Method details

RNAi

We performed feeding RNAi experiments using the method previously described (74). The RNAi plasmids with targeted genes were constructed. For single gene knockdown, we inserted DNA fragment of targeted genes into the vector pL4440. For double depletion, the same DNA fragment was inserted into the npp-3(RNAi) vector. Primers containing restriction enzyme (RE) for cloning the gene fragment are listed in Table S2.

Clones were verified by single clone PCR of NPP-3 and candidate gene and Sanger Sequencing. The RNAi plasmid was then transformed into HT115 E. coli competent cells. The HT115 bacteria with the RNAi plasmid were then seeded on RNAi plates (NGM plates with 100μg/ml ampicillin and 0.1 mM IPTG).

For feeding RNAi experiments, L4 larvae were transferred to NGM plates seeded with corresponding RNAi bacteria for 26 hrs, except for npp-7(RNAi), where late L4 larvae were picked and incubated for 18 hrs. For baf-1(RNAi), we pickedL4 on the RNAi plates and then incubated at 20°C for 48 hrs before microscopy imaging (59).

Embryonic viability and brood size assay

The embryonic viability and brood size assays were performed on the worms fed with HT115 bacteria containing pL4440, and pL4440 inserted NPP-3, MDF-1, or ligated NPP-3 and MDF-1 plasmid to knock down single or double genes. The protocol was adapted from (75). The worms were transferred to fresh plates every 12 hours and the plates were scored. The brood size is defined by the total numbers of embryos and larva on the plates. Embryonic inviability is defined by the number of embryos that did not hatch in 24 hours. There are 3 biological replicates in each experiment, and the experiments were repeated twice.

Sample preparation for RNA Sequencing

Samples for the RNA sequencing are extracted from the embryos (Adapted from Zamanian Lab’s protocol https://www.zamanianlab.org/ZamanianLabDocs/protocols/Caenorhabditis_elegans/RNA_Extra ction/RNA_Extraction/#rna-isolation-from-trizol-samples). The RNA samples were sent to BGI for RNA sequencing and analyses.

Immunofluorescence

N2 fed with L4440(RNAi) and npp-3(RNAi) bacteria for 26 hrs were prepared. The gravid hermaphrodites were dissected, and embryos were released. The immunofluorescence steps were previously described (76). Primary antibody H3K9me3 (1:500 diluted with antibody dilution buffer, Abcam ab8898) and secondary antibody: goat-anti-Mouse IgG- FITC (1:500, diluted with 1X PBST, Jackson Immuno Research 115-096-062).

Confocal microscopy

Embryos were dissected and mounted in M9 buffer on the 2% agarose pad. The slides were then sealed with Vaseline to prevent evaporation.

For the time-lapse images, images were captured using a Nikon spinning disk confocal microscope equipped with a 100×, 1.4 NA objective lens, and images were acquired with MetaMorph software. To ensure consistency across experiments, parameters such as laser power, exposure time, time interval, and z-stack interval were kept constants for all groups.

The permeability ability experiment (Figure S2) was carried out with DeltaVision (DV) imaging system (60×1.42 NA oil objective).

Image quantification and Imaris simulation

Quantification of chromosome condensation during cell cycle:

The chromosome condensation assay was based on Maddox’s previous study (41). Z-stack images of GFP::H2B including whole nucleus (12 μm) were acquired from telophase of P0 cell to metaphase of P1 cell. Maximum intensity projection was applied to each frame. As nucleus of P1 cell enlarges during prophase, individual scaling was applied to square region that fit within the nucleus during each time point. This approach ensures that the intensity distribution (0–255) at each timepoint remains independent of the nucleus size or photobleaching effects. The values of pixels in each intensity (0–255) can be acquired by the histogram in ImageJ software and then the total values within various thresholds can be acquired. The condensation parameter under the specific threshold can be calculated as the total values of pixels (below the threshold) / total values of pixel in the square. 35% threshold (gray values in 0-90) was the optimal one to present the difference between the control and npp-3(RNAi) groups.

Quantification of nuclear envelope rupture size:

The middle plane of nucleus expressing NPP-1::GFP and H2B::mCherry was selected for analysis. The raw confocal 16-bit images were input to ImageJ. We scaled the intensity to 8-bit, such that the minimum intensity is 0 and maximum intensity is 255 at sites with the highest intensity. By drawing the circle fitting the nuclear envelope, and then using the function (Area to line) to transfer the circle into a line (1-pixel width). The intensity of NPP-1::GFP can be profiled along the line. The gap size was measured by the distance between the neighboring peaks at the value of 255. For the statistics, gap sizes within 2 pixels were discarded. The nuclear gap per nucleus was calculated as the total gap size along the line over the total distance of the line.

Quantification of fluorescence intensity:

For the quantification of MDF-1, MDF-2, HCP-3, KNL-1, HCP-1, and BUB-1 intensities, all images were acquired under identical microscopy parameters to ensure consistency. Z-stack images were processed using maximum intensity projection (58). For analysis, a 40×40 pixel region encompassing the nucleus (containing the MDF-1 signal) was cropped, as well as an adjacent 80×80 pixel region encompassing the nucleus, with surrounded by background.

The mean intensity of background was calculated as:

The integrated intensity of the target gene signal was determined by: (Mean intensity of small area- mean intensity of background)*1600 pixel square.

Statistical analysis

All analyses were done using GraphPad Prism. For comparison between two normally distributed datasets, Student’s t-test was used. For comparison between multiple datasets, one-way ANOVA non-parametric test was used, otherwise two tailed Fisher’s Exact Test was used in the Figure 6D. In all cases, the statistical tests used, the number of samples analyzed, and the p-values are indicated in the figure legends.

Supplemental information

Figure S1.

Figure S2.

Figure S3.

Figure S4.

Figure S5.

Figure S6.

Figure S7.

Supplemental Table S1:

Supplemental Table S2:

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files.

Acknowledgements

We thank the Yuen lab and Tse lab members for suggestions. We thank the Karen Oegema and Arshad Desai labs for providing strains. We thank the University of Hong Kong School of Biological Sciences Core Facilities, the Li Ka Shing Faculty of Medicine Core Facility, and SUSTech Core Research Facilities for providing equipment for this study. Financial support for this research was provided by General Research Fund (GRF) 17118623 from the Research Grant Council (RGC) of Hong Kong.

Additional information

Author contributions

L.J., Y.C.T. and K.W.Y.Y. conceived the study. L.J. performed the methodology and acquired the data. L.J., Y.C.T. and K.W.Y.Y. analyzed and interpreted the data. L.J. wrote the original draft of the manuscript. L.J., Y.C.T. and K.W.Y.Y. reviewed and edited the manuscript. Y.C.T. and K.W.Y.Y. supervised the study.

Funding

Research Grants Council, University Grants Committee (General Research Fund (GRF) 17118623)

• Karen Wing Yee Yuen