RanBP2-dependent annulate lamellae drive nuclear pore assembly and nuclear expansion

Domain: biology
Published: 2026-03-25
Editorial outcome: major_revision

Abstract
Nuclear pore complexes (NPCs) enable nucleocytoplasmic transport. While NPCs primarily localize to the nuclear envelope (NE), they also appear in cytoplasmic endoplasmic reticulum (ER) membranes called annulate lamellae (AL). Though discovered in the mid-20th century, AL’s function and biogenesis remain unclear. Previously considered exclusive to embryonic and malignant cells, we find AL in somatic mammalian cells. Under normal conditions, AL store pre-assembled AL-NPCs that integrate into the NE, producing approximately one-third of newly formed nuclear pores and supporting nuclear expansion during G1. Upon pathological stimuli, AL transfer to the NE is impaired, leading to their cytoplasmic accumulation. RanBP2 (Nup358) is essential for AL biogenesis, with its phenylalanine-glycine repeats promoting AL-NPC scaffold oligomerization. ER-associated Climp63 (CKAP4) directs AL-NPCs to ER sheets and the NE. This AL-driven nuclear pore formation is complementary to the canonical routes, constituting a distinct NPC assembly pathway. Our work uncovers the biogenesis mechanism of AL and the nuclear function of this key cellular organelle.

Introduction
Annulate lamellae (AL) are specialized subdomains within the endoplasmic reticulum (ER), characterized by a variable array of parallel-arranged layers. These assemblies feature a regular distribution of pore structures embedded in the membranes, which closely resemble nuclear pore complexes (AL-NPCs)1,2. AL were commonly observed in rapidly developing, differentiating, germ and malignant cells3–11. Despite their early discovery by McCullough in 195212 and designation by Swift in 195613, and subsequent reports describing AL in specific cell types3,7,14–21, the universal and evolutionarily conserved function as well as direct biogenesis mechanisms of AL have remained enigmatic.

It could be hypothesized that AL might be utilized by rapidly growing cells to increase the pool of nuclear envelope (NE) NPCs (NE-NPCs) and sustain optimal levels of nucleocytoplasmic transport. This process is vital for normal cellular function, and abnormal expression or localization of nucleoporins (Nups), the building units of NPCs, have been observed in cancer and neurodegenerative disorders22–25. However, it is currently unknown if the Nups localization defects, often visualized by low-resolution microscopy techniques as foci or granules, are linked to the presence of AL and if AL regulate nuclear function under physiological conditions.

Here, we address this long-standing knowledge gap and uncover both the universal cellular function of AL and the direct mechanisms underlying their biogenesis. We demonstrate that AL are more abundant than previously recognized and are present in the cytoplasm of various somatic cells under normal physiological conditions. In normally proliferating cells, AL contain pre-assembled AL-NPCs that contribute to nuclear pore assembly and promote nuclear expansion during G1 phase, revealing a distinct NPC assembly pathway complementary to the two previously characterized routes26. Under pathological conditions or stress stimuli, this supply is disrupted, leading to the clustering and expansion of AL-NPCs in the cytoplasm. Importantly, the component of the NPC cytoplasmic filaments RanBP2 (also known as Nup358) is required for the formation of AL in normal cells and for their clustering under stress conditions. N-terminal unstructured phenylalanine-glycine (FG) repeat region of RanBP2 promotes the oligomerization of NPC scaffold components, leading to AL-NPC formation in the cytoplasm. In addition, we identify the ER-resident protein Climp63 (CKAP4) that turns out to ensure the proper localization of AL-NPCs to ER sheets and their integration into the NE. Disrupting these biogenesis mechanisms inhibits AL-NE merging, nuclear pore assembly and nuclear expansion during G1 phase, illustrating the essential role of AL in normal cycling cells.

Results
AL are present in diverse somatic cells
To determine whether cytoplasmic Nup foci observed in various stressed cells represent AL, we used cellular models that have linked microtubules (MTs), fragile X-related proteins (FXRPs; FXR1, FXR2 and FMRP), and ubiquitin-associated protein 2-like (UBAP2L) to aberrant Nup accumulation in the cytoplasm. Indeed, previous findings demonstrated an important role of dynein-mediated MT-based transport as well as chaperoning proteins FXRPs and UBAP2L in the localized assembly of Nups at the NE during interphase25,27,28. Nup foci were also seen in the context of a human disease such as fragile-X syndrome (FXS)25, which is characterized by the absence of FMRP and in several other human disorders14. Although FXRPs-UBAP2L models do not capture all perturbations that generate cytoplasmic Nup foci, they provide a well-established framework for assessing whether these structures correspond to AL.

As expected, acute (90 minutes) MT depolymerization or deletion of UBAP2L in mCherry-Nup133 knock-in (KI) HeLa cells (Supplementary Fig. 1a-d) led to the formation of cytoplasmic mCherry-Nup133 foci that also contained Nup62 (Supplementary Fig. 1e). Correlative light and electron microscopy (CLEM) analysis showed these foci to be localized to parallel-aligned ER membrane structures containing stacked nuclear pore-like complexes, suggesting that they represent AL (Fig. 1a).

To overcome existing limitations in the microscopy analysis of AL, we utilized single molecule localization microscopy29,30 (SMLM) to generate super-resolution images of the AL foci. In particular, we applied a multi-color variant based on a dichroic image splitter and spectral demixing (splitSMLM)31. Via the simultaneous imaging of multiple species of fluorophores, the method enables high-precision observations of the structural organization of NPCs and other multi-component complexes, reaching a resolution of 20 nm31. This approach is easily applicable under various experimental conditions and can provide greater details on the distribution and composition of AL. We confirmed the presence of highly organized cytosolic assemblies containing tightly packed NPC structures, likely corresponding to AL, upon disruption of the FXRPs-UBAP2L pathway (Fig. 1b, Supplementary Fig. 1f-h).

Surprisingly, small AL containing fewer NPCs (on average 1.65) could be observed in the cytoplasm of wild-type (WT) HeLa cells (Fig. 1b-c, Supplementary Fig. 1i), whereas in cells treated with the MT depolymerizing agent nocodazole AL were larger (average of 13.8 NPCs) (Fig. 1c). Accordingly, when imaged with conventional (diffraction-limited) fluorescence microscopy, AL foci also appeared smaller for WT HeLa interphase cells (average size 0.090 µm2) compared to cells with depolymerized MTs (average size 0.213 μm2, Supplementary Fig. 2a-b). Similar small cytoplasmic Nup foci were previously postulated to represent AL in normal interphasic cells32. We observed a relatively broad distribution range of AL foci sizes (Supplementary Fig. 2a-b) in both WT and nocodazole-treated cells, confirming the reported diversity of AL morphologies and abundance33. Hereafter, AL foci considered “large” have a size bigger than 0.3 μm2.

Importantly, small AL could be detected not only in a variety of common cancer cell lines including U2OS, DLD-1, HCT116, but also in the non-transformed cell lines such as RPE-1, FB789 and MRC5 (Supplementary Fig. 3a, 4a). Likewise, small AL structures were observed in normally proliferating human fibroblasts, while large AL were found in FXS patient-derived fibroblasts (Fig. 1d). Finally, AL structures were identified in human induced pluripotent stem cells (hiPSCs)-derived neurons (Fig. 1e, Supplementary Fig. 3a, 4a), corroborating our findings on the widespread existence of AL in somatic cells. Although AL abundance varied across different cell types (Fig. 1f), AL size consistently increased following nocodazole treatment (Supplementary Fig. 3b, 4b). These results illustrate the strength of splitSMLM as a powerful method to study AL and reveal the widespread existence of small AL in somatic, normally proliferating mammalian cells.

AL cluster in the cytoplasm upon pathological stimuli
Small and large AL-NPCs were composed of most of the tested Nups that form different subcomplexes, with the exception of the nuclear basket nucleoporin Nup153 and ELYS, under various analyzed conditions (Fig. 1g, Supplementary Fig. 2c-f). Interestingly, RanBP2 was localized symmetrically on both sides of AL-NPCs (Supplementary Fig. 1f), consistent with previous reports28,34. In Drosophila embryos, AL-NPCs contain ELYS, but not Nup153 and Nup627 and in Xenopus egg extracts, AL-NPCs did not contain ELYS, while Nup153 and Nup62 were identified35,36. These results suggest that AL consist of pre-assembled NPCs, displaying compositional variations across different species. Nucleocytoplasmic transport factors Exportin-1, Importin β, and Ras-related nuclear protein (Ran) likewise localized to AL foci (Supplementary Fig. 5a), as previously reported16, suggesting that they may be involved in AL-mediated NPC assembly, analogous to the established roles of these factors in NPC assembly pathways10,36. NE-resident proteins SUN1 and SUN2, but not Lamin A, Lamin B1, Nesprin, Emerin, and Lap2b, co-localized with AL foci (Supplementary Fig. 5b).

Since AL exist in various sizes and acute MT disruption leads to large AL, we studied their possible dynamic nature. Spinning disk confocal live video microscopy of WT mEGFP-Nup107 HeLa cells, synchronized in interphase, revealed a progressive increase in size and cytoplasmic clustering of AL foci over time following MT depolymerization (Supplementary Fig. 5c and Supplementary Video 1). Similar clustering events were observed upon depletion of FXR1 or in UBAP2L knockout (KO) cells (Supplementary Fig. 5d, e). These results demonstrate that small AL present in normal cells form large AL and accumulate in the cytoplasm when exposed to specific stimuli or in pathological states such as FXS (Fig. 1d).

AL are dynamic and fuse with the NE during G1
The existence of small AL in normal cells prompted us to investigate their physiological role in normally proliferating cells and to address why pathological stimuli, such as MT disruption, lead to the cytoplasmic accumulation of AL. While the conserved role of AL remains unclear, early studies in Drosophila embryos demonstrated that AL integrate into the NE to supply additional membranes and NPCs to support rapid nuclear growth, representing the first indication of the functional role of AL in the context of development7. To determine whether the more widespread, small AL described herein serve a similar role in non-stressed somatic cells, we first examined the abundance of AL structures during different cell cycle stages. Analysis of phosphorylated retinoblastoma (p-Rb; a G1/S phase transition marker) and cyclin B (G2 marker) revealed that small AL foci are present throughout interphase, but predominantly in the G1 and S phases. Large AL foci induced by acute MT disruption followed the same cell cycle distribution pattern (Supplementary Fig. 6e-j). It is therefore possible that nuclear growth during the G1 phase of somatic cells is supported by AL, which primarily exist during G1 (Fig. 2a, b, Supplementary Fig. 6a-d) and which could contribute to the incorporation of new NPCs into the NE.

A quantitative analysis of fixed cells estimated that AL-NPCs within the cytoplasmic AL account for approximately 3% of total nuclear NPCs, increasing to 10% following microtubule depolymerization (Fig. 2c), potentially challenging this hypothesis. However, a simple static comparison of the NPC numbers in two compartments cannot accurately determine the contribution of AL-NPC to the NE. A more time-resolved analysis of the putative dynamic nature of AL structures is required.

For this reason, we used spinning disk live-cell imaging with short time intervals between the acquisition points. We found that AL foci are highly dynamic under physiological conditions, undergoing frequent fusion and fission events (Fig. 2e, Supplementary Video 2, 3), unlike the continuous fusion observed upon microtubule depolymerization (Supplementary Fig. 5c and Supplementary Video 1). Notably, small AL foci fused more rapidly with the NE than large AL (33 and 192 seconds, respectively), and a subset of small AL originated through the fragmentation of large AL and subsequently fused with the NE (81 seconds; Fig. 2d, e, and Supplementary Video 4). After contacting the NE, AL occasionally undergo multiple cycles of detachment and reattachment before ultimately fusing completely with the NE (Fig. 2d, e, and Supplementary Video 5).

In unstressed cells, small AL structures contain an average of 1.65 AL-NPCs (Fig. 1c). Given the observed net AL-NPC fusion frequency with the NE (14 events per 15 minutes; Fig. 2d), we estimate that approximately 924 AL-NPCs are incorporated into the NE during a single G1 phase of the cell cycle (10 hours). Considering that the total number of nuclear pores in HeLa cells ranges from 2000 to 400037, the contribution of AL may in fact be substantial, accounting for an estimated 22–45% of total nuclear NPCs.

To provide additional evidence for the AL-dependent nuclear delivery of NPCs, we generated photoconvertible mEos2-Nup133 KI HeLa cells (Supplementary Fig. 6k-m). The mEos2 is a green-to-red photoconvertible fluorescent protein; although its green fluorescent state lacks sufficient photostability for long-term imaging38. Conversion of mEos2-Nup133 at the NE from green to red fluorescence at a defined interphase time point revealed no detectable diffusion of red signal from the NE into the cytoplasm (Supplementary Fig. 6n, and Supplementary Video 6). Interestingly, photoconversion of cytoplasmic mEos2-Nup133-positive AL foci resulted in the progressive accumulation of a weak but detectable red signal at the NE (Fig. 2f, g, and Supplementary Video 7), indicating that AL-NPCs can be incorporated into the nucleus. The weak nuclear signal is likely due to progressive photobleaching of the fluorescence signal in the course of the experiment and dynamic remodeling of AL foci, which flattened and dispersed as a rim around the nuclei after migration towards the NE (Fig. 2f). Future development of photoconvertible tags with improved long-term stability and brightness will be required to investigate the dynamics of AL incorporation into the NE in live cells in greater detail. Nevertheless, taken together our data show that AL structures observed in normally proliferating cells can attach to the NE during G1.

AL-NPCs promote nuclear pore formation and nuclear expansion
To provide morphological evidence for AL incorporation into the NE, we next used splitSMLM to examine the structural organization and composition of AL-NPCs. This analysis visualized symmetric AL-NPCs in the proximity of or directly attached to the NE containing asymmetric NE-NPCs in WT interphase cells (Fig. 3a panels 1-2). AL-NPC fusion events into the NE were observed where symmetric AL-NPCs were positioned laterally onto the NE (Fig. 3a panels 2-4). Some fusion events were accompanied by the formation of lateral NE openings containing both AL-NPCs and NE-NPCs next to each other (Fig. 3a panels 5-6).

Analysis of Lamin A and B1 by splitSMLM revealed that despite the presence of these NE incisions, the underlying nuclear lamina remained intact (Fig. 3b). Importantly, the analysis of the ER marker PDI confirmed that the sites of AL-NPC insertion within these NE openings colocalized with the ER and ER-NE contact sites (Fig. 3c). Thus, the splitSMLM analysis confirms the observed AL structures in normal cells as specialized sub-compartments of the ER, which is known to be continuous with the outer NE and contribute to its formation39. These observations are also in agreement with a previously proposed model suggesting “en bloc” insertion of AL into the NE and remodeling of AL-NPCs by GTPase Ran to become asymmetric functional NPC structures in fly embryos7,10,34, demonstrating evolutionary conservation of this process. Although 20 nm resolution images from splitSMLM analysis strongly suggest insertion of intact AL into NE, at this point we cannot formally exclude the possibility that AL-NPCs undergo local disassembly or that AL act solely as donors of NPC subcomplexes prior to their NE insertion.

Importantly, spinning disk confocal live video microscopy of the ER marker mScarlet-ER confirmed that AL foci move along the ER to merge with the NE in WT interphase cells (Fig. 3d, and Supplementary Video 8). MT depolymerization is predicted to modulate ER dynamics40, damaging reticulated ER tubules and leading to vesiculation and thickening of cisternal sheets (Supplementary Fig. 7a), which could explain the inhibition of AL foci transfer to the NE and their clustering (Fig. 3d, Supplementary Fig. 5c and Supplementary Video 9). Interestingly, AL-NPCs were also often localized near microtubules (Supplementary Fig. 7b). Live-cell imaging further revealed a consistent spatial association between AL and the microtubule network (Supplementary Video 10), suggesting that they may utilize MT-ER interactions to merge with the NE and to increase the pool of NE-NPCs. Our results indicate that MT depolymerization in somatic cells disrupts ER dynamics, thereby impairing the transfer of AL-NPCs to the NE, resulting in their cytoplasmic accumulation. However, we cannot exclude the possibility that the transport of soluble Nups along microtubules also contributes to AL-NPC assembly. In support of this, studies in Drosophila oocytes have shown that MT mediate the delivery of distinct Nup condensates to AL-NPC assembly sites a process essential for efficient AL-NPC formation10.

Since acute MT depolymerization inhibited AL-NPC transfer to the NE, we set out to analyze the physiological consequences of this inhibition. The density of NE-NPCs (Fig. 3e, f) and the level of Nups at NE (Supplementary Fig. 7c, d) decreased upon acute nocodazole treatment. The short treatment excluded any possible indirect effects of nocodazole on mitotic progression. Strikingly, when cells are arrested in the G1/S phase by sustained addition of thymidine, the nucleus continues to grow beyond its normal size, and MT depolymerization blocks this nuclear growth in G1/S-arrested WT cells (Fig. 3g-j). Nuclear expansion is sustained by a constant supply of many proteins and lipids from the ER to the NE41, a process which could also be disrupted by MT depolymerization. The insertion of AL-NPCs may support nuclear expansion by potentiating nucleocytoplasmic transport rates, or the supply of lipids and proteins required for NE expansion may occur simultaneously with AL insertion into the NE. Taken together, AL are more widespread than previously anticipated and exist in WT somatic cells. AL act to supply a relatively large pool of AL-NPCs (estimated 924) to the NE, thereby promoting the nuclear expansion specifically during G1 phase of the cell cycle. AL are inserted into the NE at the ER-NE junctions through ER dynamics, and this process requires an intact microtubule network. These findings not only establish AL as functional determinants of nuclear architecture during normal mammalian cell proliferation but also indicate existence of an additional specialized pathway for NPC assembly during normal cell cycle progression.

RanBP2 is required for clustering of AL under stress
Having demonstrated a cellular function of AL in normally growing cells, we next aimed to uncover the mechanisms driving AL biogenesis. The component of NPC cytoplasmic filaments RanBP2 (also known as Nup358) has been shown to localize to AL structures and it has been proposed to regulate a Nup condensate fusion mechanism supporting AL biogenesis during Drosophila oogenesis10. However, direct evidence for RanBP2’s role in AL formation is missing. It is also unknown if the role of RanBP2 is conserved in somatic cells and in other species, and what the molecular basis for possible RanBP2 function on AL is. RanBP2 was required for the formation of large AL foci induced by MT depolymerization (Fig. 4a-c, Supplementary Fig. 7e-l) and by inactivation of FXRPs-UBAP2L-dynein pathway in several human cell lines tested (Supplementary Fig. 7m-r). Another component of NPC cytoplasmic filaments, Nup214, was not required for the formation of large AL foci (Supplementary Fig. 8a-c). Higher eukaryotes utilize two NE-NPC assembly pathways, each associated with different cell cycle stages: the postmitotic and the interphase pathway26. We found that downregulation of ELYS (required for postmitotic NE-NPC assembly), or Nup153 or POM121 (driving interphase NE-NPC assembly) did not inhibit AL foci formation when induced by acute nocodazole treatment Supplementary Fig. 8a-f). In contrast, defects in NE-NPC biogenesis were reported to induce AL24 and as expected, downregulation of ELYS or Nup153 led to the formation of large AL foci in untreated cells (Supplementary Fig. 8a, d, g). Downregulation of RanBP2 strongly inhibited the formation of large AL foci induced by Nup153 or ELYS depletion (Fig. 4d-i), suggesting its universal and widespread role in AL clustering.

Is RanBP2 also required for the maintenance of the AL structures? Downregulation (Supplementary Fig. 9a-f) or auxin-inducible depletion (Supplementary Fig. 9g-k) of RanBP242 after nocodazole treatment severely decreased the number of large AL foci and their clustering observed upon MT depolymerization. Thus, RanBP2 is required for the formation and the maintenance of large AL. This process is likely to be independent of reported NPC degradation mechanisms involving autophagy43,44, since RanBP2 downregulation inhibited large AL foci formation also in the presence of lysosomal inhibitors (Supplementary Fig. 10a-h).

Previous studies have shown that RanBP2 knockdown reduces translation of reporter constructs45, whereas we show that deletion or downregulation of RanBP2 did not alter the protein levels of multiple Nups across various cell lines (Supplementary Fig. 10i-k). Notably, the use of auxin-inducible RanBP2 depletion minimizes the confounding effects of chronic translational defects (Supplementary Fig. 10k). Therefore, the observed reduction in AL is unlikely to result from global translational regulation of Nups by RanBP2 under these conditions. The RanBP2 function on AL could also not be explained by possible effects on levels of FXR1 and UBAP2L, the factors driving spatial assembly of Nups during early interphase28, as they were not affected by auxin-inducible deletion of RanBP2 in DLD-1 (Supplementary Fig. 10l) and in HCT116 cells (Supplementary Fig. 10m). Thus, RanBP2 is required for the clustering of small AL into large AL and for AL maintenance but does not regulate protein levels of Nups or Nup-interacting factors.

RanBP2 supports small AL biogenesis and nuclear function in G1
Is RanBP2 also required for the formation and function of small AL that we identified under normal growing conditions? The splitSMLM analysis revealed a relatively broad distribution of the number of NPCs in a single AL-NPC structure (Fig. 4j-l) and as expected, acute MT depolymerization increased both the number of individual AL-NPCs in single AL foci and the total number of AL-NPCs per cell relative to WT cells (Fig. 4j-l). Importantly, RanBP2 was required for the formation of individual AL-NPCs, as well as their clustering both in WT and in nocodazole-treated cells (Fig. 4j-l). AL-NPCs were reported to lack nuclear basket components7,35,36, whereas RanBP2 appears to be localized symmetrically on both sides of AL-NPCs28 (Fig. 3a, b, Supplementary Fig.1f) and RanBP2 can oligomerize to form multimers45, providing a possible explanation for its ability to cluster individual AL-NPCs. In accordance with its role in AL-NPC biogenesis, RanBP2 was required for the formation of small AL foci in WT cells from G1 to S phase but not in the G2 stage (Fig. 4m, Supplementary Fig. 11a) and RanBP2 downregulation by siRNA decreased levels of Nups at the NE (Supplementary Fig. 11b, c). Small AL foci and the levels of Nups at the NE during interphase also decreased following auxin-inducible deletion of RanBP2 (Supplementary Fig. 11d-h), suggesting that RanBP2-mediated formation of small AL is a prerequisite for their merging with the NE and increasing the pool of NE-NPCs. Short-term depletion of RanBP2 reduced levels of several Nups in isolated nuclear fractions and increased their abundance in the cytoplasm in DLD-1 (Fig. 4n) and HCT116 cells (Fig. 4o), consistent with the idea that RanBP2 does not regulate the total protein levels of Nups (Supplementary Fig. 10i-k) but their localization to the NE. Indeed, RanBP2 deletion resulted in reduced NE-NPC density (Fig. 4p, 4r).

Can RanBP2 also contribute to the nuclear pore function? We analyzed the nucleocytoplasmic distribution of endogenous Ran, as previously reported28,46, focusing specifically on Ran protein localization rather than the nucleotide-dependent Ran-GTP gradient. Ran is actively imported into the nucleus by transport factors and exported following cargo release, allowing continuous cycling between nuclear and cytoplasmic compartments. Downregulation of RanBP2 (Supplementary Fig. 12a,b) increased the nuclear-to-cytoplasmic (N/C) ratio of Ran, consistent with impaired nuclear export of Ran protein. Acute depletion of RanBP2 similarly increased the N/C ratio of Ran, with stronger effects observed in G1 compared to G2 cells (Supplementary Fig. 12c,e), suggesting that AL- and RanBP2-dependent NPC assembly contributes to efficient Ran export during early interphase. In contrast, prolonged RanBP2 depletion led to more pronounced defects in G2 cells (Supplementary Fig. 12d,e), likely reflecting cumulative perturbations in nucleocytoplasmic transport. Importantly, we note that the canonical Ran-GTP gradient is determined not solely by Ran localization but by the coordinated activities of RCC1, RanGAP, and the nucleotide state of Ran. Moreover, the Ran-specific transport factor NTF2, which has been implicated in nuclear size scaling47,48, was not directly examined here. Whether cell cycle-dependent regulation of these components contributes to the observed changes in Ran distribution remains an open question. Consistent with impaired Ran export, the use of a light-inducible nuclear export system (LEXY)49 confirmed export defects upon acute RanBP2 depletion (Supplementary Fig. 12f,g ).

Our data in normally proliferating cells demonstrated a role for AL in supplying the NE-NPC pool, thereby supporting the nuclear expansion during early interphase. Since RanBP2 was required for AL-NPC formation in WT cells, we next analyzed the role of RanBP2 in nuclear function. Importantly, short-term depletion of RanBP2 was sufficient to reduce nuclear size (Fig. 4q, 4s), expanding our observations in nocodazole-treated cells (Fig. 3g-i). We conclude that RanBP2-mediated AL-NPC assembly can preserve nuclear pore density as well as nuclear growth during interphase, confirming the critical role of cytosolic NPCs in the nuclear function. Collectively, RanBP2 plays direct and widespread roles in AL, being essential for the biogenesis of single AL-NPCs, their clustering and AL maintenance in multiple stressed and normally proliferating somatic cells.

RanBP2 FG repeats mediate AL assembly and nuclear function
Next, we set out to study the precise molecular mechanisms of RanBP2-mediated AL biogenesis. First, to understand if the function of RanBP2 on AL is specific and which functional domains of this large protein (Fig. 5a) are required for AL formation, we performed rescue experiments using RanBP2 fragments. The full-length (FL; aa 1-3224) and the N-terminal (NT) fragment of RanBP2 (aa 1-1171) efficiently reversed the inhibition of nocodazole-induced large AL foci observed upon downregulation of RanBP2, and the FG repeat region (832-1171) was essential for the RanBP2 AL clustering function (Fig. 5a-e). The effects of auxin-inducible deletion of RanBP2 on AL formation could also be efficiently rescued by the NT fragment of RanBP2 (aa 1-1171) but not by the version lacking the FG repeat region (1-832; Supplementary Fig. 13a-e).

Importantly, full-length RanBP2 with a deletion of the FG region, which is not expected to affect other cellular functions of RanBP2, failed to restore large AL foci in RanBP2-depleted cells by either auxin-inducible degron (Fig. 5f-i) or RanBP2-specific siRNAs (Supplementary Fig. 13f-i), confirming an essential role of the RanBP2 FG repeats in AL accumulation. In contrast, mutation of the LIQIML motif, which is important for RanBP2 targeting to the NE and its function in NE-NPCs assembly45, inhibited efficient NE localization of full-length RanBP2 leading to its nuclear and cytoplasmic localization, as expected, but successfully rescued AL defects observed under both RanBP2 deletion strategies (Fig. 5f-I, Supplementary Fig. 13f-i). These results further support the conclusion that the FG region of RanBP2 regulates AL formation independently of other known cellular functions of this protein and identify a mutant form of RanBP2 which can separate its functions at the NE and on AL.

These findings enabled us to further assess the role of AL in nuclear expansion in untreated WT cells. The full-length WT and the RanBP2 LIQIML mutant, but not the full-length RanBP2 lacking the FG region, successfully rescued the nuclear expansion defect caused by RanBP2 depletion by auxin-inducible degron or siRNA (Supplementary Fig. 13j-l, Fig. 5j-k). Taken together, these results demonstrate the important and specific role of the FG region of RanBP2 in AL formation and AL-driven nuclear function.

RanBP2 N-terminus drives AL-NPC oligomerization and assembly
To corroborate the role of the NT region of RanBP2, we set out to study its molecular interactions relevant for AL biology. Ectopic expression of NT RanBP2 (aa 1-1171; Fig. 6a, 6b, Supplementary Fig. 14a) but not of FL Nup85 or FL Nup133 (Supplementary Fig. 14b,14c), induced AL foci where multiple Nups, with the exception of ELYS and Nup153 (Fig. 6c), could co-localize, relative to controls. CLEM analysis (Fig. 6d) and splitSMLM (Fig. 6e) suggested that these foci represent AL. Ectopic expression of NT RanBP2 increased the number of individual AL-NPCs per cell and the size of AL (Fig. 6e-g). We conclude that elevated levels of the NT RanBP2 fragment can induce AL under normal growth conditions.

Since RanBP2 was required for the assembly of small AL foci and individual AL-NPCs in the cytoplasm (Fig. 4j-l), we next asked how RanBP2 NT regulates this crucial function at the molecular level. Interestingly, structural analysis of the NPC demonstrated that the N-termini of five RanBP2 molecules and one Nup93 molecule bind to the overlapping region of two Y complexes (also named Nup107-Nup160 complexes) in the scaffold’s outer ring45, and deletion of RanBP2 resulted in an outer ring that lacked Y complexes on the cytoplasmic side 50. To test if RanBP2 can promote the oligomerization state of scaffold components of AL-NPCs in a similar fashion, we analyzed separated cytoplasmic and nuclear cellular fractions by gel-filtration chromatography (Supplementary Fig. 14d). Deletion of RanBP2 inhibited the formation of oligomers of Y complexes and Nup96-labelled scaffold in the cytoplasm but not in the nucleus (Fig. 6h).

Intrinsically disordered FG repeats have been reported to stabilize subcomplexes within NE-NPCs51, therefore we examined if similar interactions between the RanBP2 NT fragment (aa 1-1171) and other Nups could play a role in AL-NPC assembly. The outer ring of the NPC is mainly composed of the evolutionarily conserved Y complex. We found that the N-terminal fragment of RanBP2 could bind the Y complex, as well as Nup205 and Nup93 (Fig. 6i), which belong to the inner ring and have recently been reported to also reside on the cytoplasmic face of NPCs45. The interaction of these Nups with NT RanBP2 was dependent on the FG repeat domain (Fig. 6i), suggesting that it facilitates the assembly of single AL-NPCs. Deletion of RanBP2 did not affect the formation of the Y complex (Supplementary Fig. 15a) both in the cytoplasm and in the nucleus (Supplementary Fig. 15b), but it restricted the binding of the Y complex to Nup93 (Fig. 6j, Supplementary Fig. 15c) specifically in the cytoplasm (Supplementary Fig. 15c). This suggests that RanBP2 may stabilize interactions between the outer and inner rings of AL-NPCs in agreement with reported high-resolution NPC structures45, and thereby promote AL-NPC assembly in the cytoplasm. Indeed, Nup93 was required for the formation of large AL-foci (Supplementary Fig. 15d-f), and the increase of AL foci abundance by ectopic expression of NT RanBP2 was dependent on Nup93 (Supplementary Fig. 15g, 15h, 15j, 15k,15m). Downregulation of Nup93 also led to the formation of Nup62-positive nuclear foci (Supplementary Fig. 15j), which did not co-localize with other Nups, suggesting that they may not represent AL. Co-localization of NT RanBP2 with Nup133 (Supplementary Fig. 15i) and Nup62 (Supplementary Fig. 15l) in the cytoplasm required Nup93, in line with reports showing that Nup93 helps to connect the central channel to the NPC scaffold52. We conclude that the function of the NT part of RanBP2 in AL-NPCs can be attributed to its interactions with Nup93 and its ability to stabilize connections between NPC subcomplexes.

Taken together, these results show that RanBP2 drives AL formation through two distinct mechanisms. Under physiological conditions, RanBP2 contributes to scaffold assembly within individual AL-NPCs, including organization of Y-complex components and interactions with Nup93, while under stress conditions it promotes the expansion and clustering of AL-NPCs in the cytoplasm.

Climp63 directs AL-NPCs to ER sheets and the NE
Our findings on the molecular mechanism of AL-NPC assembly and the direct role of RanBP2 in this process create a unique opportunity to identify additional factors involved in AL biology, in particular those facilitating AL-NPC insertion into the ER membranes. Since the NT RanBP2 fragment can induce the formation of AL (Fig. 6a-g), we analyzed the proteins interacting with HA-tagged NT RanBP2 (aa 1-1171) compared to HA-tag only, using liquid chromatography-mass spectrometry (LC-MS). Of total 97 proteins specifically bound to NT RanBP2, 20 were associated with the NPC, as expected. Additionally, numerous ER-associated proteins were also identified (Supplementary Fig. 16a-c). Among them, Climp63 (also known as CKAP4), a type II membrane protein, emerged as an interesting candidate for the regulation of AL because it has been implicated in maintaining ER morphology, being important for the biogenesis of ER sheets and regulation of their width, and for coordinating the formation and dynamics of ER nanoholes53,54. Moreover, its N-terminus binds to MTs, linking the ER to the cytoskeleton55. Thus, it is particularly interesting that we indeed detected an interaction of the HA-tagged NT RanBP2 with endogenous RanBP2 and Climp63 (Fig. 7a, 7b, Supplementary Fig. 17a). Since stringent cell lysis conditions were used for these experiments and several other ER-resident proteins were not found to interact with RanBP2 (Fig. 7a, Supplementary Fig. 17a), this argues against nonspecific co-purification of bulk ER membranes and supports a more specific association between RanBP2 and Climp63. In contrast to the interaction of RanBP2 with Nups (Fig. 6i), our current data do not allow us to determine whether the association of Climp63 depends on the FG-rich region of the NT part of RanBP2, and mapping the direct Climp63-RanBP2 binding sites therefore represents an important avenue for future work.

In interphase cells, RanBP2-labeled AL foci preferentially localized to ER regions positive for Climp63 (Fig. 7c-e, Supplementary Fig. 17 b, c), residing closer to the nucleus relative to the entire ER network (Supplementary Fig. 17d). These Climp63-positive regions likely correspond to ER sheets, in line with published findings54. This suggests that, under normal growth conditions, AL may primarily reside within sheet-like regions of the ER rather than in its tubules. The tendency of the ER sheets to localize close to the nucleus may facilitate AL merging with the NE under physiological conditions.

Downregulation of Climp63 (Fig. 7f-k) led to the dispersion of ER sheets throughout the cytoplasm (Fig. 7g), in accordance with previous reports54, and strongly altered the morphology and distribution of AL foci. AL foci increased in both abundance (Fig. 7g, 7h) and size (Fig. 7i), relocating to the cell periphery, regions that originally corresponded to ER tubules (Fig. 7g), in Climp63-downregulated cells. Depletion of several ER-shaping and ER-associated proteins including RTN4, ATL1, ATL3, LNPK, KTN1 and P180 did not reproducibly affect AL formation (Supplementary Fig. 17e-g). Instead, their knockdown resulted in highly variable AL phenotypes, likely reflecting general perturbations of ER morphology rather than a specific role in AL biology. Thus, Climp63, and possibly a restricted subset of ER factors, but not ER-associated proteins in general, are specifically involved in the AL pathway. Additionally, the nuclear intensity of RanBP2 was reduced upon Climp63 downregulation (Fig. 7j). SplitSMLM analysis confirmed that Nup foci in Climp63-downregulated cells correspond to AL (Fig. 7k). One can speculate that the reported ability of Climp63 to connect the ER to MTs could explain the observed phenotype and that the absence of Climp63 would inhibit MT-mediated transport of AL towards the NE. Alternatively, Climp63-mediated regulation of ER dynamics and nanomorphology could explain these effects. These observations also suggest that RanBP2-mediated biogenesis of AL-NPCs in the cytosol can be separated from their proper localization at ER membranes, a process directly dependent on Climp63 and that in the absence of Climp63, cells attempt to compensate for these defects by increasing the formation of AL-NPCs.

Irrespective of the underlying mechanisms, which could be a topic for future investigations, downregulation of Climp63 decreased Nup levels at the NE (Fig. 7g, 7j) and inhibited nuclear expansion (Fig. 7l-m), suggesting that Climp63-mediated ER dynamics is an important prerequisite for the integration of AL-NPCs into the NE in normal cells. Taken together, our results identify the molecular mechanism of AL biogenesis in normal cells, where RanBP2 is crucial for the formation of AL-NPCs in the cytoplasm and Climp63 acts as a factor targeting AL-NPCs to the proper membrane compartment, the ER sheets. These mechanisms ensure nuclear pore assembly and function as well as nuclear growth specifically during the G1 phase of the cell cycle.

AL-dependent NPC assembly is additive to canonical pathways
What is the relationship of AL-dependent nuclear pore assembly to the canonical assembly pathways acting during two stages of the cell cycle?

Indeed, the postmitotic and the interphase pathways have been primarily described to drive NPC assembly at the NE26. Interestingly, in contrast to RanBP2, downregulation of essential upstream components ELYS (postmitotic pathway) or Nup153 (interphase pathway) did not inhibit AL formation but instead strongly induced it (Supplementary Fig. 8a-f)24. Our measurements in the G1 cell cycle stage estimated that the contribution of AL to pore assembly is substantial, accounting for 22–45% of total nuclear NPCs, suggesting that somatic cells may utilize an additional assembly pathway governed by RanBP2.

To test this hypothesis, we analyzed the complementarity of the RanBP2-driven AL pathway with the two canonical NPC assembly pathways. Downregulation of ELYS or Nup153 reduced NE-associated Nup levels and NPC density in the nucleus (Fig. 8a-e) in HeLa cells, as expected. Strikingly, additional knockdown of RanBP2 further exacerbated these effects (Fig. 8a-e). The same results were obtained in DLD-1 cells where RanBP2 was depleted for only 4 hours using auxin-inducible degradation (Fig. 8f-j). These findings suggest that RanBP2-dependent AL do not act redundantly with the classical NPC assembly pathways but instead contribute independently to the biogenesis of a substantial pool of NPCs at the NE. Taken together, mammalian somatic cells may utilize three distinct nuclear pore assembly pathways, in contrast to the two previously described26,56–58.

Methods
Antibodies
The following primary antibodies were used: rabbit monoclonal anti-Nup133 (Abcam, ab155990, 1:1000 for WB, 1:500 for IF), mouse monoclonal anti-Nup133 (E-6, Santa Cruz Biotechnology, sc-376763, 1:1000 for WB, 1:500 for IF), mouse monoclonal anti-Nucleoporin p62 (BD Biosciences, 610497,1:1000 for WB, 1:200 for IF), rabbit polyclonal anti-RanBP2 (Abcam, ab64276, 1:1000 for WB, 1:1000 for IF), rabbit polyclonal anti-Nup214 (Abcam, ab70497, 1:1000 for WB, 1:500 for IF), rabbit polyclonal anti-Nup85 (Bethyl, A303-977A, 1:1000 for WB, 1:500 for IF), mouse monoclonal anti-Pericentrin 1 (D-4, Santa Cruz Biotechnology, sc-376111, 1:1000 for WB, 1:500 for IF), rabbit monoclonal anti-SEC13 (R&D Systems, MAB9055, 1:1000 for WB, 1:500 for IF), rabbit polyclonal anti-ELYS (Bethyl, A300-166A, 1:500 for WB, 1:500 for IF), mouse monoclonal Nup205 (H-1, Santa Cruz Biotechnology, sc-377047, 1:1000 for WB, 1:500 for IF), mouse monoclonal anti-Nup93 (E-8, Santa Cruz Biotechnology, sc-374399, 1:1000 for WB, 1:500 for IF), rabbit monoclonal anti-Nup98 (C39A3, Cell Signaling Technology, 2598, 1:1000 for WB, 1:500 for IF), rabbit polyclonal anti-Nup96 (Bethyl, A301-784A, 1:1000 for WB, 1:500 for IF), rabbit polyclonal anti-POM121 (GeneTex, GTX102128, 1:1000 for WB, 1:250 for IF), rabbit polyclonal anti-Nup153 (Abcam, ab84872, 1:1000 for WB, 1:500 for IF), rabbit polyclonal anti-CRM1/Exportin 1 (Novus Biologicals, NB100-79802, 1:500 for IF), mouse monoclonal anti-NTF97/Importin beta (Abcam, ab2811, 1:500 for IF), mouse monoclonal anti-Ran (BD Biosciences, 610340, 1:1000 for WB, 1:500 for IF), rabbit monoclonal anti-SUN1 (Abcam, ab124770, 1:500 for IF), rabbit monoclonal anti-SUN2 (Abcam, ab124916, 1:500 for IF), rabbit polyclonal anti-Lamin A (C-terminal) (Sigma, L1293, 1:1000 for WB, 1:500 for IF), rabbit polyclonal anti-Lamin B1 (Abcam, ab16048, 1:1000 for WB, 1:500 for IF), rabbit monoclonal anti-Nesprin1 (Abcam, ab192234, 1:500 for IF), rabbit polyclonal anti-Emerin (Abcam, ab40688, 1:500 for IF), rabbit polyclonal anti-LAP2 (Proteintech, 14651-1-AP, 1:500 for IF), rabbit polyclonal anti-Phospho-Rb (Ser807/811, Cell Signaling Technology, 9308, 1:500 for IF), mouse monoclonal anti-Cyclin B1 (G-11, Santa Cruz Biotechnology, sc-166757, 1:1000 for WB, 1:500 for IF), rabbit polyclonal anti-Cyclin B1 (GeneTex, GTX100911, 1:1000 for WB, 1:500 for IF), rat monoclonal anti-HA (Roche, 11867423001, 1:500 for WB, 1:500 for IF), rabbit polyclonal anti-GAPDH (Sigma, G9545, 1:10000 for WB), mouse monoclonal anti-β-Actin (Sigma, A2228, 1:10000 for WB), mouse monoclonal anti-mCherry (4B3, Thermo Scientific, MA5-32977, 1:1000 for WB, 1:500 for IF), rabbit polyclonal anti-UBAP2L (Abcam, ab138309, 1:1000 for WB, 1:500 for IF), mouse monoclonal anti-FXR1 (Millipore, 03-176, 1:1000 for WB, 1:500 for IF), rabbit polyclonal anti-α-tubulin (Abcam, ab18251, 1:10000 for WB, 1:500 for IF), mouse monoclonal anti-α-tubulin (Sigma, T9026, 1:5000 for WB, 1:500 for IF), mouse monoclonal anti-Nuclear Pore Complex Proteins (mAb414, Abcam, ab24609, 1:1000 for WB, 1:500 for IF), rabbit polyclonal anti-LC3B (Novus Biologicals, NB100-2220SS, 1:1000 for WB), rabbit polyclonal anti-LC3A (Novus Biologicals, NB100-2331, 1:1000 for WB), rabbit polyclonal anti-p62 (GeneTex, GTX100685, 1:1000 for WB, 1:500 for IF), rat monoclonal anti-GFP (3H9, ChromoTek, 3h9-100, 1:1000 for WB, 1:500 for IF), rabbit polyclonal anti-GFP (Abcam, ab290, 1:1000 for WB, 1:500 for IF), rabbit polyclonal anti-Aurora B (Abcam, ab2254, 1:500 for IF), rabbit polyclonal anti-cyclin A (H-432, Santa Cruz Biotechnology, sc-751, 1:1000 for WB, 1:500 for IF), mouse monoclonal anti-PDI (Abcam, ab2792, 1:500 for IF), mouse monoclonal anti-Climp63 (Enzo, ALX-804-604, 1:1000 for WB, 1:500 for IF), rabbit polyclonal anti-CKAP4 (Proteintech, 16686-1-AP, 1:1000 for WB, 1:500 for IF), rat monoclonal α-tubulin-conjugated to Alexa Fluor® 647 (Abcam, ab195884, 1:500 for IF), rabbit polyclonal anti-RTN4/NOGO (Proteintech, 10950-1-AP, 1:1000 for WB), rabbit polyclonal anti-RRBP1 (Proteintech, 22015-1-AP, 1:1000 for WB), rabbit polyclonal anti- KTN1 (Proteintech, 19841-1-AP, 1:1000 for WB), rabbit polyclonal anti-ATL1(OriGene, ta332595, 1:1000 for WB), rabbit polyclonal anti-ATL3 (Proteintech, 16921-1-AP, 1:1000 for WB), rabbit polyclonal anti-LNPK (Atlas Antibodies, HPA014205, 1:1000 for WB).
Secondary antibodies used were the following (HRP-conjugated antibodies were used at a dilution of 1:5000, while fluorescent-labelled antibodies were used at a dilution of 1:500): goat anti-mouse IgG antibody (HRP) (GeneTex, GTX213111-01), goat anti-rabbit IgG antibody (HRP) (GeneTex, GTX213110-01) and goat anti-rat IgG antibody (HRP) (Cell Signaling Technology, 7077S), goat polyclonal anti-mouse AF647 (Thermo Fisher Scientific, A-21236), goat polyclonal anti-mouse AF568 (Thermo Fisher Scientific, A-11031), goat polyclonal anti-mouse AF555 (Thermo Fisher Scientific, A-21424), goat polyclonal anti-mouse AF488 (Thermo Fisher Scientific, A-11029), goat polyclonal anti-rabbit AF647 (Thermo Fisher Scientific, A-21245), goat polyclonal anti-rabbit AF568 (Thermo Fisher Scientific, A-11036), goat polyclonal anti-rabbit AF555 (Thermo Fisher Scientific, A-21429), goat polyclonal anti-rabbit AF488 (Thermo Fisher Scientific, A-11034), goat polyclonal anti-mouse CF680 (Sigma, SAB4600199), goat polyclonal anti-chicken CF660C (Sigma, SAB4600458), goat polyclonal anti-rat CF660C (Sigma, SAB4600193).

Plasmid and siRNA transfections
The following plasmids were used: pX330-P2A-EGFP/RFP and pUC57 was a generous gift from Zhirong Zhang (Romeo Ricci laboratory IGBMC). pQCXIP-mScarlet-ER was a generous gift from Julie Eichler 77 (Catherine-Laure Tomasetto laboratory IGBMC). pEF-HA-MCS2, pEF-HA-RanBP2 (1-3224), pEF-HA-RanBP2 (1-2448), and pEF-HA-RanBP2 (1-1171) were generous gifts from Ralph Kehlenbach78 (University of Göttingen). pEF-HA-RanBP2 (1-832) and pEF-HA-RanBP2 (FL without FG repeat) was obtained by inserting the target sequence, obtained by PCR using pEF-HA-RanBP2 as a template, into pEF-HA-MCS2. pCDNA 3.1 3XHA NUP358 FL (LIQIML) was generous gifts from André Hoelz (California Institute of Technology). The siRNA-resistant RanBP2 plasmid was obtained by PCR and DpnI (Thermo) treatment. NLS-mCherry-LEXY (pDN122) (Plasmid #72655) and mEos2-N1 (Plasmid #54662) was purchased from Addgene. The primers used for plasmid generation are provided in the Supplementary Table 1.

The following siRNA oligonucleotides were used: Non-targeting individual siRNA (Dharmacon), FXR1 siRNA (Dharmacon), UBAP2L siRNA (Dharmacon), RanBP2 siRNA (Dharmacon), Nup153 siRNA (Dharmacon), ELYS siRNA (Dharmacon), POM121 siRNA (Life Technologies), Nup214 siRNA (Eurogenetec), Nup93 siRNA (Horizon), CLIMP-63(Dharmacon), RTN4 (Horizon), p180/RRBP1 (Horizon), KTN1 (Horizon), ATL1 (Horizon), ATL3 (Horizon), LNPK (Horizon). The sequences of siRNA are provided in the Supplementary Table 1.

X-tremeGENETM 9 DNA Transfection Reagent (Roche) was used to perform transient plasmid transfections according to the manufacturer’s instructions. Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) was used to deliver siRNAs for gene knock-down (KD) according to the manufacturer’s instructions.

Generation of cell lines and cell culture
HeLa Kyoto (human cervix carcinoma) cells and U2OS (human bone osteosarcoma) cells were purchased from ATCC. HeLa Kyoto GFP-Nup107 derived stable cell were purchased from CSL cell bank. AID-NUP358 DLD-1 (colorectal adenocarcinoma epithelial cells) cells and AID-NUP358 HCT116 (human colon cancer cells) cells were generous gifts from Mary Dasso42,45 (National Institutes of Health). hTERT-RPE1 (Human retinal pigment epithelial-1) cells was a generous gift from Juliette Godin (IGBMC). FB789 (Human Fibroblast) cells was a generous gift from Luca Proietti (IGBMC). MRC5 (Human Fibroblast) cells was a generous gift from Thierry Seroz (IGBMC).Control human fibroblasts and FXS patient-derived fibroblasts were previously reported25.

mCherry-Nup133 and mEos2-Nup133 knock-in HeLa Kyoto cell lines were generated using CRISPR/Cas9 genome editing system as described previously78. The guide RNAs (gRNA) were designed as described previously79, and cloned into pX330-P2A-EGFP through ligation using T4 ligase (New England Biolabs). The donor constructs used as templates for homologous recombination to repair the Cas9-induced double-strand DNA breaks were generated by cloning the Nup133 DNA fragments upstream and downstream of the CRISPR target sequences and the sequence for mCherry or mEos2, assembled using ExonucleaseⅢ (Takara) method, into a pUC57 vector. HeLa Kyoto cells were transfected for 24h and GFP positive cells were collected by FACS (BD FACS Aria II), cultured for 2 days and seeded with FACS into 96-well plates. Single-cell clones of mCherry-Nup133 knock-in or mEos2-Nup133 knock-in were validated by Western blot and sequencing of PCR-amplified targeted fragment by Sanger sequencing (GATC). The sequences of gRNA and the primers used for plasmid generation are provided in the Supplementary Table 1.

UBAP2L knock-out (KO) in mCherry-Nup133 knock-in HeLa Kyoto cell lines were generated using CRISPR/Cas9 genome editing system as described previously78. Two guide RNAs (gRNA) were designed using the online software Benchling (https://www.benchling.com/), and cloned into pX330-P2A-EGFP/RFP through ligation using T4 ligase (New England Biolabs). mCherry-Nup133 knock-in HeLa Kyoto cells were transfected for 24h and GFP and RFP double positive cells were collected by FACS (BD FACS Aria II), cultured for 2 days and seeded with FACS into 96-well plates. UBAP2L KO single-cell clones were validated by Western blot and sequencing of PCR-amplified targeted fragment by Sanger sequencing (GATC). The sequences of gRNA are provided in the Supplementary Table 1.

All cell lines were cultured at 37°C in 5 % CO2 humidified incubator. HeLa Kyoto and its derived cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (4.5 g/L glucose) supplemented with 10 % fetal calf serum (FCS), 100 U/ml Penicillin + 100 µg/ml Streptomycin. U2OS cell lines were cultured in DMEM (1 g/L glucose) supplemented with 10 % FCS, Non-Essential Amino Acids + Sodium Pyruvate 1 mM + Gentamicin 40 µg/mL. DLD-1 cell lines were cultured in DMEM (1 g/L glucose) supplemented with 10 % FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. HCT116 cell lines were cultured in McCoy’s 5A medium + 10 % FCS + 100 U/ml penicillin, and 100 µg/ml streptomycin. hTERT-RPE1 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) F-12 supplemented with 10% FCS, 0.01 mg/ml hygromycin B. FB 789 cell lines were cultured in MEM w/Earles's salts supplemented with 15 % FCS, + Vitamines + AANE + Gentamicine 40 µg/ml + Penicillin 100 UI/ml + Streptomycin 100 µg/ml. MRC5 cell lines were cultured in DMEM (1 g/L glucose) supplemented with 10 % FCS, + Gentamicine 40 µg/ml.

Culture and differentiation of hiPSCs into neurons
Human induced pluripotent stem cells (hiPSCs, GM8330-8; kindly given by Michael E. Talkowski, Center for Genomic Medicine, Massachusetts General Hospital) were cultured in mTeSR1, following the manufacturer’s instructions, and maintained on Cultrex-BME. hiPSCs were cultured at 37°C with 5% CO₂ under humidified conditions. Colonies were passaged every 3 to 4 days by manual cutting. For the generation of inducible rtTA/Ngn2 hiPSC lines, Lentiviral vectors (PCW57) containing the human NGN2 gene cassette under the control of the TRE promoter, along with a puromycin resistance gene linked to rTetR via a T2A sequence under a constitutive promoter, were used to transduce hiPSCs. The construct was cloned and packaged into lentiviruses by the Molecular Biology Platform of the IGBMC, hereafter named TetO-NGN2 lentivirus.

hiPSCs were dissociated using Accutase and seeded in 12-well plates at a density of 6 × 10⁴ cells per well in mTeSR1 supplemented with 10 μM Rocki (STEMCell), and incubated overnight at 37°C. The next day, the medium was removed, and the cells were incubated for 6 hours in DMEM/F12 containing 8 μg/ml Polybrene, 10 μM Rocki, and 4 × 10⁴ TU of TetO-NGN2 lentivirus. After 6 hours, cells were washed with DPBS (Thermo Fisher) and returned to mTeSR1 at 37°C and 5% CO₂. Puromycin selection began on day 1 post-transduction, with 0.5 μg/ml from day 1 to 2, 1 μg/ml from day 3 to 4, and then 0.5 μg/ml again from day 5 to 7, with daily medium changes using mTeSR1. Selected hiPSC colonies were subsequently expanded in new Cultrex-BME-coated plates after Accutase dissociation and maintained under standard conditions.

For the differentiation of rtTA/Ngn2 hiPSCs into induced neurons, the differentiation was adapted from reported protocol without addition of astrocytes80. On day 0, TetO-NGN2 hiPSCs were dissociated with Accutase and seeded at a density of 4 × 10⁵ cells/cm² on Poly-L-Ornithine (50 μg/cm²) + Laminin (7 μg/cm²)-coated petri dishes and in mTeSR1 supplemented with 10 μM Rocki and 3 μg/ml doxycycline. On day 1, media was replaced with DMEM/F12 + Glutamax 1X + NEAA 1X + B27 1X + N2 1X + doxycycline 3 μg/ml. On day 3, media was replaced with Neurobasal + Glutamax 1X + NEAA 1X + B27 1X + NT-3 (10 ng/mL) + BDNF (10 ng/mL) + doxycycline 3 μg/ml + cytosine β-D-arabinofuranoside (AraC) 5 μM. From day 5 to day 20, media was half-changed each 3-4 days with Neurobasal + Glutamax 1X + NEAA 1X + B27 1X + NT-3 (10 ng/mL) + BDNF (10 ng/mL) + doxycycline 3 μg/ml. Doxycycline was maintained until day 14 of differentiation. Neurons were fixed/immunostained at 20 DIV.

Cell treatments and cell cycle synchronization
To induce the formation of the cytoplasmic AL foci by microtubule depolymerization, cells were incubated with 10 μM nocodazole (Sigma M1404-50MG) in culture media for 90 min at 37°C.
To measure nuclear growth, cells were arrested in G1/S phase by sustained addition of thymidine (Sigma, T1895) at 2 mM. After 16 hours cell were treated with 10 μM nocodazole and collected at different time points. To test the role of autophagy in AL foci abundance, cells were incubated with 30 nM Bafilomycin (Sigma, B1793-10UG) in culture media for 6h at 37°C.

For cell cycle synchronizations double thymidine block and release (DTBR) protocol was used. Cells were treated with 2 mM thymidine for 16h, washed out (three times with warm thymidine-free medium), then released in fresh thymidine-free culture medium for 8h, treated with 2 mM thymidine for 16h again, washed out, and then released in fresh thymidine-free culture medium for 9h (mitosis) or 12 hours early G1 phase.

Immunofluorescence
Cells were plated on 9-15 mm glass coverslips (Menzel Glaser) in 12- or 24- well tissue culture plates. For immunofluorescence, cells were washed twice with PBS before being fixed with 4% paraformaldehyde (PFA, Electron Microscopy Sciences 15710) in PBS for 15-20 min at room temperature. Cells were washed three times for 5 min in PBS before permeabilization for 5 min with 0.5% NP-40 (Sigma) in PBS. Cells were washed three times for 5 min in PBS before blocking for 1h at room temperature with 3 % BSA in PBS-Triton 0.01 % (Triton X-100, Sigma, T8787). Cells were then incubated with primary antibody in blocking buffer (3 % BSA in PBS-Triton 0.01 %) for 1h at room temperature. Cells were washed three times with PBS-Triton 0.01 % with gentle shaking for 10 min before being incubated with secondary antibody in blocking buffer for 1h at room temperature in the dark. After incubation, cells were washed 3 times with PBS-Triton 0.01 % for 10 min each time with gentle shaking in the dark, and then glass coverslips were mounted on glass slides using MOWIOL containing 0.75 μg/ml DAPI (Calbiochem) and imaged with 100X, 63X or 40X objectives using a Zeiss epifluorescence microscope or confocal microscope a Leica Spinning Disk Andor/Yokogawa confocal microscope.

Live-cell imaging
For live-cell microscopy, cells were grown on 35/10 mm 4 compartment glass bottom dishes (Greiner Bio-One, 627871) or μ-Slide 8 well glass bottom (Ibidi, 80827). Before photography, cells were treated with SiR-DNA (Spirochrome, SC007) according to the manufacturer's instructions. Live cell microscopy was performed using a confocal microscope Leica/Andor/Yokogawa spinning disk, Leica CSU-W1 spinning disk, or Nikon PFS spinning disk with a 40X or 63X objectives with Live Data Mode equipped with automated temperature control.

In particular, for AL foci’ dynamics assays under normal growth conditions, HeLa Kyoto GFP-Nup107 cells were analyzed by Leica CSU-W1 spinning disk (63X NA 1.4 oil objective) for 9h. Z-stacks (10 μm range, 1 μm step) were acquired every 5 min and videos were made with maximum intensity projection images for every time point shown at a speed of 7 frames per second. For AL foci’ dynamics assays after microtubule depolymerization, HeLa Kyoto GFP-Nup107 cells were arrested in S phase by thymidine block and microtubule depolymerization and nucleoporin aggregation were induced by addition of 10 μM nocodazole addition. For AL foci’ dynamics assays under FXR1 knockdown (KD) or UBAP2L KD conditions, HeLa Kyoto GFP-Nup107 derived stable cell were treated with the indicated siRNAs for 48 h before imaging.

For rapid live-cell imaging, HeLa Kyoto GFP-Nup107 cells were imaged for 15 min using a Yokogawa X1 spinning disk equipped with Nikon TI2 microscope and photometrics prime 95B camera and a 100x NA 1.4 oil-immersion objective using Live SR module from GATACA system. Z-stacks spanning 3-5 μm with a step size of 1 μm were acquired every 3 s. Videos were generated from maximum-intensity projections of each time point and displayed at 7 frames per second. Quantification was performed by focusing specifically on regions adjacent to the nuclear envelope (NE). Because attachment and detachment events could not be reliably recorded across the entire nuclear circumference due to the limited field of view, each nucleus was divided into multiple NE-adjacent sections. Foci dynamics were tracked frame by frame within each section, and attachment and detachment events were recorded. Events from all sections were subsequently pooled for statistical analysis.

For AL foci and microtubules dynamics assays, cells were treated with SiR-tubulin (Spirochrome, SC002) according to the manufacturer's instructions.

For AL foci and ER’ dynamics assays, HeLa Kyoto GFP-Nup107 were transfected with the pQCXIP-mScarlet-ER for 24 h and analyzed by Nikon PFS spinning disk (100X NA1.4 oil objective) for 5h where images were acquired every 20s (without Z-stacks) or were analyzed for 9h where images were acquired every 2 min (without Z-stacks).

For photoactivatable nucleocytoplasmic shuttling, AID-NUP358 DLD-1 cells were transfected with the NLS-mCherry-LEXY (pDN122) for 24h and analyzed by a Leica Sp5 confocal inverted microscope (DMI6000) system (40X NA1.3 oil objective). The circular region of interest (ROI; ~38 µm2) was placed onto single, selected cells. The ROI was scanned with a 458-nm laser beam for 10 min followed by a 20-min dark-recovery phase. The mCherry signal was imaged in parallel every 10s for 30 min using the 561-nm laser line for excitation.

For photoconversion assays, HeLa Kyoto mEos2-Nup133 cells were analyzed by Yokogawa X1 spinning disk equiped with Nikon TI2 microscope, photometrics prime 95B camera and ILas Head for photomanipulation (100X NA1.4 oil objective). The modular software part developed by the GATACA system was used to set the acquisition of the selected area to be photoconverted with applied repeats of blue light for a period of 200 seconds (typical value, depending on the size of the area) by the 500 mW laser diode at 100%. A 30-second acquisition pre-sequence and with acquisition every 10 seconds in the green and red channels has been performed. Two post sequences were carried out after acquisition, the first for 30 seconds with an interval every 10 seconds in order to quickly observe the effect of photoconversion and the second sequence for 12 hours every 15 minutes to see the effect over a longer period. The software was set up to use spinning with the 491 nm 50 mW laser diode at 20% power with an acquisition time of 150 ms for the green channel and the 561 nm 50 mW laser diode at 30% power with 150 ms exposure time for the red channel.

Single molecule localization microscopy
For super-resolution imaging using single molecule localization microscopy (SMLM), cells were plated on 35 mm glass bottom dishes with a 14 mm micro-well #1.5 cover glass (Cellvis). Samples were prepared as described above for immunofluorescence but were not mounted using MOWIOL and were stored in PBS.

The imaging was performed as previously described, using the splitSMLM method81 for multi-color single molecule localization microscopy (SMLM) based on a dichroic image splitter. In brief, the immunolabeled samples were mounted in an imaging buffer containing 200 U/ml glucose oxidase (G2133, Sigma), 1000 U/ml catalase (C1345, Sigma), 10 % w/v glucose, 200 mM Tris-HCl pH 8.0, 10 mM NaCl, 50 mM MEA (30080, Sigma), with addition of 2 mM COT (138924, Sigma). The buffer optimizes the performance of the fluorophores AF647, CF660C and CF680 for three-color direct stochastic optical reconstruction microscopy (dSTORM). Imaging was then performed on a modified Leica SR GSD system, with a HCX PL APO 160X/1.43 Oil CORR TIRF PIFOC objective. The acquisitions began with a pumping phase, during which the sample was illuminated with the 642 nm 500 mW fiber laser (MBP Communication Inc.) but the fluorescence was not recorded due to a very high density of fluorophores in a bright state. The image collection started when the density of fluorophores dropped to a level that allowed observation of individual molecules and in case of emitter depletion, the sample was additionally illuminated with a 405 nm diode laser (Coherent Inc.) to increase the emitter density (“back-pumping”). The resulting emission was split with an Optosplit II (Cairn Research) image splitter, attached to a camera port of the microscope, and containing a Chroma T690LPXXR dichroic mirror. This creates a long and short wavelength channel on the Andor iXon Ultra 897 EMCCD camera attached to the image splitter. Each fluorophore thus shows a characteristic brightness ratio between the two channels, enabling the separation of spectrally different fluorophore species (i.e., spectral demixing)81. Localization of the single molecules in both channels was performed using the Leica LAS X software with the “direct fit” fitting method. The localization tables were then exported and further processed using the SharpViSu software29,82,83 and the SplitViSu plugin81. This accounts for spectral demixing, correction of chromatic errors, drift-correction, and the reconstruction of super-resolution images. A pixel size of 10 nm was used to reconstruct the super-resolution images from 2D histograms of single-molecule coordinates. For the single-color imaging without an image splitter, the images were recorded with an Andor iXon + EMCCD camera, attached to the second camera port of the microscope. Data were then processed as described, using the Leica LAS X and SharpViSu software, while omitting the spectral demixing steps in the SplitViSu plugin.

The magnified NPC regions shown in the article were upsampled by bilinear interpolation for improved visualization.

Correlative light and electron microscopy
Cells were grown on carbon coated sapphire disks (3 mm diameter, 50 μm thickness, Wohlwend GmbH, art. 405) and high pressure frozen (HPM010, AbraFluid) in their culture medium. Freeze substitution was performed in a Leica EM-AFS2 with 0.1 % uranyl acetate in dry acetone for 24h at -90°C. The temperature was then raised to -45°C over 9h (slope 5 degrees / hour) and the samples were further incubated at this temperature for 5h to increase electron contrast. After rinsing in acetone, the samples were infiltrated with increasing concentrations of Lowicryl HM20 (Polysciences Inc.), while raising the temperature to -25°C. Finally, the blocks were polymerized using UV light. After removal of the sapphire disks from the block surface, 300 nm sections were cut parallel to the block surface using an ultramicrotome (UC7, Leica Microsystems). The sections were collected on carbon coated mesh grids (S160, Plano).

Fluorescence imaging of the sections was carried out with a widefield microscope (Olympus IX81), equipped with a 100X 1.40 NA Plan-Apochromat objective, placing the grids in water in a glass-bottom dish (Mattek). After light microscopy acquisition, the grids were post-stained with 2 % uranyl acetate in 70 % methanol and Reynold’s lead citrate. Electron microscopy (EM) was performed using a Tecnai F30 transmission electron microscope (Thermo Fisher) at 300kV acceleration voltage. The grid squares that were previously imaged at the light microscope (LM) were mapped at low mag. After registration with the LM image, TEM tomography was performed on the areas of interest using the software package SerialEM84. Tomograms were reconstructed with IMOD85. Correlation between LM and EM images was done with the plugin ec-CLEM86 of the software platform Icy87, using features of the samples that could be identified in both imaging modalities.

Image analysis
Image quantification analysis was performed using ImageJ or CellProfiler software. In particular, the presence of Nups AL foci was visually confirmed by two individuals.

For nuclear size or nuclear envelope intensity analysis of Nups, CellProfiler software was used. Briefly, the Prewitt Edge Finder method was used to enhance the edges in a DAPI image, and then a nucleus size threshold was set to automatically identify nuclei. This allowed identification and measurement of nuclei area, morphology factor and mean nuclear intensity for the desired channel. The software's parameter measurements were exported to an Excel file and statistically analyzed.

For the size of Nups foci, ImageJ software was used. In brief, the Nups channel was thresholded in ImageJ and then the size of the Nups foci was calculated by analyzing particles (size 1-100). AL foci that were considered “large” had a size bigger than 0.3 μm2.

For co-localization analysis of RanBP2 and Nups in the cytoplasm, CellProfiler software was used. The Prewitt Edge Finder method was used to enhance the edges in a DAPI image, and then nuclei, RanBP2 and different Nups were identified according to the corresponding channels. The cytoplasmic fraction of RanBP2 and Nups was identified using the MaskObjects module. Co-localization analysis of RanBP2 and Nups in cytoplasm was conducted by the MeasureObjectOverlap module. The software's parameter measurements were exported to an Excel file and statistically analyzed. For nuclear envelope or cytoplasm intensity of Ran, ImageJ was used. The ROIs of the cell and nuclei were selected based on tubulin and DAPI channels, and the ROI of the cytoplasm was selected by the XOR function of cell and nuclei. Finally, the mean gray value of the nuclei and cytoplasm was measured.

For AL-NPCs analysis in the cytoplasm, AL-NPCs were recognized by the Nup133 signal surrounding Nup62 signals. The number of AL-NPCs was counted visually by two individuals.

For the measurement of NPCs density on the nuclear membrane, ImageJ software was used. Briefly, the images of the Nup62 channel were converted to 32 bits using ImageJ and then filtered using Gaussian blur (sigma = 2). The nuclear region was manually selected and then the ‘Find Maxima’ function (noise tolerance 10) was performed to obtain the area of the selected region and the number of AL-NPCs.

For protein import and export assay analysis, ImageJ software was used. Briefly, the ROI within the uniform distribution of NLS-mCherry-LEXY signal was selected in the nuclear region, and the measurement of the mean gray value of the same ROI in each frame (sequential time points) was performed by the multi-measure option.

For calculation of the mean fluorescence intensity of Nups as a function of distance from the nucleus a custom Python script was used with help of the scikit-image module88. The nucleus and the nuclear envelope were identified using the RanBP2 channel following well-established procedures89,90. Similarly, the region corresponding to the ER was determined from the mScarlet-ER channel. Regions corresponding to RanBP2 foci were detected using the scikit-image method blob_log for blob detection. The analyzed region for Climp63 and mScarlet-ER corresponded to the ER region excluding the nucleus. The analyzed region for the RanBP2 foci corresponded to the blob regions that fall within the ER region excluding the nucleus. For each pixel in the analyzed regions, the minimum Euclidean distance to any nuclear envelope pixel was taken as the distance from the nucleus. Given the intensity-distance pairs for each pixel, the pixels were binned for the distance and the mean pixel intensity determined for each bin. The obtained histograms were finally normalized for the purpose of comparison.

For colocalization analysis, to assess pixel colocalization/correlation, we used correlation measurement within CellProfiler. Briefly, we used the "MaskImage" module to obtain Climp63, mScarlet-ER, and RanBP2 located in the cytoplasm. “Measure Colocalization” modules were used to study the colocalization and correlation between intensities in different images (different color channels) on a pixel-by-pixel basis across an entire image. The number of cells measured per condition was listed in the corresponding figure’s legend.

For nuclear height measurement, confocal microscopy was used to perform z-sampling of the DAPI channel of the sample, with a z-step of 0.5 μm. Finally, Fiji analysis was used to obtain an approximate nuclear height based on the Z-Extent.

Protein sample preparation and western blotting
After centrifugation at 250 g for 3 minutes at 4°C, cells were washed twice with cold PBS, and lysates were prepared using cell lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 % sodium deoxycholate, 0.1% SDS, 0.5% sodium deoxycholate, 1 % Triton X-100, 1 mM EDTA, 1 mM EGTA, 2 mM Sodium pyrophosphate, 1 mM Na3VO4 and 1 mM NaF) supplemented with protease inhibitor cocktail (Roche) and incubated on ice for 30-60 min. After centrifugation at 16 000 g for 15 min at 4°C, the supernatant was transferred to the new clean Eppendorf tubes, and the total protein concentration was measured with a Bio-Rad Protein Assay kit (Bio-Rad). Separation of cytoplasmic and nuclear fractions was done using the NE-PER nuclear and cytoplasmic extraction reagent kit (Thermo Scientific™, 78833). Protein samples were boiled for 10 min at 95°C in 1X Laemmli buffer (LB) with β-Mercaptoethanol (BioRad, 1610747).

SDS-PAGE of proteins was done using pre-cast 4-12 % Bis-Tris gradient gels (Thermo Scientific, NW04120BOX) or pre-cast NuPAGE™ 3-8 % Tris-Acetate gradient Gels (Thermo Scientific, EA0378BOX). Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, IPFL00010) using wet transfer modules (BIO-RAD Mini-PROTEAN® Tetra System). Membranes were blocked in 5 % bovine serum albumin (BSA, Millipore , 160069), or 5% non-fat milk powder mixed with 3 % BSA resuspended in TBS-T for 1h at room temperature, followed by incubation with primary antibodies diluted in TBS-T 5 % BSA/5 % milk overnight at 4°C or 1h at room temperature and secondary antibodies diluted in TBS-T 5 % BSA/5 % milk at room temperature. Membranes were imaged using SuperSignal West Pico (Pierce, Ref. 34580). Western blotting images were acquired by GE Healthcare_Amersham Imager 600 or Invitrogen iBright 1500. The grayscale value of protein bands was quantified using ImageJ software.

Immunoprecipitation
Cell lysates were prepared for immunoprecipitations (IP) as described above. Lysates were adjusted to equal volume and concentration. For endogenous IP experiments, lysates were incubated with IgG or target-specific antibodies overnight at 4°C with rotation, and then incubated with protein G sepharose 4 Fast Flow beads (GE Healthcare Life Sciences) for 4h at 4°C with rotation. Before using, beads were blocked with 3 % BSA diluted in 1X cell lysis buffer and incubated for 2h at 4°C with rotation. The incubated IgG/ specific antibodies-samples-beads were washed with washing buffer (25 mM Tris-HCl pH 7.5, 300 mM NaCl, 0.5 % Triton X-100, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM Sodium pyrophosphate, 0.5 mM Na3VO4 and 0.5 mM NaF) or TBS-T 4 to 6 times.

For GFP-IP/HA-IP experiments, GFP-Trap A agarose beads (Chromotek) or Pierce™ anti-HA Magnetic Beads (Thermo Scientific 88836) were used. Cells expressing GFP- or HA-tagged plasmids for at least 24h before lysis were used. Lysates were incubated with beads for 4h at 4°C with rotation, and then washed with washing buffer (25 mM Tris-HCl pH 7.5, 300 mM NaCl, 0.5 % Triton X-100, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM Sodium pyrophosphate, 0.5 mM Na3VO4 and 0.5 mM NaF) or TBS-T 4 to 6 times.

Gel fractionation
To calibrate the Superose 6 Increase 3.2/300 column, the gel filtration standard (Bio-Rad, Catalog # 1511901) was rehydrated and diluted to 1.5 mg/mL according to the instruction of the manufacturer. The column was equilibrated using buffer comprising 15 mM pH 7.5 Tris, 15 mM NaCl, 60 mM KCl, 340 mM sucrose, 0.15 mM spermine, 0.5 mM spermidine, 0.5 % Triton X-100, 1 mM NaF, 10 mM DTT. The standard sample (50 µL) was separated by a flow rate of 0.015 ml/min. Kav values for the standards were calculated. The calibration curve of Kav versus the logarithm of their molecular weights was determined as follows:
Kav = (Ve - Vo) / (Vt - Vo)
where Ve = elution volume of the protein
Vo = column void volume = 0.8 mL
Vt = total bed volume = 2.4 mL

HeLa cell cytosolic and nuclear fractions were prepared as described above. For the gel filtration of the HeLa cell cytosol extraction sample, a 2.4-mL Superose 6 Increase 3.2/300 column (Cytiva) was equilibrated using buffer comprising 15 mM pH 7.5 Tris, 15 mM NaCl, 60 mM KCl, 340 mM sucrose, 0.15 mM spermine, 0.5 mM spermidine, 0.5 % Triton X-100, 1 mM NaF, 10 mM DTT. For the gel filtration of the HeLa cell nuclear fraction sample, a 2.4-ml Superose 6 Increase 3.2/300 column (Cytiva) was equilibrated using buffer comprising 50 mM Tris 7.5, 1 % NP-40, 150 mM NaCl, 1mM EDTA, 0.1 % SDS, 0.5 % sodium deoxycholate. The cytosolic and nuclear fractions (typically 1~3 mg/ml) were clarified by centrifugation for 15 minutes at 12,000 X g before loading to the equilibrated gel filtration column. Samples were separated by a flow rate of 0.017 ml/min, and 50 µL fractions were collected for Western blot analysis (typically collects from 1.2mL to 2.4mL).

LC-MS/MS analysis
MS grade Acetonitrile (ACN), MS grade H2O and MS grade formic acid (FA) were from ThermoFisher Scientific (Waltham, MA, USA). Sequencing-grade trypsin/Lys C mix was from Promega (Madison, WI, USA). Trifluoroacetic acid (TFA) and ammonium bicarbonate (NH4HCO3) were from Sigma-Aldrich (Saint-Louis, MO, USA).

For sample preparation prior to LC-MS/MS analysis beads from immunoprecipitation experiments were incubated overnight at 37°C with 20 μL of 50 mM NH4HCO3 buffer containing 1 µg of sequencing-grade trypsin/Lys C mix. The digested peptides were loaded and desalted on evotips provided by Evosep (Odense, Denmark) according to manufacturer’s procedure before LC-MS/MS analysis. For LC-MS/MS acquisition samples were analyzed on a timsTOF Pro 2 mass spectrometer (Bruker Daltonics, Bremen, Germany) coupled to an Evosep one system (Evosep, Odense, Denmark) operating with the 30SPD method developed by the manufacturer. Briefly, the method is based on a 44-min gradient and a total cycle time of 48 min with a C18 analytical column (0.15 x 150 mm, 1.9µm beads, ref EV-1106) equilibrated at 40°C and operated at a flow rate of 500 nL/min. H2O/0.1 % FA was used as solvent A and ACN/ 0.1 % FA as solvent B. The timsTOF Pro 2 was operated with a DIA-PASEF method comprising 12 pydiAID frames with 3 mass windows per frame resulting in a cycle time of 0.975 seconds as described in Bruker application note LCMS 218.

For data analysis MS raw files were processed using Spectronaut 18 (Biognosys, Switzerland). Data were searched against the SwissProt Homo Sapiens database (downloaded 07 2023, 20423 entries). Specific tryptic cleavages were selected and a maximum of 2 missed cleavages were allowed. The following post-translational modifications were considered for identification: Acetyl (Protein N-term) and Oxidation (M) as variables. Identifications were filtered based on a 1 % precursor and protein Qvalue cutoff threshold. The protein LFQ method was set to automatic and the quantity was set at the MS2 level with a cross-run normalization applied. Multivariate statistics on protein measurements were performed using Qlucore Omics Explorer 3.9 (Qlucore AB, Lund, SWEDEN). A positive threshold value of 1 was set to allow a log2 transformation of abundance data for normalization i.e. all abundance data values below the threshold are replaced by 1 before transformation. The transformed data were finally used for statistical analysis i.e. the evaluation of differentially present proteins between two groups using a bilateral Student’s t-test. A p-value better than 0.05 was used to filter differential candidates.

Data acquisition, statistical analysis and reproducibility
All experiments were performed in a strictly double-blinded manner. All experiments were performed with at least three independent biological replicates, unless stated otherwise in the figure legends and image quantifications were carried out in a blinded manner. Curves and graphs were made using GraphPad Prism and Adobe Illustrator software. Data were analyzed using a one-sample two-tailed T-test or a two sample unpaired two-tailed T-test (two-group comparison or folds increase relative to the control, respectively) or one-way ANOVA. A p-value less than 0.05 (typically ≤ 0.05) was considered statistically significant and stars were assigned as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is a revised manuscript, transferred to Nature Communications after review and consideration at [REDACTED]. The\noriginal manuscript was extensive, robust and used state of the art microscopical approaches and orthogonal methods of\nintervention and observation to reveal the role for RanBP2 in clustering and incorporating cytoplasmic aggregates of NPCs\nin Annulate Lamellae (AL) into the primary nucleus. In revision, the authors have addressed all the Nat Cell Biol comments\nand incorporated substantial amounts of new data into the manuscript - whilst it was a mammoth story before, it is doubly-\nmammoth here! This is a massive amount of work and the authors ought be lauded for their efforts here. Whilst the narrative\nand structure has improved substantially since the original submission, it is quite a challenging manuscript to carry in your\nhead whilst reading. I think the manuscript is suitable for publication.\nRegards my specific comments, the authors have demonstrated physiological contribution of the AL-insertion pathway to\npopulating the interphase nucleus with NPCs and allowing the nucleus to expand. The new timelapse imaging of AL-\ninsertion into the NE is a strong addition, as is the separation of Nup153/ELYS effects from a RanBP2-dependent insertion.\nThey have clarified issues about the RanBP2-CLIMP63 interaction, and whilst not showing it is direct, they have included\ncontrols to highlight the specificity of the co-precipitation and made it clearer how the IP was performed.\nMechanistically, the mapping of the AL-clustering activity to the FG domain of RanBP2 is a helpful addition.\nThe G1-restricted RanBP2 contribution to nuclear expansion is interesting, but I wonder if the authors could explain how the\ncells remain in G1 for the imaging period (48h); from the methods, the thymidine arrest is only for 16h, then dmso/noc for 90\nminutes and then imaging. The restriction on nuclear expansion is clear, but I can't work out why the cells don't reenter\nthe cycle and divide over the next 2 days? The details of this assay could be added to the methods as I don't think this is\nincluded.\nA second element that could be clarified in the text a little is the role of RanBP2 in oligomerising Y-complexes within\nindividual NPCs and binding Nup93 (Fig 6E-G) from the RanBP2-dependent clustering of individual NPCs into larger AL\naggregates. I think these are two separate things, and I'm not sure that the Y-complex and Nup93 binding is needed for\nthe narrative.\nLastly, the cartoon at the end really helped with understanding; however, it is a little stylised. Rather than a grey block for the\nER, I wonder if individual INM, ONM and ER membranes could be depicted. It is a little hard to understand what the authors\nthink is happening to accommodate this insertion. There will certainly be some gymnastics required to place these AL-NPCs\nacross the INM/ONM for contribution to transport activity, but it isn't clear from the cartoon how this would happen. To help\nothers follow up on this work, I would encourage the authors to outline how this might happen so that subsequent\nexperimental followups can be planned.\nMinor\nL214, underlying nuclear membrane should probably be underlying nuclear lamina.", "reviewer_2": "Overall assessment of revision: The revised manuscript addresses my major concerns with the initial submission. The most\ncritical aspect related to the membrane topology of the nuclear envelope at sites that the authors interpret as insertion events\nfor AL into the NE. The new data acquired using splitSMLM that suggests openings in the nuclear envelope at the sites of\nAL integration is the most critical. While this addresses my major concern it should be more robustly presented (see below).\nIn addition, the cartoon model should incorporate these new insights. One other issue related to the newly added data\nrelates to interpretation of the described cell cycle differences in Ran distribution.\nSpecific Points:\n1. Related to the gaps in the nuclear envelope: In Fig. 3c it would be helpful to demarcate the edges of the PDI signal that\nthe authors interpret as gaps. Because of the importance of this finding, either a quantitative representation of how\ncommonly this was observed and/or additional examples should be included – even if just from the timepoint where the gap\nand apposed AL are viewable.\n2. The cartoon model in 7k still fails to capture the topological requirements for the incorporation of AL into the NE – this\nneeds to be addressed as for non-experts this will be how they conceive of the study’s conclusions. As drawn the\nembedding of the NPC into the two lipid bilayers needs to be accurately represented and the NE must have a gap into which\nthe NPC could move from the AL. The intermediate steps should be shown.\n3. The authors have added new data assessing the distribution of the Ran GTPase and interpret the findings as functionally\nrelevant. However, this line of investigation is too speculative at this stage. The authors discuss a “gradient” of Ran, but they\nare looking only at Ran distribution. The Ran gradient relates to the nucleotide state of Ran-GTP and is a sum effect of Ran,\nthe RanGEF RCC1 and RanGAP. If this is to interpreted it is essential to understand if the Ran GTPase gradient (of which\nthere are biosensors) is actually cell cycle regulated. Also, it would be important to assess if Ntf2 (the dedicated transport\nfactor for Ran) levels are different – but Ntf2 is not even mentioned in the manuscript. Last, Ntf2 is particularly relevant to the\nstudy because it has previously been tied to nuclear size scaling, which the authors report to be affected (PMIDs: 20946986,\n26823604).", "reviewer_3": "In this revised submission, the authors have improved the manuscript, where particularly the splitSMLM data in Fig. 3 are a\nconvincing complement to demonstrate fusion of NPCs from cytoplasm to NE. Overall, with reference to my previous report\non this manuscript, I'd have a few remaining comments/questions, the responses to which, as far as I am concerned may\nnext be assessed by the editor.\n2nd paragraph of Results section: CLEM analysis \"showed these foci to be localized ..., demonstrating that they are AL (Fig.\n1a)\". The amount of images shown is too small to be conclusive about this. More data would need to be shown and ideally\nquantified to support this conclusion. Alternatively, this statement should be toned down to a less conclusive, more\nsuggestive variant.\nRelated to this, the caption of Fig. 1a denotes the shown CLEM images as being \"representative\". At the risk of further\nincreasing the length of this already voluminous submission: Could the authors complement these images with additional\nbiological repeats in an SI Figure, to demonstrate that these observations can at least be made in triplicates? That way, even\nwhen full quantification may be too ambitious, the authors would not need to state anything about representativeness of the\nshown image, but can just demonstrate it with data.\nA similar observation can be made about the CLEM data in Fig. 6a.\nIn the section \"AL are highly dynamic and can fuse with the NE...\", its title, main text and figure labelling: Fig. 2e and SI\nvideos 2,3 are said to show that there are fusion and fission events of AL foci with the NE. The shown images, even for Z\nslices in confocal microscopy, at this scale still represent 2D projections of a 3D reality, which may complicate interpretation.\nIs it obvious, depending on the depth resolution in these experiments, that the foci are not drifting behind/in front of the view\nof the nucleus?\nWhat data is the quantification in Fig. 2d based on? Having looked at Fig. 2e and SI videos 2,3, I struggle to identify the\nnumbers of attachment/detachment events noted in Fig. 2d, and from the underpinning data shown, it seems that their\nidentification is not that unambiguous. Could the authors explain more explicitly how this (per se very welcome)\nquantification results from the microscopy data?\nData in Fig. 2f are shown to further substantiate the claim of AL-insertion into NE, albeit with the authors' caveat about long-\nterm stability and brightness of the fluorophores in these data. Is there a negative control for these experiments?\nMinor:\nFig. 1b, top-left panel: A \"2\" label appears to be missing in this image."}, "author_response": "Point-by-point response to Reviewers' Comments\nReviewer #1\nThis is a revised manuscript, transferred to Nature Communications after review and\nconsideration at [REDACTED]. The original manuscript was extensive, robust and used\nstate of the art microscopical approaches and orthogonal methods of intervention and\nobservation to reveal the role for RanBP2 in clustering and incorporating cytoplasmic\naggregates of NPCs in Annulate Lamellae (AL) into the primary nucleus. In revision, the\nauthors have addressed all the Nat Cell Biol comments and incorporated substantial amounts\nof new data into the manuscript - whilst it was a mammoth story before, it is doubly-mammoth\nhere! This is a massive amount of work and the authors ought be lauded for their efforts here.\nWhilst the narrative and structure has improved substantially since the original submission,\nit is quite a challenging manuscript to carry in your head whilst reading. I think the manuscript\nis suitable for publication.\nWe thank Reviewer 1 for their very positive assessment of the revised manuscript and their\nrecognition of the extensive work involved. In response to the comments, we have made\nfurther efforts to improve clarity by simplifying the narrative and structure and by distributing\nthe data across separate figures (now nine main figures). We appreciate the reviewer’s\nsupport for the publication of this study.\nRegards my specific comments, the authors have demonstrated physiological contribution of\nthe AL-insertion pathway to populating the interphase nucleus with NPCs and allowing the\nnucleus to expand. The new timelapse imaging of AL-insertion into the NE is a strong addition,\nas is the separation of Nup153/ELYS effects from a RanBP2-dependent insertion. They have\nclarified issues about the RanBP2-CLIMP63 interaction, and whilst not showing it is direct,\nthey have included controls to highlight the specificity of the co-precipitation and made it\nclearer how the IP was performed.\nMechanistically, the mapping of the AL-clustering activity to the FG domain of RanBP2 is a\nhelpful addition.\nWe thank Reviewer 1 for these comments and for acknowledging the additional mechanistic\ninsights provided in the revised manuscript.\nThe G1-restricted RanBP2 contribution to nuclear expansion is interesting, but I wonder if\nthe authors could explain how the cells remain in G1 for the imaging period (48h); from the\nmethods, the thymidine arrest is only for 16h, then dmso/noc for 90 minutes and then imaging.\nthe cycle and divide over the next 2 days? The details of this assay could be added to the\nmethods as I don't think this is included.\nWe thank the reviewer for pointing this out and apologize for the lack of clarity. In these\nexperiments, cells were maintained at the G1/S boundary by sustained thymidine treatment,\nfollowed by microtubule depolymerization or RanBP2 depletion, which both prevent nuclear\ngrowth (Fig. 3g-i and Supplementary Fig. 9). We have updated the experimental schemes,\nthe Results text, and the corresponding figure legends to clarify this point.\nA second element that could be clarified in the text a little is the role of RanBP2 in\noligomerising Y-complexes within individual NPCs and binding Nup93 (Fig 6E-G) from the\nRanBP2-dependent clustering of individual NPCs into larger AL\naggregates. I think these are two separate things, and I'm not sure that the Y-complex and Nup93 binding is needed for\nthe narrative.\nWe agree with Reviewer 1. We have revised the text to clearly distinguish these two\nprocesses and to emphasize that RanBP2 plays distinct roles during AL assembly. Under\nphysiological conditions, RanBP2 contributes to scaffold assembly within individual AL-NPCs,\nincluding organization of Y-complex components and interactions with Nup93, whereas\nunder stress conditions it promotes the expansion and clustering of AL-NPCs in the\ncytoplasm.\nLastly, the cartoon at the end really helped with understanding; however, it is a little stylised.\nRather than a grey block for the ER, I wonder if individual INM, ONM and ER membranes\ncould be depicted. It is a little hard to understand what the authors think is happening to\naccommodate this insertion. There will certainly be some gymnastics required to place these\nAL-NPCs across the INM/ONM for contribution to transport activity, but it isn't clear from the\ncartoon how this would happen. To help others follow up on this work, I would encourage the\nauthors to outline how this might happen so that subsequent experimental followups can be\nplanned.\nWe thank the reviewer for these helpful suggestions and agree that the previous schematic\nmodel did not sufficiently reflect the complexity of the underlying data. We have therefore\nprepared a revised model that explicitly depicts the inner nuclear membrane (INM),\nouter\nnuclear membrane (ONM), and endoplasmic reticulum (ER), and illustrates how AL-NPC\ninsertion across these membranes may occur. In addition, the revised schematic model\nhighlights open questions to guide future experimental investigations. This updated model is\nnow presented in Fig. 9.\nL214, underlying nuclear membrane should probably be underlying nuclear lamina.\nWe thank the reviewer for pointing out this error, which has now been corrected.\nReviewer #2\nOverall assessment of revision: The revised manuscript addresses my major concerns with\nthe initial submission. The most critical aspect related to the membrane topology of the\nnuclear envelope at sites that the authors interpret as insertion events for AL into the NE.\nThe new data acquired using splitSMLM that suggests openings in the nuclear envelope at\nthe sites of AL integration is the most critical. While this addresses my major concern it should\nbe more robustly presented (see below). In addition, the cartoon model should incorporate\nthese new insights. One other issue related to the newly added data relates to interpretation\nof the described cell cycle differences in Ran distribution.\nWe thank Reviewer 2 for their assessment of the revised manuscript and for highlighting the\nimportance of the new splitSMLM data. In response, we have strengthened the presentation\nof these data and revised the schematic model to incorporate the new insights. The updated\nmodel is now shown in Fig. 9. We also address the comments regarding the interpretation of\ncell cycle-dependent Ran distribution below.\nSpecific Points:\n1. Related to the gaps in the nuclear envelope: In Fig. 3c it would be helpful to demarcate\nthe edges of the PDI signal that the authors interpret as gaps. Because of the importance of\nthis finding, either a quantitative representation of how commonly this was observed and/or\nadditional examples should be included – even if just from the timepoint where the gap and\napposed AL are viewable.\nWe thank the reviewer for this suggestion. We have included additional examples in Fig. 3c\nshowing gaps in the PDI signal with apposed annulate lamellae to further support this\nobservation.\n2. The cartoon model in 7k still fails to capture the topological requirements for the\nincorporation of AL into the NE – this needs to be addressed as for non-experts this will be\nhow they conceive of the study’s conclusions. As drawn the embedding of the NPC into the\n3\ntwo lipid bilayers needs to be accurately represented and the NE must have a gap into which\nthe NPC could move from the AL. The intermediate steps should be shown.\nWe thank the reviewer for this comment. As outlined above, we have prepared a revised\nschematic model that explicitly incorporates the required nuclear envelope topology,\nincluding membrane gaps, accurate representation of the inner and outer nuclear\nmembranes, and intermediate steps of AL-NPC incorporation into the nuclear envelope. This\nupdated model is now presented in Fig. 9.\n3. The authors have added new data assessing the distribution of the Ran GTPase and\ninterpret the findings as functionally relevant. However, this line of investigation is too\nspeculative at this stage. The authors discuss a “gradient” of Ran, but they are looking only\nat Ran distribution. The Ran gradient relates to the nucleotide state of Ran-GTP and is a sum\neffect of Ran, the RanGEF RCC1 and RanGAP. If this is to interpreted it is essential to\nunderstand if the Ran GTPase gradient (of which there are biosensors) is actually cell cycle\nregulated. Also, it would be important to assess if Ntf2 (the dedicated transport factor for Ran)\nlevels are different – but Ntf2 is not even mentioned in the manuscript. Last, Ntf2 is\nparticularly relevant to the study because it has previously been tied to nuclear size scaling,\nwhich the authors report to be affected (PMIDs: 20946986, 26823604).\nWe agree with Reviewer 2 and have toned down the interpretation of these data accordingly.\nWe have revised the text to explicitly state that our analysis addresses Ran localization rather\nthan the Ran-GTP gradient. We now clarify that the canonical Ran-GTP gradient depends\non the coordinated activities of RCC1, RanGAP, and the nucleotide state of Ran, and not\nsolely on Ran distribution. In addition, we now discuss the role of Ran-associated factors,\nnoting that the Ran-specific transport factor NTF2, which has been implicated in nuclear size\nscaling was not directly examined in this study. We emphasize that whether cell cycle-\ndependent regulation of these components contributes to the observed changes in Ran\ndistribution remains an open question.\nReviewer #3\nIn this revised submission, the authors have improved the manuscript, where particularly the\nsplitSMLM data in Fig. 3 are a convincing complement to demonstrate fusion of NPCs from\ncytoplasm to NE. Overall, with reference to my previous report on this manuscript, I'd have a\nfew remaining comments/questions, the responses to which, as far as I am concerned may\nnext be assessed by the editor.\nrecognizing the value of the splitSMLM data in Fig. 3. We are happy to address the remaining\ncomments and questions below.\n2nd paragraph of Results section: CLEM analysis \"showed these foci to be localized ...,\ndemonstrating that they are AL (Fig. 1a)\". The amount of images shown is too small to be\nconclusive about this. More data would need to be shown and ideally quantified to support\nthis conclusion. Alternatively, this statement should be toned down to a less conclusive, more\nsuggestive variant.\nWe thank the reviewer for this comment. We have toned down the statement to a more\nsuggestive wording to avoid overinterpretation and have added additional representative\nCLEM examples to further support the identification of these structures as annulate lamellae.\nRelated to this, the caption of Fig. 1a denotes the shown CLEM images as being\n\"representative\". At the risk of further increasing the length of this already voluminous\nsubmission: Could the authors complement these images with additional biological repeats\nin an SI Figure, to demonstrate that these observations can at least be made in triplicates?\nThat way, even when full quantification may be too ambitious, the authors would not need to\nstate anything about representativeness of the shown image, but can just demonstrate it with\ndata.\nA similar observation can be made about the CLEM data in Fig. 6a.\nWe thank the reviewer for this suggestion. We have added additional CLEM examples in Fig.\n1a and in the new Fig. 6d (formerly Fig. 6a) and have included the number of biological\nreplicates (n = 3). We have retained the term “representative” in the figure legends while\nexplicitly indicating the number of independent biological repeats.\nIn the section \"AL are highly dynamic and can fuse with the NE...\", its title, main text and\nfigure labelling: Fig. 2e and SI videos 2,3 are said to show that there are fusion and fission\nevents of AL foci with the NE. The shown images, even for Z slices in confocal microscopy,\nat this scale still represent 2D projections of a 3D reality, which may complicate interpretation.\nIs it obvious, depending on the depth resolution in these experiments, that the foci are not\ndrifting behind/in front of the view of the nucleus?\nWe thank the reviewer for raising this important point. Although we occasionally observed\nfoci drifting behind or in front of the nuclear plane at low frequency, we minimized this\n5\npossibility by acquiring smaller Z-stacks (3–5 μm) rather than the standard 10 μm. Importantly,\nnone of the examples or videos presented in the manuscript show foci drifting behind or in\nfront of the nucleus. In addition, during quantification, potential overlapping foci were\nexcluded from the analysis rather than included as insertion or fusion events.\nWhat data is the quantification in Fig. 2d based on? Having looked at Fig. 2e and SI videos\n2,3, I struggle to identify the numbers of attachment/detachment events noted in Fig. 2d,\nand from the underpinning data shown, it seems that their identification is not that unambiguous.\nCould the authors explain more explicitly how this (per se very welcome)\nquantification results from the microscopy data?\nWe thank the reviewer for this comment. The quantification shown in Fig. 2d is based on fast\nlive-imaging microscopy data, with Supplementary Video 3 providing a representative\nexample. Quantification was performed by focusing specifically on regions adjacent to the\nnuclear envelope (NE). Because recording attachment and detachment events across the\nentire nuclear circumference was not feasible due to the limited field of view, each nucleus\nwas divided into multiple NE-adjacent sections. Foci dynamics were then tracked frame by\nframe within each section, and attachment and detachment events were recorded\naccordingly. These events were subsequently pooled for statistical analysis. A detailed\ndescription of the image acquisition and quantification procedure has been added to the\nMethods section.\nData in Fig. 2f are shown to further substantiate the claim of AL-insertion into NE, albeit with\nthe authors' caveat about long-term stability and brightness of the fluorophores in these data.\nIs there a negative control for these experiments?\nWe thank the reviewer for this comment. We have included a -40 s time point prior to\nphotoconversion, which does not show an increase in nuclear signal at -20 s. In contrast, 20\ns after photoconversion, a statistically significant increase in the red fluorescent signal is\nobserved. This pre-photoconversion time point serves as an internal negative control for the\nexperiment.\nMinor:\nFig. 1b, top-left panel: A \"2\" label appears to be missing in this image.\nWe thank the reviewer for pointing this out. The missing label has now been added.\n6"}]

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