Tpr is a conserved nuclear pore complex (NPC) protein implicated in the spindle assembly checkpoint (SAC) by an unknown mechanism. This study shows that Tpr is required for normal SAC response by stabilizing Mad1 and Mad2 before mitosis. Tpr coimmunoprecipitates with Mad1 and Mad2 (hereafter designated as Tpr/Mad1/Mad2 or TM2 complex) during interphase and mitosis, and is required for Mad1-c-Mad2 recruitment to NPCs. Interestingly, Tpr is normally undetectable at kinetochores and dispensable for Mad1, but not for Mad2, kinetochore localization, which suggests that SAC robustness depends on Mad2 levels at kinetochores. Protein half-life measurements demonstrate that Tpr stabilizes Mad1 and Mad2, ensuring normal Mad1-c-Mad2 production in an mRNA- and kinetochore-independent manner. Overexpression of GFP-Mad2 restores normal SAC response and Mad2 kinetochore levels in Tpr-depleted cells. Mechanistically, evidence is provided that Tpr might spatially regulate SAC proteostasis through the SUMO-isopeptidases SENP1 and SENP2 at NPCs. Thus, Tpr is a kinetochore-independent, rate-limiting factor required to mount and sustain a robust SAC response (Schweizer, 2013).

The GGGGCC (G4C2) repeat expansion in a noncoding region of C9orf72 is the most common cause of sporadic and familial forms of amyotrophic lateral sclerosis and frontotemporal dementia. The basis for pathogenesis is unknown. To elucidate the consequences of G4C2 repeat expansion in a tractable genetic system, this study generated transgenic fly lines expressing 8, 28 or 58 G4C2-repeat-containing transcripts that do not have a translation start site (AUG) but contain an open-reading frame for green fluorescent protein to detect repeat-associated non-AUG (RAN) translation. These transgenic animals were shown to display dosage-dependent, repeat-length-dependent degeneration in neuronal tissues and RAN translation of dipeptide repeat (DPR) proteins, as observed in patients with C9orf72-related disease. This model was used in a large-scale, unbiased genetic screen, ultimately leading to the identification of 18 genetic modifiers that encode components of the nuclear pore complex (NPC), as well as the machinery that coordinates the export of nuclear RNA and the import of nuclear proteins. Consistent with these results, morphological abnormalities were found in the architecture of the nuclear envelope in cells expressing expanded G4C2 repeats. Moreover, a substantial defect in RNA export was identified that resulted in retention of RNA in the nuclei of Drosophila cells expressing expanded G4C2 repeats and also in mammalian cells, including aged induced pluripotent stem-cell-derived neurons from patients with C9orf72-related disease. These studies show that a primary consequence of G4C2 repeat expansion is the compromise of nucleocytoplasmic transport through the nuclear pore, revealing a novel mechanism of neurodegeneration (Freibaum, 2015).

In addition to their eminent role in transport, NE-NPCs organize the nuclear periphery by delineating zones of active euchromatin as compared to transcriptionally repressed heterochromatin in between pores. Crucial to this is that NPCs are laterally immobile within the NE, which was shown to depend on the nuclear lamina. Lamins are nuclear intermediate filament proteins and come in two major types: B-type Lamins are ubiquitous, while A-type Lamins are expressed exclusively when cells differentiate. Both proteins engage in distinct meshworks and also impact on NPC insertion rate. This work puts NPC organization and the mode of pore insertion into a developmental context. It is proposed that in Drosophila AL insertion is innate to earliest embryogenesis and diminishes when pores get laterally restricted and cluster at the NE. There are no A-type lamins expressed at that stage, and specifically expressed INM proteins could be crucial. Intriguingly, the formation of immobile pore clusters coincides with the transcriptional upregulation of hundreds of genes at zygotic induction, a developmental transition present in all metazoan that is accompanied by characteristic changes in chromatin signatures. This study revealed that the zygotically upregulated INM protein LBR, a developmentally controlled INM tether of peripheral heterochromatin, is sufficient to prematurely aggregate NPCs in blastoderm interphases, when artificially expressed earlier in embryogenesis. This also leads to larger AL likely because LBR counteracts AL insertion for which lateral NPC mobility is required. The data suggest a zygotically induced regulation that links pore insertion and organization, NE composition and ultimately also chromatin organization at the nuclear periphery. All of these events eventually contribute to the commitment of originally pluripotent somatic nuclei into distinct lineages (Hampoelz, 2016).

The molecular events that direct nuclear pore complex (NPC) assembly toward nuclear envelopes have been conceptualized in two pathways that occur during mitosis or interphase, respectively. In gametes and embryonic cells, NPCs also occur within stacked cytoplasmic membrane sheets, termed annulate lamellae (AL), which serve as NPC storage for early development. The mechanism of NPC biogenesis at cytoplasmic membranes remains unknown. This study shows that during Drosophila oogenesis, Nucleoporins condense into different precursor granules that interact and progress into NPCs. Nup358 is a key player that condenses into NPC assembly platforms while its mRNA localizes to their surface in a translation-dependent manner. In concert, Microtubule-dependent transport, the small GTPase Ran and nuclear transport receptors regulate NPC biogenesis in oocytes. This study has delineated a non-canonical NPC assembly mechanism that relies on Nucleoporin condensates and occurs away from the nucleus under conditions of cell cycle arrest (Hampoelz, 2019).

Nuclear pore complexes (NPCs) bridge the nuclear envelope (NE) and mediate nucleocytoplasmic exchange. They are giant assemblies of about 110 MDa in animals with an elaborate structure and composition. About 30 different genes encode for NPC components, termed nucleoporins (Nups). Those are subclassified into scaffold Nups that assemble into a cylindrical architecture with a ~50 nm wide central channel; and intrinsically disordered phenylalanine-glycine rich FG-Nups that line this channel. Scaffold Nups assemble into the so-called Y and inner ring complexes that form the outer and inner rings, respectively (see The Nuclear Pore Complex as a Flexible and Dynamic Gate). FG-Nups (containing phenylalanine-glycine repeats) have the capacity to phase separate in vitro. In vivo, they establish a unique biophysical milieu within the central channel that is impermeable to inert molecules. FG-Nups transiently interact with nuclear transport receptors (NTRs, also called importins, exportins, or karyopherins) that form complexes with cargo and cross the permeability barrier. Transport directionality across the NE is ensured by the small GTPase Ran. RCC1, the RanGTP exchange factor (RanGEF) is chromatin associated and maintains a high RanGTP concentration in the nucleus. The Ran GTPase activating protein (RanGAP) binds to Nup358 (also called RanBP2) at the cytoplasmic face of the NPC and ensures high RanGDP levels in the cytosol. Although RanGTP displaces cargo from import complexes in the nucleoplasm, GTP hydrolysis disassembles export complexes once they arrive at the cytoplasmic face. Nup358 is absent from lower eukaryotes but essential in animals and involved in active nuclear transport, cell cycle progression, malignant transformation, and viral infection (Hampoelz, 2019).

NPC assembly is an intricate process. In multicellular organisms, two assembly pathways were described. First, the relatively rapid assembly of NPCs from pre-existing building blocks concomitantly with nuclear envelope (NE) reformation at the end of open mitosis is referred to as 'post-mitotic' assembly. This pathway is spatially directed to chromatin by the Nup Elys. Temporal control is provided by cell-cycle-dependent kinases and phosphatases. Second, interphase assembly is a relatively slow process that generates NPCs from scratch in order to double their number for the next mitosis. It proceeds from inside out through the NE and requires the active nuclear import of Nups. Here, Nup153 spatially directs the Y complex to the inner nuclear membrane (Vollmer, 2015; Hampoelz, 2019 and references therein).

Little is known about the early steps of NPC assembly that occur prior to membrane association. FG-Nups serve as a velcro for scaffold Nups. They, however, have a considerable aggregation propensity in isolation that has to be controlled during NPC biogenesis in vivo. Non-NPC-associated Nups are chaperoned by importin β. RanGTP dissociates importin β complexes and thereby releases Nups for interphase and post-mitotic NPC assembly. Likewise, RanGTP induces NPC assembly in vitro, but the in vivo relevance of this finding remains to be tested (Hampoelz, 2019).

In multicellular organisms, nuclear pores also reside in stacked membrane sheets of the endoplasmic reticulum (ER), termed annulate lamellae (AL). Those are particularly prominent in gametes and embryos of a multitude of species including Drosophila. In early fly embryos, AL insert into the NE in order to supply the rapidly growing nuclei with additional membranes and NPCs (Hampoelz, 2016). AL are therefore thought to be maternally provided NPC storage pools. How AL assemble in the absence of a nuclear compartment, which spatially coordinates the process in case of the two previously characterized pathways, remains elusive. This study has investigated AL-NPC biogenesis in vivo during Drosophila melanogaster oogenesis. AL-NPC biogenesis was shown to be vastly abundant during oogenesis. It depends on the condensation of Nups into compositionally different granules that are transported along microtubules (MTs) and regulated by Nup358 in concert with Ran and NTRs. This NPC biogenesis is mechanistically distinct from both canonical NPC assembly pathways and progresses away from chromatin. It is proposed that instead, Nup358 condensates fulfill the role of spatially directing NPC biogenesis, in the absence of a bona fide nuclear compartment (Hampoelz, 2019).

Little has been known about the biogenesis of AL and the spatial cues that allow NPC formation away from the nuclear compartment. This work addresses these questions during Drosophila oogenesis and suggests a third, non-canonical NPC assembly mechanism. Already in nurse cells, Nup358 condenses into large granules (see A Model for NPC Biogenesis beyond the Nuclear Compartment). Condensation might be fostered by local translation of nup358 transcripts that enrich at the surface of Nup358 granules in a translation dependent manner. In nurse cells, AL biogenesis is suppressed, and only limited NPC assembly is observed within Nup358 granules. This could be due to the available amount or configuration of ER membranes or because high cytoplasmic concentrations of RanGDP promote the formation of Importin-Nup complexes that prevent other Nups from condensation. Nup358 granules become assembly platforms for AL-NPCs once they travel through ring canals into the ooplasm. Scaffold and FG-Nups condense into oocyte specific granules, possibly facilitated by elevated levels of RanGTP that dissociates the respective Nups from Importin. In the oocyte, NPC precursor granule interactions are promoted by MT dependent granule dynamics. Upon interaction, granules transfer material and assemble Nups onto available ER membrane, ultimately leading to the formation of larger stacks with multiple membrane sheets. Those are inherited to the embryo where they supplement dividing nuclei with NPCs throughout early embryogenesis (Hampoelz, 2019).

The phase-separating properties of FG-Nups have been subject to extensive research in vitro. This study provides evidence for condensation of Nups in vivo. Several properties, namely the coalescence of Nup358 granules, the transfer of material between granules, the high molecular mobility within granules, and the contact shapes observed upon granule interactions, are hallmarks of biomolecular condensates. Such condensates are defined as 'non-membranous organelles'. Although AL inherently contain stacked membrane sheets, they retain at least some characteristics of a phase separated condensate such as a milieu that is distinct from the surrounding cytoplasm. These findings underline the importance of phase separation at membranes that was also observed in other biological systems (Hampoelz, 2019).

Several lines of evidence, namely 1,6-hexanediol treatment, depletion of BicD and embargoed, and the interference with the Ran nucleotide status suggest that condensation of Nups into NPC precursor granules is critical for AL biogenesis. It is further underlined by the fact that Colchicine treatment counteracts the RanQ69L phenotype by reducing the number of MT-promoted granule interactions. Condensation concentrates NPC constituents in a constrained volume within the large ooplasm and might prevent unspecific interactions of soluble Nups. MT dynamics enhances interactions of otherwise unmixed, compositionally heterogeneous NPC precursor condensates and is a prerequisite for NPC assembly from condensed granules. It also prevents unwanted fusion and relaxation of compositionally homogeneous condensates of the same type. Facilitated interactions of granules could be of particular importance in the highly viscous ooplasm, where cytoskeleton-induced streaming is critical for the efficient distribution of various components (Hampoelz, 2019).

The two canonical NPC assembly pathways rely on the stepwise and orchestrated assembly of soluble Nups or subcomplexes onto either anaphase chromatin or the NE surface during interphase, respectively. However, these spatial cues are absent in the ooplasm and alternative mechanisms to locally concentrate assembly modules must be important. It is believed that the condensation of Nups replaces the canonical cues, in line with previous work that had shown functions of natively unfolded FG-Nups to stabilize each other but also NPC scaffold components during yeast NPC assembly. Controlled interactions and material transfer between condensates might account for specific steps of assembly and even provide a certain order, although this concept remains to be further tested (Hampoelz, 2019).

The data strongly suggest Nup358 granules as assembly platforms, where NPCs are seeded onto ER membranes. Nup358 has no reported role in initiating the assembly process in both previously described pathways (Weberruss, 2016). On the contrary, during interphase Nup358 assembles rather late onto the NPC scaffold. Although such information is not available for post-mitotic assembly, its mitotic localization to kinetochores could indicate an early role for Nup358. Indeed, Nup358 is of structural importance for the pore scaffold, given that its loss destabilizes the outer Y complex at the cytoplasmic ring at NE-NPCs. It is thus conceivable that Nup358 could not only stabilize but also recruit scaffold components onto membranes. Post-mitotic and interphase NPC assembly are initiated by two distinct Nups, Elys and Nup153, respectively. Elys localizes the Y complex onto anaphase chromatin and is dispensable for interphase assembly but also for AL formation, given that its depletion induces AL. In contrast, Nup153 seeds NPCs during interphase assembly onto the inner nuclear membrane, and it has been suggested to have a similar role at the ER during AL biogenesis. This study, however, found that Nup153 is absent from AL in oocytes(Hampoelz, 2019).

Despite all molecular and conceptual differences, the common driving force for NPC biogenesis at and beyond the nucleus is Ran that coordinates the availability of Nups for assembly by dissociating them from NTRs. Nup358 binds RanGAP and directly links the NPC to Ran activity. At the NE this is eminent to ensure a sharp Ran gradient and thus efficient nucleocytoplasmic transport. This study shows that this interaction is preserved beyond nuclei, because RanGAP strongly enriches at Nup358 granules in a Nup358-dependent manner. One might speculate that within the RanGTP milieu of the ooplasm, RanGAP induces a local Ran gradient at Nup358 granules that drives NPC biogenesis; conceptually similar to the nuclear compartment for interphase or postmitotic NPC assembly (Figure 7C and 7C'). Thereby, the observed progressive dilution of Nup358 and RanGAP at Nup358 granules in the oocyte could be important to drive their progression into AL. It might be caused by ooplasmic RanGTP that favors complex formation between Crm1 and Nup358. Although this would be consistent with the observed embargoed gene silencing phenotype, the enhanced condensation of Nup358 upon global induction of RanGTP argues for an alternative interpretation: Nup358 functionally interacts with both, Importins and Crm1 under specific conditions. It is thus not clear how exactly it is being chaperoned. The phenotype observed under embargoed gene silencing conditions might be indirectly caused by disturbance of the spatial distribution of Ran and NTRs across nurse cells and oocytes, as indicated by the variety in phenotype across individuals. Yet it stresses the importance of spatially controlled Nup condensation to assemble AL-NPCs. In any scenario, NTR-mediated de-condensation of Nup358 and the consequent reduction of local RanGAP activity would regulate the progression of NPC biogenesis by determining the availability of soluble, 'assembly prone' Nups and the degree of mixing at granule interfaces (Hampoelz, 2019).

Various aspects of AL-NPC biogenesis are markedly different from both canonical NPC assembly pathways. During oogenesis, Nup condensation, local translation and MT dependent dynamics interplay with Ran activity in order to faithfully assemble AL in oocytes. They are inherited to the embryo where this pool of ready-made NPCs supplements nuclei during the rapid interphases of the blastoderm stage. Because AL are present in a plethora of species, similar mechanisms are likely to operate throughout animals (Hampoelz, 2019).

Furthermore, genome-wide analysis of Nup binding revealed that nuclear pores target both active and silenced loci in similar proportions. PcG-bound regions, marked by the H3K27Me3 mark, appear to be common targets of the stable NPCs, which is supported by a previous report of Nup153 playing a role in PcG-mediated gene silencing. On the basis of the rest of the current findings, it is proposed that a specific subset of genes associates with NPCs independently of expression, for the purposes of stabilizing possible or future activation-induced long-range contacts. Although the transcriptional memory function of Nup98 is not limited to NPC targets and has been shown to apply to intranuclear genes, it is possible that NPC binding may be particularly beneficial for genes with complex regulatory landscapes, such as ecdysone-inducible or Hox genes, where pre-scaffolding and epigenetic maintenance of enhancer-promoter architecture is most critical (Pascual-Garcia, 2017).

The nuclear pore complex (NPC) tethers chromatin to create an environment for gene regulation, but little is known about how this activity is regulated to avoid excessive tethering of the genome. Tethering specific genomic loci to the NPC appears to contribute to transcriptional activation. Also, the NPC has been further implicated in creating a repressive environment or retaining genes at the periphery after repression, possibly contributing to epigenetic transcriptional memory. This paper proposes a negative regulatory loop within the NPC controlling the chromatin attachment state, in which Nup155 and Nup93 recruit Nup62 to suppress chromatin tethering by Nup155. Depletion of Nup62 severely disrupts chromatin distribution in the nuclei of female germlines and somatic cells, which can be reversed by codepleting Nup155. See a model for the chromatin attachment state controlled by an internal regulatory circuit in the NPC. Thus, this universal regulatory system within the NPC is crucial to control large-scale chromatin organization in the nucleus (Breuer, 2015).

Cytological study of the chromatin attachment state to the nuclear envelope is experimentally challenging, as chromatin usually occupies the entire nucleus. However, meiotic chromatin becomes fully detached from the nuclear envelope and compacted into a spherical structure, the karyosome, after recombination in Drosophila oocytes. Chromatin detachment and karyosome formation are crucial to make a single spindle and allow subsequent chromosome segregation and are conserved features also seen in mammalian oocytes. By taking advantage of this unique nuclear organization in oocytes, this study sought factors required for chromatin detachment from the nuclear envelope by individually knocking down various nuclear proteins in the female germline (the oocyte and nurse cells) by RNAi (Breuer, 2015).

Strikingly, the depletion of either of two nuclear pore proteins, Nup62 or Nup93, led to disruption of the compact karyosome morphology, while the depletion of several other pore proteins did not. The chromatin shifted near the nuclear periphery, resulting in strong (Nup62) or partial (Nup93) overlap with a nuclear pore marker in the oocytes in comparison with the control RNAi, which was confirmed by superresolution microscopy. Nup93 is a linker scaffold protein known to be required for the recruitment of Nup62, one of the central channel proteins containing FG repeats (Sachdev, 2012). It was confirmed that the defect is not an off-target effect by rescue experiments using RNAi-resistant transgenes. In addition, similar karyosome defects were observed in female sterile Nup62 mutants. In nurse cells (polytenized germline cells that support oocyte growth), chromatin also distributed irregularly and more toward the nuclear periphery after RNAi of these genes. This demonstrates a general role for both genes in global chromatin organization rather than being restricted to oocytes (Breuer, 2015).

To identify the cause of the karyosome defect upon Nup62 or Nup93 RNAi, the structural integrity and transport function of NPC was tested. RNAi of Nup62 or Nup93 did not disrupt the overall structural integrity of the NPC, as judged by the localization of FG-containing subunits and the core scaffold subunit Nup107. The active import function of the NPC showed small differences as assessed by fluorescence recovery after photobleaching (FRAP) of GFP fused with a nuclear localization signal (NLS). There was a significant increase in the nuclear size of early oocytes, which may be caused by a reduced ability of the nuclear pore to act as a diffusion barrier (Breuer, 2015).

Next, a relationship was examined with the meiotic recombination checkpoint, which is known to disrupt karyosome formation in the presence of unrepaired double-strand breaks (DSBs) in oocytes. Inactivation of the checkpoint did not suppress the karyosome defects of Nup62 or Nup93 RNAi, demonstrating that the defect is independent of the meiotic recombination checkpoint in oocytes (Breuer, 2015).

Considering the above results, it was hypothesized that chromatin is excessively anchored to the NPC in RNAi of Nup62 or Nup93. If this was the case, it was predicted that chromatin specifically interacting with the NPC must be preferentially accumulated at the nuclear periphery rather than random chromatin. In order to test this, previously identified genomic loci bound to another nuclear pore component, Nup98, were used in Drosophila S2 culture cells. Nup98 has two distinct populations -- one at nuclear pores and the other in the nucleoplasm -- that bind distinct genomic loci in S2 cells. Nurse cells were subjected to fluorescence in situ hybridization (FISH) using individual probes corresponding to genomic loci known to be associated with Nup98 within the NPC or located in the nucleoplasm in S2 cells and were costained with a DNA dye. A proportion was measured of the total DNA signals in the nuclear periphery region, defined by a distance from the nuclear lamina of <10% of the nuclear radius, which occupies ~20% of the nuclear area. In control RNAi, ~16%-17% of the total DNA (propidium iodide or DAPI signal) was located in the nuclear periphery region. For all genomic loci (three NPC-bound and four nucleoplasmic), 17%-25% of the signal foci were found in the nuclear periphery. This indicates that there is no preference for periphery locations of the total DNA or of these specific genomic regions in wild-type nurse cells. When Nup93 was knocked down, there was a small increase (from 16%-17% to 20%-24%) in the total DNA that occupies the nuclear periphery (Nup93 RNAi was used, since it gives a milder phenotype than Nup62 RNAi). Strikingly, a strong, consistent redistribution of all NPC-bound genomic loci to the periphery (from 17%-25% to >40%) was observed, whereas the nucleoplasmic loci showed smaller variable changes. The increases for NPC-bound loci were significantly higher than the increases for both total DNA and the nucleoplasmic loci, supporting the hypothesis that depletion of Nup62 or Nup93 results in an excessive attachment of specific chromatin regions to the NPC (Breuer, 2015).

The results suggest that Nup62 and Nup93 suppress the interaction between chromatin and another NPC subunit. If this is the case, codepletion of this hypothetical NPC subunit that mediates chromatin attachment to the nuclear pore should restore detachment of chromatin in Nup62- or Nup93-depleted oocytes. Several NPC subunits have previously been shown to have chromatin-binding activity, including Nup155, Nup50, and ELYS/Mel-28. Flies expressing two shRNAs were generated: one for Nup62 and the other for each of the aforementioned chromatin-binding NPC subunits, the non-chromatin-bound Nup160, or a control. Codepletion of Nup155 specifically restored normal karyosome morphology and detachment from the nuclear periphery in Nup62-depleted oocytes. Furthermore, in nurse cells, simultaneous RNAi of Nup155 also restored normal chromatin distribution caused by Nup62 RNAi. Crucially, codepletion of Nup155 did not rescue the larger nuclear size in Nup62-depleted oocytes. This demonstrates that Nup62's function on chromatin organization is independent of its function on nuclear size maintenance, which may reflect its function as a diffusion barrier (Breuer, 2015).

It was also found that single depletion of Nup155 led to a large reduction of Nup62 (one of the FG-containing subunits) from the nuclear envelope and its accumulation in the cytoplasm. However, it did not significantly reduce the total amount of the FG-containing subunits at the nuclear envelope in both Nup155 and Nup62/Nup155 double RNAi. This demonstrates that Nup155 is required for Nup62 recruitment, and the apparent rescue of the Nup62 depletion defect by Nup155 codepletion is not due to a loss of integrity or a reduced number of nuclear pores. Taken together, the results suggest a negative regulatory loop in which Nup155 recruits Nup62 to the nuclear pores, and, in turn, Nup62 suppresses chromatin anchoring by Nup155 (Breuer, 2015).

A potential negative regulatory circuit was uncovered within the NPC that controls the chromatin attachment state to the nuclear pores in the oocytes and nurse cells. Therefore, attempts were made to test whether a common regulatory system also controls chromatin organization in somatic cells. Using the Drosophila S2 cell line, Nup62 or Nup155 were depleted individually and simultaneously by RNAi. Control RNAi cells showed a relatively even distribution of chromatin within the nucleus except for a dense region that corresponds to heterochromatin. In contrast, Nup62 RNAi resulted in an uneven distribution of chromatin within the nucleus. To quantify this, the area that chromatin occupies relative to the nuclear area was measured. The cells depleted of Nup62 showed a significant decrease in chromatin occupancy compared with a control RNAi. Strikingly, double depletion of Nup62 and Nup155 showed a chromatin occupancy similar to the control. This rescue was reversed by RNAi-resistant full-length Nup155 but not by resistant Nup155 lacking the chromatin-binding region. No significant change in chromatin occupancy was observed upon Nup155 depletion alone. This demonstrated the presence of a common negative loop within the NPC that controls the global chromatin distribution between female germline cells and somatic cells (Breuer, 2015).

Recent reports described the role of the NPC to tether chromatin and thus create an environment for gene regulation. While recruitment mechanisms for specific genes have been described, very little is known about whether or how this tethering is regulated. This study makes two major conceptual advances in understanding of global chromatin organization, especially the critical role and regulation of the NPC-mediated tethering. First, it highlights a far greater role of the NPC in large-scale chromatin organization than previously anticipated. Second, it points to a universal regulatory circuit inside the NPC that controls the attachment state of chromatin to the nuclear pore. This consists of a negative regulatory loop in which chromatin-binding Nup155 recruits the central channel protein Nup62, which in turn suppresses chromatin binding. As nuclear pore components associate with the genome to positively or negatively influence gene expression, this regulatory loop might be part of a wider network for the NPC to control gene expression, depending on the cellular and developmental context. Although a genuine and direct regulatory role of this loop has yet to be demonstrated, its intrinsic capacity supplies the NPC with a key mechanism to globally or locally organize the metazoan genome. On the other hand, any change or imbalance in this regulatory network might have dramatic effects for the nuclear architecture and, concomitantly, the expression profile of the cell. This may have a significant medical implication, as nuclear pore components not only are known to deteriorate with age but are also affected in several tissue-specific human diseases (Breuer, 2015).

The niche critically controls stem cell behavior, but its regulatory input at the whole-genome level is poorly understood. This study elucidated transcriptional programs of the somatic and germline lineages in the Drosophila testis and genome-wide binding profiles of Zfh-1 and Abd-A expressed in somatic support cells and crucial for fate acquisition of both cell lineages. Key roles were identified of nucleoporins and V-ATPase proton pumps, and their importance was demonstrated in controlling germline development from the support side. To make the dataset publicly available, an interactive analysis tool was generated, that uncovered conserved core genes of adult stem cells across species boundaries. The functional relevance of these genes was tested in the Drosophila testis and intestine, and a high frequency of stem cell defects was found. In summary, this dataset and interactive platform represent versatile tools for identifying gene networks active in diverse stem cell types (Tamirisa, 2018).

Using cell-type-specific transcriptome profiling and in vivo TF binding site mapping together with an interactive data analysis tool, this study comprehensively identified genes involved in controlling proliferation and differentiation within a stem cell support system. Importantly, many candidates that were functionally tested not only were required within the soma, but also had non cell-autonomous functions in the adjacent (germline) stem cell lineage (Tamirisa, 2018).

An interconnected network of TFs was identified that plays an important role in the maintenance and differentiation of both germline and somatic cell populations, signal processing V-ATPase proton pumps, and nuclear-transport-engaged Nups as regulators in the Drosophila male stem cell system. V-ATPases have been implicated in the regulation of various cellular processes in not only invertebrates but also vertebrates. For example, the V-ATPase subunit V1e1 was previously shown to be essential for the maintenance of NBs in the developing mouse cortex, as loss of this subunit caused a reduction of endogenous Notch signaling and a depletion of NBs by promoting their differentiation into neurons. Furthermore, two independent studies revealed that V-ATPase subunits and their isoforms are required for proper spermatogenesis in mice, in particular for acrosome acidification and sperm maturation. Thus, it is tempting to speculate that these proton pumps also have important functions in the stem cell pool of the mammalian testis and very likely many other stem cell systems, and some evidence is provided for their crucial role also in ISCs (Tamirisa, 2018).

This work also uncovered nuclear transport associated proteins, the Nups, as important control hubs in the somatic lineage of the Drosophila testis. This is of particular interest, since cell-type-specific functions of Nups have been identified only recently and may represent a critical feature of different stem cell systems. Examples include Nup153, one of the Nups expressed in all four stem cell systems, which interacts with Sox2 neural progenitors and controls their maintenance as well as neuronal differentiation; Nup358, which plays a role at kinetochores, and Nup98, which regulates the anaphase promoting complex (APC) and mitotic microtubule dynamics to promote spindle assembly. Interestingly, it has been shown just recently that Nups play a critical role in regulating cell fate during early Drosophila embryogenesis, thereby contributing to the commitment of pluripotent somatic nuclei into distinct lineages. Current results suggest that they may play a similar role in controlling the transition of continuously active adult stem cells toward differentiation. The next challenge will be to unravel how variations in the composition of an essential and basic protein complex like the NPC causes differential responses of cells, in particular in stem cells and their progenies (Tamirisa, 2018).

The datasets in conjunction with the versatile and easy-to-use analysis tool allowed identification of a substantial number of stem cell regulators for detailed mechanistic characterization. Importantly, this analyses have shed first light on processes and genes shared between diverse invertebrate and vertebrate stem cell systems and uncovered functionally relevant differences. Owing to its flexibility and the option to include datasets from any species, the online tool represents a valuable resource for the entire stem cell community. It not only provides an open platform for data analysis but also leverages the power of comparative analysis to enable researchers mining genomic datasets from diverse origins in a meaningful and intuitive fashion (Tamirisa, 2018).

PIWI-interacting RNAs (piRNAs) guide transposon silencing in animals. The 22-30 nt piRNAs are processed in the cytoplasm from long non-coding RNAs that often lack RNA processing hallmarks of export-competent transcripts. By studying how these transcripts achieve nuclear export, this study uncovered an RNA export pathway specific for piRNA precursors in the Drosophila germline. This pathway requires Nxf3-Nxt1, a variant of the hetero-dimeric mRNA export receptor Nxf1-Nxt1. Nxf3 interacts with UAP56, a nuclear RNA helicase essential for mRNA export, and CG13741/Bootlegger, which recruits Nxf3-Nxt1 and UAP56 to heterochromatic piRNA source loci. Upon RNA cargo binding, Nxf3 achieves nuclear export via the exportin Crm1 and accumulates together with Bootlegger in peri-nuclear nuage, suggesting that after export, Nxf3-Bootlegger delivers precursor transcripts to the piRNA processing sites. These findings indicate that the piRNA pathway bypasses nuclear RNA surveillance systems to export unprocessed transcripts to the cytoplasm, a strategy also exploited by retroviruses (ElMaghraby, 2019).

Integrity of the germline genome is essential for the survival of multicellular species. In animal gonads, a class of small regulatory RNAs, called PIWI-interacting RNAs (piRNAs), guide Argonaute effector proteins to silence transposable elements, thereby counteracting their mutagenic impact. piRNAs are processed from single-stranded precursors, which are transcribed by RNA polymerase II from transposon-rich loci termed piRNA clusters. As piRNA biogenesis occurs in peri-nuclear processing centers, the long non-coding precursor transcripts must be exported from the nucleus to the cytoplasm (ElMaghraby, 2019).

mRNAs, the most prominent RNA polymerase II transcripts, are exported through nuclear pore complexes primarily by the nuclear RNA export factor 1 (Nxf1/Tap) and its binding partner Nxt1/p15. The Nxf1-Nxt1 heterodimer is recruited to export-competent mRNAs after successful completion of RNA-processing events such as 5' capping, splicing, and 3' end formation (Heath, 2016). Nuclear mRNA surveillance systems ensure that unprocessed transcripts are not exported but instead are degraded within the nucleus. mRNA processing is therefore a critical step in licensing export of RNA polymerase II transcripts to the cytoplasm (ElMaghraby, 2019).

In Drosophila, only few piRNA precursors are spliced and poly-adenylated, and these require the mRNA export receptor Nxf1-Nxt1 for nuclear exit. Most piRNA precursors instead lack RNA processing marks characteristic for mRNAs. These precursors originate from heterochromatic loci, whose expression depends on Rhino, a variant of the conserved heterochromatin protein 1 (HP1). Rhino, via its adaptor protein Deadlock, recruits effector proteins that stimulate transcription initiation within heterochromatin (through the TFIIA-L homolog Moonshiner) (Andersen, 2017), and that suppress co-transcriptional RNA processing events such as splicing or 3' cleavage and polyadenylation (through the Rai1 homolog Cutoff). Rather than imposing transcriptional silencing similar to HP1, Rhino thereby facilitates non-canonical transcription of heterochromatic piRNA source loci to allow the production of non-coding precursors for transposon-targeting piRNAs. How the resulting unprocessed piRNA precursors escape nuclear RNA quality control and how they are transported to cytoplasmic piRNA processing centers are central open questions (ElMaghraby, 2019).

This study has identified a nuclear export pathway dedicated to piRNA precursors. It involves Nuclear export factor 3 (Nxf3), which is required for piRNA production, transposon silencing, and fertility and which evolved from the principal mRNA-export receptor Nxf1. Instead of relying on RNA processing events for recruitment, Nxf3 is targeted to nascent piRNA precursors via Rhino, Deadlock, and CG13741/Bootlegger, a previously unknown effector protein of Rhino-dependent heterochromatin expression. After cargo binding, Nxf3 mediates piRNA precursor export in a Crm1/Xpo1-dependent manner, allowing Nxf3 and Bootlegger to deliver piRNA precursors to peri-nuclear nuage where piRNA biogenesis factors accumulate. This dedicated RNA export pathway (formed by Nxf1 duplication and neo-functionalization) therefore bypasses nuclear RNA surveillance systems and transports piRNA precursors from their heterochromatic origins to their cytoplasmic processing sites (ElMaghraby, 2019).

This work uncovered the Nxf3-Bootlegger pathway, which transports RNA Polymerase II-generated transcripts emanating from heterochromatin to peri-nuclear nuage where they are processed into piRNAs. This pathway (1) bypasses nuclear RNA surveillance mechanisms that would otherwise degrade the unprocessed precursor RNAs and (2) enables nuclear export and delivery of the piRNA precursors to nuage. Retroviruses also must bypass nuclear RNA quality-control mechanisms to export their unprocessed genomic RNA. Specific hairpins in the retroviral RNA recruit the mRNA export receptor Nxf1/Tap (e.g., Mason-Pfizer monkey virus) or exploit the exportin Crm1 through a virally encoded adaptor protein (e.g., HIV). Due to their high sequence diversity, piRNA precursors are unlikely to recruit export machinery via sequence or structural features. Instead, this work suggests that specificity in piRNA precursor export is achieved through Rhino, which recruits the specialized NXF variant, Nxf3, to nascent transcripts at heterochromatic piRNA clusters. The Nxf3-Bootlegger pathway highlights Rhino's central role in facilitating productive expression of transposon sequence information within heterochromatin. Via its adaptor protein Deadlock, Rhino connects H3K9me3 marks at piRNA clusters to a set of effector proteins that allow transcription initiation (through the TFIIA-L paralog Moonshiner), prevents transcription termination (through the Rai1/DXO paralog Cutoff), and directs nuclear export of the emerging unprocessed RNAs (through the Nxf1 paralog Nxf3). In this, Rhino provides an epigenetic specificity anchor at heterochromatic piRNA clusters where the canonical layers of specificity are either inaccessible (transcription factor binding sites for transcription initiation) or absent (RNA maturation signatures for nuclear export). Thus, central dogmas of gene expression control are bypassed at piRNA clusters through the action of physically connected paralogs of core gene expression factors (ElMaghraby, 2019).

Notably, another NXF paralog, Drosophila Nxf2, is also an essential piRNA pathway factor as it licenses heterochromatin formation downstream of nuclear Piwi. This points to the NXF protein family as a hotspot for genetic innovation through gene duplication and neo-functionalization. In vertebrates, Nxf1/Tap has also diversified into multiple NXF variants. Mammalian NXF2 and NXF3 (both not directly related to Drosophila Nxf2 and Nxf3) are preferentially expressed in gonads, and Nxf2 mutant mice are male sterile. It is proposed that neo-functionalized NXF variants have evolved throughout the animal kingdom at least in part to serve key functions in genome defense (ElMaghraby, 2019).

In order to modulate gene expression control, neo-functionalized protein paralogs must connect to the central gene expression machinery. Indeed, Nxf3 and Bootlegger co-purify with UAP56, a nuclear DEAD-box ATPase that facilitates loading of Nxf1-Nxt1 onto export-competent mRNA. In the context of mRNA export, UAP56 functions together with the THO complex. Recent work indicates a role for UAP56 and THO in piRNA cluster transcription and in defining Rhino's chromatin occupancy (Zhang, 2018). Based on the current findings, it is speculated that UAP56 and THO, analogous to their function in mRNA export, also license loading of Nxf3-Nxt1 onto piRNA precursors. In this process, Bootlegger might function analogous to SR proteins, canonical export adaptors that recruit Nxf1-Nxt1 to export-competent mRNA, or analogous to the adaptor Aly/Ref that interacts with UAP56 and Nxf1-Nxt1. It is proposed that the mechanistic investigation of specialized paralog-based gene expression pathways, such as the Nxf3-Bootlegger pathway, will provide important insight also into the molecular principles of canonical gene expression (ElMaghraby, 2019).

While the Nxf3-Bootlegger pathway evolved from the general mRNA export machinery, it also shares similarities to pre-microRNA export, which is dependent on Exportin-5. In both cases, the export machinery not only facilitates passage through the NPC, but binding of the export factors to cargo RNA also protects against nuclear RNA degradation. Similarly, and in contrast to mRNA export, both pathways utilize the Ran-GTP gradient, either directly (Exportin-5) or indirectly via Crm1 (Nxf3). As the Ran-GTP gradient confers directionality to nuclear transport pathways (here via dissociation of Crm1 from Nxf3 in the cytoplasm), it is suggested that Crm1-mediated export allows Nxf3 to remain bound to its own RNA cargo after nuclear export. Staying RNA bound enables Nxf3 and Bootlegger to escort piRNA precursors to nuage, a peri-nuclear biomolecular condensate (Nott, 2015) where the piRNA biogenesis machinery is concentrated. The identification of the Nxf3-Bootlegger pathway thus provides key insight into the long sought-after mechanism that contributes specificity to piRNA biogenesis. Remarkably, human NXF3 has independently evolved an export dependency on Crm1, suggesting that it also holds on to its RNA cargo after NPC translocation. NXF variants might therefore more broadly enable delivery of RNA cargo to specific subcellular compartments. The Nxf3-Bootlegger pathway provides a glimpse into how biological information is sorted and spatially distributed in cells. It is noted that similar findings on the Nxf3-Bootlegger pathway have been published by Kneuss and colleagues (Kneuss et al., 2019) (ElMaghraby, 2019).

Interactions between the genome and the nuclear pore complex (NPC) have been implicated in multiple gene regulatory processes, but the underlying logic of these interactions remains poorly defined. This study reports high-resolution chromatin binding maps of two core components of the NPC, Nup107 and Nup93, in Drosophila cells. This investigation uncovered differential binding of these NPC subunits, where Nup107 preferentially targets active genes while Nup93 associates primarily with Polycomb-silenced regions. Comparison to Lamin-associated domains (LADs) revealed that NPC binding sites can be found within LADs, demonstrating a linear binding of the genome along the nuclear envelope. Importantly, this study identified a functional role of Nup93 in silencing of Polycomb target genes and in spatial folding of Polycomb domains. These findings lend to a model where different nuclear pores bind different types of chromatin via interactions with specific NPC sub-complexes, and a subset of Polycomb domains is stabilized by interactions with Nup93 (Gozalo, 2019).

Spatial architecture in the nucleus is set up by interactions between the genome and protein components of nuclear macro-complexes and scaffolds. The most prominent nuclear scaffold is the nuclear envelope (NE), which consists of a double membrane interspersed by a variety of trans-membrane and closely associated proteins. Chromatin re-organization and gene re-positioning during cellular differentiation involves losing or gaining interactions between the genome and the NE, and such rearrangements can influence gene expression programs. For instance, the nuclear lamina, which is a filamentous protein network underlying the NE, has been extensively implicated in setting up tissue-specific genome organization by sequestering genes destined for silencing. Genome-wide mapping of Lamin-associated domains (LADs), as well as related functional studies, have led to the current view of the nuclear lamina as a compartment for stable gene repression. Another major component of the NE is the nuclear pore complex (NPC), which consists of multiple copies of approximately 30 different proteins termed nucleoporins (Nups) and is responsible for selective nucleo-cytoplasmic transport. In addition to transport-related functions, NPCs and individual Nups are also involved in genome organization and gene regulation through physical interactions with the genome. Yet unlike the nuclear lamina, the functional relationship between NPCs and genome regulation appears to be considerably more varied and remains less understood (Gozalo, 2019).

Given the close proximity of nuclear pores to the underlying chromatin, it is not surprising that multiple studies have now identified binding of Nups to subsets of genes and regulatory elements in a number of species. Many of these studies have reported preferential association of particular Nups with actively transcribing genes or re-localization of genes to the NPCs during activation. These findings have led to the predominant view of the NPC as a nuclear compartment for active processes, functionally opposed to those of the nuclear lamina. However, at least in metazoan systems, this view is confounded by the reported intranuclear presence of Nups that have been classified as dynamic. The ~30 conserved Nups that comprise the NPC can be either dynamic, meaning they are able to come on and off the NPC during interphase, or stable, meaning they are core components of the NE-embedded NPC for the majority of the cell cycle. Currently, many of the reported contacts between active genes and Nups have been described for dynamic Nups, such as Nup98, Nup153, and Nup62, and can frequently occur in the nucleoplasm. Consequently, it is unclear whether genomic binding to actual NPCs is functionally distinct from intranuclear Nup binding (Gozalo, 2019).

Genomic binding to actual NPCs can be determined by mapping chromatin-binding patterns of stable Nups, which are components of the outer-ring Nup107-Nup160 and the inner-ring Nup93-Nup205 sub-complexes. Interestingly, previous studies that profiled chromatin binding of stable Nups did not identify enrichment for transcribing loci and reported prevalence of repressive chromatin. Similarly, DamID profiling of Nup98, artificially tethered to the NPC and thus used as a marker for actual NPC binding, showed no enrichment for active genes and instead exhibited high incidence of motifs for the architectural protein Su(Hw) in Drosophila cells. These studies conflict with the simplified view of the NPC as a scaffold for gene activation and highlight the complexity of NPC-genome interactions. It should be noted that the majority of the NPC genome-binding datasets, mentioned above, were produced using either the DamID technique, which tends to generate wide binding peaks, or the lower-resolution ChIP-chip approach, and thus may have given an incomplete picture of the locations and functions of NPC-genome contacts (Gozalo, 2019).

One hypothesis, which can explain this dichotomy of both active and silent regions at the NPC, is that individual stable Nups bind distinct regions of the genome and regulate distinct chromatin-associated processes. This study set out to explore this hypothesis by generating precise binding maps of stable Nups, using an optimized ChIP-seq approach. ChIP-seq maps revealed that Nup107, a core component of the outer ring sub-complex, and Nup93, a core component of the inner ring sub-complex, bind highly non-overlapping regions of the genome. Specifically, while Nup107 preferentially targets active promoters, as has been reported for other Nups, Nup93 associates primarily with silenced regions bound by Polycomb group (PcG) proteins. PcG proteins are conserved regulators of epigenetically maintained gene repression, which often bind the genome in long Polycomb (Pc) domains. In agreement with its binding pattern, this study found that Nup93 plays a functional role in the silencing and long-range interactions of Pc targets. Together, the results emphasize the concept that different sub-complexes of the nuclear pore interact with and influence distinct chromatin states, revealing a complex landscape of NE-genome interactions (Gozalo, 2019).

The results provide high-resolution chromatin binding maps of stable NPC components and offer a resource for future comparisons to a variety of genomic features. These maps and analysis contribute several insights into the nuclear organization field. First, it was found that representative members of the two core sub-complexes of the NPC, Nup107 and Nup93, bind to active and silenced regions, respectively. This differential binding helps explain the variability in previous conclusions on NPC-genome contacts and extends understanding of how NPC-genome contacts shape three-dimensional genome architecture. ChIP-seq maps of Nup107 are consistent with the predominant view of the NPC as a place for targeting active genes and suggest that this function is carried out primarily through associations of the genome with the outer-ring NPC sub-complex. Although currently it is not possible to definitively prove that all identified binding peaks of Nup107 and Nup93 represent NPC binding, comparison to LADs and immunofluorescence localization analysis, as well as DNA FISH analysis of select loci, suggest that a large proportion of these binding peaks represent regions present at the nuclear periphery, at actual NPCs (Gozalo, 2019).

Second, the findings describe a functional connection between Nup93, a conserved subunit of the inner-ring NPC sub-complex, and Polycomb complexes, which are key regulators of developmental gene silencing. ChIP-seq map of Nup93 demonstrates preferential targeting of Nup93 to a large subset of PcG domains, particularly those that exhibit the highest level of Pc binding. Importantly, it was found that lowering levels of Nup93 leads to de-repression of Pc targets in both cultured cells and fly tissues. These findings suggest that (1) a core NPC subunit is involved in the epigenetic maintenance of silencing via its chromatin binding role; and (2) a subclass of particularly stable PcG chromatin domains are targeted to the nuclear periphery, where they require Nup93 for optimal silencing. The function of the Nup93 sub-complex in gene repression appears to be highly conserved, as strikingly, the S. pombe homolog of Nup93 was found by Moazed and colleagues to be required for silencing and nuclear clustering of heterochromatin (Iglesias, 2019). Nup93 has also been previously shown to be required for the repression of the HoxA gene cluster in mammalian cells (Labade, 2016). This is in line with previous findings that another subunit of this sub-complex, Nup155, associates with histone deacetylases in mammalian cells and targets repressed heterochromatin in yeast. The role of Nup93 in PcG silencing is also potentially related to the previously reported link of Nup153 to PcG-mediated repression in mouse ES cells. In further support of this notion, several Nups have been previously identified in a genome-wide imaging screen for factors that affect nuclear distribution of PcG proteins (Gozalo, 2019).

Thus, a proposed model envisions that certain nuclear pores may interact with active chromatin via the Nup107 sub-complex, while other nuclear pores may associate with silent chromatin via the Nup93 sub-complex. This model is based on the ability to ChIP-seq distinct regions of the genome with different structural NPC components, and it is further supported by biochemical interaction data and the specificity of the functional effect of Nup93. The proposed model is consistent with previous findings in yeast, which reported the binding of a component of the Nup93 sub-complex to silent chromatin and the possible existence of this sub-complex as a type of an independent nuclear pore-related complex, present at the nuclear periphery. In this context, it remains to be determined whether some of the Nup93-PcG interactions similarly occur as an independent complex or if they are normally part of actual NPCs and whether the observed functional effect of Nup93 on PcG silencing always takes place at the NPC (Gozalo, 2019).

Interestingly, it was found that the Nup93-targeted PcG domains tend to preferentially interact with each other in nuclear space. It is intriguing that other Nups, such as Nup98 and Mlp1/2, have been previously shown to facilitate long-range contacts of transcribing genes, such as enhancer-promoter and 5'-3' loops. It appears that stabilization of long-range contacts, either at active or silent genes, may be generally promoted by NPC binding, but the nature of contacts depends on the particular Nup involved. Based on the combined results, it is hypothesized that Nup93 binding may promote stabilization of PcG domains that are destined to be highly repressed. This stabilization may involve promoting long-range interactions between Pc sites, as well as possibly helping sequester PcG domains into specific nuclear compartments, away from gene activity. The findings also suggest that in the case of PcG silencing, long-range interactions are more functionally involved in gene repression than localization to the nuclear periphery is, since de-repression of PcG targets is consistently associated with loss of long-range interactions (Gozalo, 2019).

Furthermore, the results suggest that some of the previously defined LADs are in fact interrupted or flanked with NPC-associated chromatin. In this manner, it appears that at least a fraction of mapped LADs may be complex, containing Nup-targeted sub-environments. These conclusions are also supported by the recent refined analysis of mammalian LADs, which revealed LAD interruptions that contain marks of active chromatin, termed 'Disruption in Peripheral signal' (DiPs). If such DiPs are biologically meaningful, the data would suggest that some such DiPs may be NPC-bound areas of the genome, characterized by functions distinct from the surrounding LADs. An intriguing conjecture is that positioning genes at NPCs within LADs may facilitate ready switching of transcriptional states, such that genes can shift between adjacent active and silent states, depending on incoming signals (Gozalo, 2019).

Finally, this analysis demonstrated widespread genomic binding by a non-stable Nup Elys, which is currently the only Nup with a known direct chromatin binding activity (Zierhut, 2014). Interestingly, Nup107 is almost exclusively found at Elys binding sites. It is tempting to speculate that Elys serves as a chromatin tethering Nup for the Nup107 sub-complex components in the interphase genome, much like it has been demonstrated to do post-mitotically, during NPC assembly. The reproducibility of Elys-Nup107 binding patterns further invokes the possibility that post-mitotic targeting of the Elys/Nup107 sub-complex to chromatin occurs at specific sites in the genome and as such, may participate in the correct re-establishment of chromatin states and nuclear architecture after mitosis. On the other hand, Nup93 similarly shares a large fraction of its binding sites with Elys, suggesting that Elys may carry a similar function in targeting the inner ring sub-complex to chromatin. Presently it remains unclear how this specificity of Nup93 versus Nup107 genome targeting may be established. But together, the findings support the model where different subunits of the NPC have evolved unique functions in chromatin regulation. Individual Nups appear to be able to facilitate either activating or repressive processes and to assist nuclear organization of chromatin domains and key proteins complexes (Gozalo, 2019).

The Nuclear Pore Complex (NPC) is the principal gateway between nucleus and cytoplasm that enables exchange of macromolecular cargo. Composed of multiple copies of ~30 different nucleoporins (Nups), the NPC acts as a selective portal, interacting with factors which individually license passage of specific cargo classes. This study shows that two Nups of the inner channel, Nup54 and Nup58, are essential for transposon silencing via the PIWI-interacting RNA (piRNA) pathway in the Drosophila ovary. In ovarian follicle cells, loss of Nup54 and Nup58 results in compromised piRNA biogenesis exclusively from the flamenco locus, whereas knockdowns of other NPC subunits have widespread consequences. This provides evidence that some nucleoporins can acquire specialised roles in tissue-specific contexts. These findings consolidate the idea that the NPC has functions beyond simply constituting a barrier to nuclear/cytoplasmic exchange, as genomic loci subjected to strong selective pressure can exploit NPC subunits to facilitate their expression (Munafo, 2021).

This study has found that Nup54 and Nup58 are specifically required for TE regulation in the follicle cells of Drosophila ovaries by enabling export and subsequent piRNA production from flam transcripts. This functional requirement is distinct from the general role of the NPC, since other Nups, even those most proximal to Nup54 and Nup58 within the pore, are broadly required for gene expression and cell survival. These findings consolidate the view of Nups as dynamic players in various cellular processes and expand the variety of roles ascribed to the Nups (Munafo, 2021).

Though flam bears canonical features common to other mRNAs, it is nonetheless specifically recognised and processed into piRNAs. A dedicated export-coupled licensing, involving Nup54 and Nup58, may allow this long transcript to be escorted directly from its genomic origin to the Yb-bodies to facilitate piRNA production. If this machinery is disrupted, for example, by loss of Nup54 and Nup58, flam transcripts are confined to the nucleus and destabilised, thus underscoring the need for a unified process from transcription to licensing. It is interesting to note that siYb caused a slight decrease of PRO-seq signal across the entire piRNA cluster locus, possibly indicating that disruption of flam export-coupled licensing negatively affects its transcription via a yet-unknown feedback mechanism. Nup54 and Nup58 interact with both the nuclear (Nxf1/Nxt1) and cytosolic (Yb) components of flam expression and thus suggest that these factors bridge nuclear export to cytosolic fate specification. Although several aspects of the molecular mechanism remain to be elucidated (i.e., how this links to upstream transcription and whether additional factors are involved in the nucleus or in the cytosol), a tentative model is proposed whereby this Nxf1-NPC-Yb axis coordinates initiation, processivity, and directionality of flam export, directly feeding the transcript into the piRNA biogenesis route via Yb-bodies. Rescue experiments and protein-protein interaction studies argue for this TE silencing function of Nup54 and Nup58 to be carried out from within the pore, especially since neither Nup is among the most dynamic components of the NPC. However, a function for Nup54 and Nup58 in the cytosol, possibly as components of Yb-bodies, cannot be completely excluded. Nup54 and Nup58 interact directly with both Yb and Nxf1; however, it is presently unclear how the transcript is released from the Nxf1/Nxt1 export complexes and handed over to Yb, especially since physical interactions between Yb and Nxf1 was not detected. It cannot be excluded that such interaction exists and remained undetected in over-expression experiments or that additional adaptor proteins take part in this process. Alternatively, Nup54/58 may form independent complexes with Yb and Nxf1; however, lack of enrichment for flam transcripts associated with Nup54/58 makes this seem unlikely. Super-resolution imaging of the NPC will be required to clarify the relative position of each component of this export route and to understand whether Nup54 and Nup58 function also outside the NPC. Of note, cytosolic Nup358 has been shown to be required for piRNA production from the dual-strand cluster 42AB in nurse cells. Its depletion leads to prominent de-localisation of Piwi, which was not observed upon loss of Nup54/Nup58, thus suggesting a different mode of action. Nonetheless, this further underscores that different NPC subunits can be co-opted for TE silencing in tissue-specific contexts (Munafo, 2021).

The proposed mechanism for flam export-coupled licensing relies on a tissue-specific effector (Yb) and could represent a broader paradigm for co-option of phenylalanine and glycine rich (FG) Nups for specific transcript trafficking routes. It is envisioned that in principle any transcript subject to strong selective pressure could evolve a dedicated export machinery via adapting FG-Nup functions in a cell-type-specific manner. Such mechanisms may have gone unnoticed previously because of the general role of the NPC. Interestingly, several Nup genes in Drosophila show signs of rapid adaptive evolution that result in hybrid incompatibilities, which are often hallmarks of genes involved in genetic conflicts, such as transposon control. It is further speculated that a tissue-specific control of export routes might contribute to explain the molecular mechanisms underlying so-called 'nucleoporopathies', human syndromes caused by mutated Nups. In these diseases, mutation or expression changes of a Nup present in all cells of the organism leads to tissue-specific phenotypes. It is hypothesised that this might stem from specific roles of those Nups in regulating genes that are essential for the functionality of that particular tissue, which would in turn make the said tissue especially susceptible to the loss of the Nup. Future investigation will shed light on how widespread these mechanisms might be (Munafo, 2021).

Nuclear envelope (NE) budding is a recently described phenomenon wherein large macromolecular complexes are packaged inside the nucleus and extruded through the nuclear membranes. Although a general outline of the cellular events occurring during NE budding is now in place, little is yet known about the molecular machinery and mechanisms underlying the physical aspects of NE bud formation. Using a multidisciplinary approach, this study identified Wash, its regulatory complex (SHRC), capping protein and Arp2/3 as new molecular components involved in the physical aspects of NE bud formation in a Drosophila model system. Interestingly, Wash affects NE budding in two ways: indirectly through general nuclear lamina disruption via an SHRC-independent interaction with Lamin B leading to inefficient NE bud formation, and directly by blocking NE bud formation along with its SHRC, capping protein and Arp2/3. In addition to NE budding emerging as an important cellular process, it shares many similarities with herpesvirus nuclear egress mechanisms, suggesting new avenues for exploration in both normal and disease biology (Verboon, 2020).

Transport of macromolecules from the nucleus to the cytoplasm is essential for all developmental processes, including the regulation of differentiation and aging, and, when mis-regulated, is associated with diseases and cancer. This indispensable process has been thought to occur exclusively through nuclear pore complexes (NPCs), channels that regulate what exits (and enters) the nucleus. Recently, nuclear envelope (NE) budding was identified as an alternative pathway for nuclear exit, particularly for large developmentally required ribonucleoprotein (megaRNP) complexes that would otherwise need to unfold/remodel to fit through the NPCs. In this pathway, large macromolecule complexes, such as megaRNPs, are encircled by the nuclear lamina (type-A and -B lamins) and the inner nuclear membrane (INM), pinched off from the INM, fuse with the outer nuclear membrane and release the megaRNPs into the cytoplasm. Strikingly, NE budding shares many features with the nuclear egress mechanism used by herpesviruses. As viruses often utilize pre-existing host pathways, the parallel between nuclear exit of herpesvirus nucleocapsids and that of megaRNPs and/or other large cargoes suggests that NE budding may be a general cellular mechanism. Indeed, this pathway has also been implicated in the removal of obsolete macromolecular complexes or other material (i.e. large protein aggregates or poly-ubiquitylated proteins) from the nucleus (Verboon, 2020).

NE budding was first demonstrated in Drosophila synapse development, proving to be essential for neuromuscular junction (NMJ) integrity. In this context, a C-terminal fragment of the Wingless receptor Fz2, Fz2C, was shown to associate with megaRNPs that formed foci at the nuclear periphery and exited the nucleus by budding through the nuclear envelope (Speese, 2012). Failure of this process resulted in aberrant synapse differentiation and impaired NMJ integrity (Speese, 2012). In a subsequent study, the NE budding pathway was shown to be necessary for the nuclear export of megaRNPs containing mitochondrial RNAs: disruption of NE budding led to deterioration of mitochondrial integrity and premature aging phenotypes that were similar to those associated with lamin mutations (i.e., laminopathies). Similar endogenous perinuclear foci/buds have been observed in plants and vertebrates, as well as other Drosophila tissues (i.e., larval salivary gland nuclei), suggesting that cellular NE budding is a widely conserved process (Verboon, 2020).

The spectrum of processes requiring this non-canonical nuclear exit pathway and the molecular machineries needed for this process, which encompasses membrane deformations, traversal across a membrane bilayer and nuclear envelope remodeling for a return to homeostasis, are largely unknown. One class of proteins that are involved in membrane-cytoskeletal interactions and organization is the Wiskott-Aldrich Syndrome (WAS) protein family. WAS protein subfamilies are involved in a wide variety of essential cellular and developmental processes, as well as in pathogen infection and disease. WAS family proteins polymerize branched actin through the Arp2/3 complex, and often function as downstream effectors of Rho family GTPases. This study identified Wash as a new WAS subfamily that is regulated in a context-dependent manner: Wash can bind directly to Rho1 GTPase (in Drosophila) or it can function along with the multi-protein WASH regulatory complex [SHRC; comprised of SWIP, Strumpellin, FAM21 and CCDC53 (also known as WASHC4, WASHC5, WASHC2 and WASHC3, respectively, in mammals)]. Wash regulation by Rho family GTPases outside of Drosophila has not yet been described; instead its regulation has been characterized in the context of its SHRC. WASH and its SHRC are evolutionarily conserved and their mis-regulation is linked to cancers and neurodegenerative disorders. Importantly, it has been shown that Wash is present in the nucleus where it interacts directly with B-type lamins and, when mutant, affects global nuclear organization/functions, as well as causing an abnormal wrinkled nucleus morphology reminiscent of that observed in diverse laminopathies (Verboon, 2015a and 2015b). Mammalian WASH proteins have also been shown to localize to the nucleus in developmental and cell-type specific manners (Verboon, 2020).

This study shows that Wash, its SHRC, capping protein and Arp2/3 are also involved in the NE budding pathway, as mutants for any of these components lack Fz2C foci/lamin buds and display the NMJ integrity and premature aging phenotypes previously associated with the loss of NE budding. In addition, this study found that CCDC53 and SWIP (SHRC subunits) colocalize with Fz2C foci/lamin buds. Wash is shown to be present in several independent nuclear complexes. The nuclear interactions of Wash with its SHRC are separate from those with B-type Lamin, leading to effects on different subsets of nuclear Wash functions. This study also found that Wash-dependent Arp2/3 actin nucleation activity is required for proper NE budding. It is proposed that Wash and its SHRC play a physical and/or regulatory role in the process of NE budding (Verboon, 2020).

NE budding is an increasingly appreciated pathway for nuclear export of large macromolecular machineries, such as megaRNPs involved in the co-regulation of major developmental pathways or unwanted RNA or protein aggregates. Previous work has shown that Drosophila Wash is present in the nucleus where it is likely involved in a number of different nuclear processes (Verboon, 2015a). This study shows that all members of the four-subunit Wash regulatory complex (SHRC), as well as the heterodimeric capping protein, are also present within the nucleus and, along with Wash, are necessary for NE budding. Wash and SHRC act early in the NE budding pathway, and this process requires actin nucleation activity of Wash achieved through its interaction with Arp2/3. Loss of Wash, any of its SHRC subunits or Arp2/3 leads to the loss of Fz2C foci/NE buds, and mutants for these factors exhibit phenotypes associated with the two cellular processes shown to require NE budding: aberrant synaptic 'ghost' bouton formation leading to disrupted NMJ integrity, and mitochondrial degeneration associated with premature aging phenotypes. This study also shows that the interaction of Wash with Lamin B results in a general disruption of the nuclear lamina and separation of the Lamin B/Lamin C homotypic meshes, leading to inefficient, rather than loss of, NE bud formation (Verboon, 2020).

While the spectrum of processes that require this alternate nuclear egress mechanism is not yet known, SHRC components are linked to neurodegenerative disorders, including hereditary spastic paraplegias, Parkinson disease, amyotrophic lateral sclerosis (ALS) and Hermansky-Pudlak syndrome. As an increasing number of neurodegenerative diseases and myopathies have been associated with the accumulation of RNA-protein aggregates in the nucleus, NE budding may be part of the endogenous cellular pathway for removing such aggregates/megaRNPs from the nucleus in normal cells (Verboon, 2020).

The parallels between the mechanism of NE budding and herpesvirus nuclear egress, as well as the presence of similar endogenous perinuclear foci/buds in other plant and animal nuclei, has suggested that NE budding is a conserved endogenous cellular pathway for nuclear export. Indeed, INM-encapsulated electron-dense granules have been identified in yeast and Torsin-deficient HeLa cells, and these show similarities to the Fz2C foci/NE buds observed in Drosophila muscle and salivary gland nuclei. While the full relationship between NPCs and NE buds is not yet known, one important difference is that the yeast and HeLa nuclear granules observed are much smaller (∼120 nm) than Fz2C foci/NE buds (∼500 nm). This identification of Wash and SHRC, proteins with the capability of remodeling cortical cytoskeleton and/or membranes, in the physical aspects of NE budding lend support for NE budding being an alternative endogenous nuclear exit pathway (Verboon, 2020).

NE budding has been proposed to occur at sites along the INM where the nuclear lamina is modified by aPKC phosphorylation. Both A- and B-type lamins play a role in NE budding and are thought to be the target of aPKC phosphorylation within the nuclear lamina, similar to the PKC-mediated phosphorylation of lamins that precedes lamina disassembly in mitotic NE breakdown (Güttinger, 2009), apoptosis (Cross, 2000) or during viral capsid nuclear egress. Viral NE budding requires a virus-encoded nuclear egress complex (NEC), which has been implicated in the recruitment of kinases to the INM. Cellular counterparts for these virally encoded NEC proteins have not yet been identified. It is also not yet known how this kinase activity is restricted to specific sites along the nuclear lamina or how those specific sites are selected (Verboon, 2020).

Previous work has shown that Wash interacts directly with Lamin B and that loss of nuclear Wash results in a wrinkled nuclear morphology reminiscent of that observed in laminopathies (Verboon, 2015a). It was reasoned that Wash-mediated disruption of the nuclear lamina may account for its NE-budding phenotypes. Consistent with this idea, it was found that Lamin B knockdown nuclei and nuclei from a wash point mutant that disrupts the interaction of Wash with Lamin B (washΔΔLamB) exhibit a wrinkled nuclear morphology, reduced Fz2C foci/NE buds and NE-budding-associated phenotypes, albeit not as strong as those observed in Wash or SHRC mutants (Verboon, 2020).

Lamin A/C and Lamin B isoforms form homotypic meshworks that interact among themselves (in as yet unknown ways), and that are somehow linked to integral membrane proteins of the INM and to the chromatin adjoining the INM. Intriguingly, the current data suggests that these lamin homotypic meshes are likely layered, rather than interwoven, and that Wash affects the anchoring of these lamin homotypic meshes to each other and/or the INM. Lamin knockdown or disruption of the Wash-Lamin B interaction leads to separated lamin isoform meshes and wrinkled nuclear morphology that are not observed in SHRC and Arp2/3 knockdown nuclei, suggesting that Wash can also affect NE budding by a means independent of disrupted global nuclear lamina integrity. Interestingly, the functions of Wash mediated with the SHRC and with Lamin B involve separate nuclear complexes. Consistent with this, it was shown previously that Drosophila Wash encodes several independent biochemical activities (actin nucleation, actin bundling, MT bundling and actin-MT crosslinking) and that the use of these activities is context dependent. In particular, when Wash interacts with Lamin, it does not require an association with SHRC or Arp2/3. It is suggested that the interaction of Wash with Lamin B is required for organizing the nuclear lamina and likely requires the actin bundling and/or cross-linking activities of Wash rather than its actin nucleation activity, such that wash mutants that cannot bind Lamin result in separation of the Lamin isoform meshes from each other. Taken together, the data suggest that loss of the interaction between Wash and Lamin B makes NE budding inefficient by generally disrupting the nuclear lamina, rather than directly disrupting NE bud formation. The role of aPKC in NE budding may also be somewhat indirect by generally disrupting the nuclear lamina thereby reducing the efficiency of NE bud formation. Alternatively, aPKC may target Wash: WASH phosphorylation by Src kinases has been shown to be necessary for regulating NK cell cytotoxicity (Verboon, 2020).

For bud formation/envelopment of a megaRNP or macromolecular cargo to occur, the INM must interact with its underlying cortical nucleoskeleton to allow the INM deformation/curvature necessary to form the physical NE bud. Force must also be generated that allows the bud to extend into the perinuclear space, as well as for the scission of the nascent bud. In the cytoplasm, WAS family proteins are often involved in membrane-cortical cytoskeleton-coupled processes, including both 'inward' membrane deformations (i.e., endocytosis) and 'outward' membrane deformations (i.e., exocytosis and cell protrusions), that are required for signal/environment sensing and cell movement during normal development, as well as during pathological conditions. Mammalian WASH, in particular, has been implicated in endosome biogenesis and/or sorting in the cytoplasm, where it, along with its SHRC, drives Arp2/3-dependent actin assembly to influence endosome trafficking, remodels membrane, and facilitates membrane scission. Thus, Wash encodes the biochemical properties needed to regulate the membrane deformation/curvature necessary to form the NE bud and/or play a role in generating the forces necessary to pinch off the NE bud from the INM. Consistent with Wash playing a role in the physical production of a NE bud, it was found that Wash acts prior to Torsin, a protein that is implicated in NE bud scission from the INM, and it requires its actin nucleation activity (Verboon, 2020).

This study has identified Wash and its SHRC as new players in the cellular machinery required for the newly described endogenous NE budding pathway. The data suggest that Wash is involved in two nuclear functions that can affect NE budding. (1) Wash is required to maintain the organization of Lamin isoforms relative to each other and the INM through its direct physical interaction with Lamin B. This Wash activity is SHRC and Arp2/3 independent, and is likely a non-specific mechanism because global disruption of the nuclear lamina/nuclear envelope would indirectly affect many nuclear processes, including NE budding. (2) Wash is specifically required for NE bud formation. This Wash activity is SHRC and Arp2/3 dependent. While the focus of NE budding research to date has centered on the composition of the megaRNPs and the spectrum of cellular/developmental processes requiring NE budding, Wash and the SHRC are likely involved in the physical aspects of NE budding. Thus, Wash and SHRC provide a molecular entry into the physical machinery that underlies NE budding. In the future, it will be exciting to further explore the roles of Wash in NE budding, and to determine how it functions to get macromolecular complexes through the INM, and how closely these nuclear roles parallel those in the cytoplasm (Verboon, 2020).

Germ cells undergo distinct nuclear processes as they differentiate into gametes. While these events must be coordinated to ensure proper maturation, the stage-specific transport of proteins in and out of germ cell nuclei remains incompletely understood. Efforts to genetically characterize Drosophila genes that exhibit enriched expression in germ cells led to the finding that loss of the highly-conserved Importin β/karyopherin family member Importin-9 (Ipo9) results in female and male sterility. Immunofluorescence and fluorescent in situ hybridization (FISH) revealed that Ipo9 (KO) mutants display chromosome condensation and segregation defects during meiosis. In addition, Ipo9 (KO) mutant males form abnormally structured sperm and fail to properly exchange histones for protamines. Ipo9 physically interacts with proteasome proteins and Ipo9 mutant males exhibit disruption of the nuclear localization of several proteasome components. Thus, Ipo9 coordinates the nuclear import of functionally related factors necessary for the completion of gametogenesis (Palacios, 2021).

Subcellular compartmentalization allows for complex modes of gene regulation in eukaryotic cells. The regulated and active transport of macromolecules between different compartments promotes cellular homeostasis and often drives differentiation. Transport of molecules from the cytoplasm to the nucleus depends on a family of proteins called karyopherins, also known as importins. The karyopherin superfamily of transporters consists of importin α and importin β subgroups. All the proteins within this karyopherin superfamily share tandem huntingtin, elongation factor 3, protein phosphatase 2A and mechanistic target of rapamycin (HEAT) repeats. These repeats allow these proteins to bind to various cargo proteins, which often, but not always, contain a nuclear localization signal within their peptide sequence. Karyopherins then transport these cargoes into the nucleus through nuclear pores (Palacios, 2021).

Another key component of the transport machinery is the small GTPase Ran (Cautain, 2015). Cytoplasmic Ran is typically maintained in a GDP-bound state, whereas nuclear Ran binds GTP. This concentration gradient of GDP and GTP bound Ran provides a directional cue for the transport of proteins between the cytoplasm and nucleus. Once importins enter the nucleus, high affinity interactions with RanGTP cause karyopherins to release their cargoes and recycle back to the cytoplasm (Palacios, 2021).

Accumulating evidence suggests that β-karyopherins do not simply function as constitutive and redundant housekeeping proteins. Interactions between different β-karyopherins with specific cargoes depend not only on their overlapping expression patterns in time and space, but also on clear differences in the affinities of the physical interactions. For example, histones can bind to multiple β-karyopherins, but their affinities vary. For example, Kapβ2 and Imp5 exhibit very strong affinity for Histone H3, whereas Impβ, Imp4, Imp7, Imp9 and Impα display weaker interactions. Additionally, a previous study identified a group of 468 cargoes for 12 β-karyopherins. Three hundred and thirty two of these cargoes were unique to one β-karyopherin family member, suggesting a division of function among these transporters. Several β-karyopherin family members have been associated with specific diseases. Accumulating evidence shows that β-karyopherins are overexpressed in multiple tumors including melanoma, pancreatic, breast, colon, gastric, prostate, esophageal, lung cancer and lymphomas. Additionally, specific karyopherin-β proteins, such as exportin-1, have been implicated in drug resistance in cancer (Palacios, 2021).

Many importins exhibit enriched expression in gonads and are functionally required during different stages of spermatogenesis and oogenesis across many species, including Drosophila. Drosophila ovaries are organized into discrete units called ovarioles, which contain a series of sequentially developing egg chambers. Each egg chamber is comprised of 16 germ cells, 15 nurse cells and one oocyte, surrounded by a layer of somatic follicle cells. The initiation of meiosis occurs early in oogenesis, marked by the formation of the synaptonemal complex (SC) and the generation of the programmed double strand breaks. After these first events, oocytes remain arrested in prophase 1 of meiosis until stage 12, followed by prometaphase 1 at stage 13 and metaphase 1 at stage 14 (Palacios, 2021).

The Drosophila testis is structured as a closed-end coiled tube. At the tip of the testis, 10-14 germline stem cells (GSCs) surround a small cluster of somatic cells called the hub. GSCs typically divide asymmetrically to produce another GSC and a gonialblast. Gonialblasts become enveloped by two somatic cyst cells, that function in an analogous manner to the Sertoli cells of the mammalian testis. The Drosophila gonialblast goes through four incomplete mitotic divisions to form an interconnected 16-cell spermatogonial cell cyst. Each spermatocyte within the cyst undergoes meiosis, resulting in the formation of cysts that contain 64 interconnected haploid cells. Immediately after the completion of meiosis, these cells enter the 'onion stage', which is marked by the appearance of the nebenkern, a two-stranded helical structure derived from mitochondria. Defects in meiosis can result in the appearance of fragmented nebenkern and alterations in the normal 1:1 ratio of nuclei and nebenkern (Palacios, 2021).

Spermiogenesis is marked by nuclear elongation and chromatin reorganization. Nuclear elongation is dependent on microtubules from the basal body that associate with the nucleus. Chromatin organization switches from a histone-based to protamine-based packaging in the late elongation stage. During elongation, the nuclear envelope that is in contact with the basal body forms a cavity that fills with microtubules while the nucleus takes on a 'canoe' shape. During chromatin reorganization, histones are ubiquitylated by an unknown ubiquitin ligase and subsequently degraded by the proteasome at the late canoe stage, immediately before protamines are incorporated into the chromatin. After histone removal, the transition like-protein (Tpl) is incorporated. This facilitates protamine incorporation. In Drosophila, mature sperm contain Mst35Ba (protamine A), Mst35Bb (protamine B) and Mst77F. Towards the end of spermiogenesis, sperm form their own membranes in a process called individualization (Fabian and Brill, 2012) (Palacios, 2021).

In Drosophila, mutants in several importins develop normally into adults, but exhibit various defects in fertility. Importin α2 mutant males exhibit a dramatic decrease in the formation of individualized and motile sperm, whereas mutant females produce small and deflated eggs with missing or fused dorsal appendages. Similarly, mutations in Importin α1 also cause male and female sterility, marked by egg-laying defects in females and the formation of spermatocytes with abnormally large round nuclei in males, and loss of Importin α3 leads to the arrest of oogenesis. The specific cargoes responsible for these phenotypes remain unknown (Palacios, 2021).

This study reports that null mutations in Ipo9 (also known as Ranbp9) cause disruption of chromosome segregation and condensation during meiosis in both female and male Drosophila. Previous results have shown that Ipo9 helps to traffic Actin, Histone H2A-H2B dimers and a variety of other factors into nuclei. This study confirmed that loss of Drosophila Ipo9 disrupts the accumulation of nuclear actin during oogenesis. In addition, it was found that Ipo9 promotes chromosome segregation during meiosis, and the exchange of histones for protamines during spermiogenesis. Biochemical experiments suggest that Ipo9 physically associates with proteasome components, and immunofluorescence studies show that loss of Ipo9 disrupts the normal trafficking of the proteasome into germ cell nuclei during spermiogenesis. Together, these data reveal new processes directly regulated by a specific nuclear transport factor during gametogenesis (Palacios, 2021).

This study provides evidence that Ipo9 specifically regulates a number of critical processes during Drosophila gametogenesis. Ipo9 null mutants survive to adulthood but exhibit female and male sterility. In the ovary, loss of Ipo9 results in defects in chromosome orientation and segregation during meiosis, resulting in mitotic catastrophes during early embryogenesis in progeny derived from Ipo9 homozygous mutant females. Ipo9 mutant males also exhibit numerous phenotypes during germ cell development, including defects in meiosis, and disruption of the nuclear shape changes and failure to fully exchange histones for protamines during spermiogenesis. Together, these represent a unique spectrum of phenotypes compared to other Drosophila β-karyopherin family members. Of the 12 Drosophila β-karyopherin genes that have been genetically characterized, loss-of-function alleles in seven result in lethality. Several other importin mutants do survive until adulthood, including ebomut, aplnull and artsnull. ebomut homozygotes display neuronal defects, aplnull mutants are male sterile, whereas artsnull mutant females produce smaller eggs that cannot be fertilized. In addition, a ketel dominant negative mutant (ketel^D) shows a female sterile phenotype and embryos derived from these flies exhibit chromosome segregation defects somewhat similar to those displayed by Ipo9KO mutants. Thus, amongst Drosophila karyopherin family members studied to date, Ipo9 is the only gene that displays specific defects during meiosis in both females and males, and in late sperm development, when mutated (Palacios, 2021).

Transgenic rescue experiments confirm that Ipo9 functions to promote the transport of proteins from the cytoplasm to the nucleus during oogenesis and spermatogenesis. A full-length Ipo9 transgene rescues most of the sterile phenotypes exhibited by Ipo9 mutants when driven in the germline. It is suspected that the failure of the Ipo9 wild-type transgene to fully rescue the male sterility of the mutant is likely due to the failure of the vasa-gal4 driver to fully recapitulate the endogenous expression pattern of Ipo9. The N-terminal domains of β-karyopherin proteins normally promote cytoplasmic-to-nuclear trafficking by contacting the nuclear pore and helping cargoes move through the nuclear pore complex. This domain also binds to RanGTP, and thus participates in the cycling of importins back-and-forth between the cytoplasm and nucleus. Strikingly, deletion of the N-terminal karyopherin domain renders the transgene non-functional, confirming that Ipo9 acts as an essential transport factor during gametogenesis in both males and females (Palacios, 2021).

The transition from histone-based to protamine-based chromatin organization is essential for the nuclear shaping that leads to a highly compact sperm nucleus. Ipo9KO nuclei are able to incorporate protamine-B, however histone H2A and H2Av are not completely removed. These results may partially explain why Ipo9KO nuclei do not elongate properly. Evidence that the ubiquitin proteasome pathway is involved in histone removal during spermiogenesis includes the histone ubiquitylation that occurs before protamine deposition and the delay in histone removal in proteasome mutants. Interestingly, Ipo9KO nuclei do not exhibit strong nuclear ubiquitylation after protamine incorporation, even though they still retain nuclear histones. Additionally, it was observed that Ipo9KO spermatids showed a significant reduction in the nuclear localization of several proteasome proteins, including Prosα6T, Prosα3T and Prosα2, compared to the control spermatids. These results suggest that the ligase(s) responsible for histone ubiquitylation and components of proteasome that ultimately degrade ubiquitylated histones are potential cargoes of Ipo9. Interestingly, Ipo9 appears to physically associate with a number of specific ubiquitin ligases, including Hyperplastic discs and KLHL10, which have been implicated in the regulation of male germ cell development, and with several components of the proteasome. Perhaps Ipo9 has evolved to temporally coordinate the import of these functionally related proteins during late sperm development. Such specialization in nuclear import may offer an economy of scale that would not exist if the responsibility of nuclear import during this critical phase of sperm development, when the cytoplasm and nuclei of sperm are becoming highly compacted, was spread across a number of potentially redundant β-karyopherins. This type of coordination in trafficking has been proposed previously in different contexts. Thus, further study of Ipo9 cargoes during sperm development may reveal critical unknown factors that play roles in meiosis, chromosome compaction and segregation, and nuclear shape changes (Palacios, 2021).

Dosage compensation in Drosophila melanogaster involves a 2-fold transcriptional upregulation of the male X chromosome, which relies on the X-chromosome-binding males-specific lethal (MSL) complex (see msl-2). However, how such 2-fold precision is accomplished remains unclear. This study shows that a nuclear pore component, Mtor, is involved in setting the correct levels of transcription from the male X chromosome. Using larval tissues, this study demonstrated that the depletion of Mtor results in selective upregulation at MSL targets of the male X, beyond the required 2-fold. Mtor and MSL components interact genetically, and depletion of Mtor can rescue the male lethality phenotype of MSL components. Using RNA fluorescence in situ hybridization (FISH) analysis and nascent transcript sequencing, this study found that the effect of Mtor is not due to defects in mRNA export but occurs at the level of nascent transcription. These findings demonstrate a physiological role for Mtor in the process of dosage compensation, as a transcriptional attenuator of X chromosome gene expression (Aleman, 2021).

Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. This study identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. Fly dTnpo (orthologous to TNPO2) was found to be expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities (Goodman, 2021).

Female reproductive behaviors and physiology change profoundly after mating. In Drosophila, the post-mating response induced by male-derived sex-peptide in females is a well-established model to elucidate how complex innate behaviors are hard-wired into the brain. This study use a genetic approach to further characterize the molecular and cellular architecture of the sex-peptide response in Drosophila females. Screening for mutations that affect the sensitivity to sex-peptide, this study identified the channel nuclear pore protein Nup54 gene as an essential component for mediating the sex-peptide response, with viable mutant alleles leading to the inability of laying eggs and reducing receptivity upon sex-peptide exposure. Nup54 directs correct wiring of eight adult brain neurons that express pickpocket and are required for egg-laying, while additional channel Nups also mediate sexual differentiation. Consistent with links of Nups to speciation, the Nup54 promoter is a hot spot for rapid evolution and promoter variants alter nucleo-cytoplasmic shuttling. These results implicate nuclear pore functionality to neuronal wiring underlying the sex-peptide response and sexual differentiation as a response to sexual conflict arising from male-derived sex-peptide to direct the female post-mating response (Nallasivan, 2021).

Single-molecule localization microscopy (SMLM) can provide nanoscale resolution in thin samples but has rarely been applied to tissues because of high background from out-of-focus emitters and optical aberrations. This study describes a line scanning microscope that provides optical sectioning for SMLM in tissues. Imaging endogenously-tagged nucleoporins and F-actin on this system using DNA- and peptide-point accumulation for imaging in nanoscale topography (PAINT) routinely gives 30 nm resolution or better at depths greater than 20 μm. This revealed that the nuclear pores are nonrandomly distributed in most Drosophila tissues, in contrast to what is seen in cultured cells. Lamin Dm0 shows a complementary localization to the nuclear pores, suggesting that it corrals the pores. Furthermore, ectopic expression of the tissue-specific Lamin C causes the nuclear pores to distribute more randomly, whereas lamin C mutants enhance nuclear pore clustering, particularly in muscle nuclei. Given that nucleoporins interact with specific chromatin domains, nuclear pore clustering could regulate local chromatin organization and contribute to the disease phenotypes caused by human lamin A/C laminopathies (Cheng, 2021).

The nuclear transport machinery is involved in a well-known male meiotic drive system in Drosophila. Fast gene evolution and gene duplications have been major underlying mechanisms in the evolution of meiotic drive systems, and this might include some nuclear transport genes in Drosophila. So, using a comprehensive, detailed phylogenomic study, this study examined 51 insect genomes for the duplication of the same nuclear transport genes. Most of the nuclear transport duplications in Drosophila are of a few classes of nuclear transport genes, RNA mediated and fast evolving. Many pseudogenes for the Ran gene were obtained. Some of the duplicates are relatively young and likely contributing to the turnover expected for genes under strong but changing selective pressures. These duplications are potentially revealing what features of nuclear transport are under selection. Unlike in flies, only a few duplications were found when the Drosophila duplicated nuclear transport genes were studied in dipteran species outside of Drosophila, and none in other insects. These findings strengthen the hypothesis that nuclear transport gene duplicates in Drosophila evolve either as drivers or suppressors of meiotic drive systems or as other male-specific adaptations circumscribed to flies and involving a handful of nuclear transport functions (Mirsalehi, 2021).

Variants in genes encoding nuclear pore complex (NPC) proteins are a newly identified cause of paediatric steroid-resistant nephrotic syndrome (SRNS). Recent reports describing NUP93 variants suggest these could be a significant cause of paediatric onset SRNS. This study report NUP93 cases in the UK and demonstrate in vivo functional effects of Nup93 depletion in a fly (Drosophila melanogaster) nephrocyte model. Three hundred thirty-seven paediatric SRNS patients from the National cohort of patients with Nephrotic Syndrome (NephroS) were whole exome and/or whole genome sequenced. Patients were screened for over 70 genes known to be associated with Nephrotic Syndrome (NS). D. melanogaster Nup93 knockdown was achieved by RNA interference using nephrocyte-restricted drivers. Six novel homozygous and compound heterozygous NUP93 variants were detected in 3 sporadic and 2 familial paediatric onset SRNS characterised histologically by focal segmental glomerulosclerosis (FSGS) and progressing to kidney failure by 12 months from clinical diagnosis. Silencing of the two orthologs of human NUP93 expressed in D. melanogaster, Nup93-1, and Nup93-2 resulted in significant signal reduction of up to 82% in adult pericardial nephrocytes with concomitant disruption of NPC protein expression. Additionally, nephrocyte morphology was highly abnormal in Nup93-1 and Nup93-2 silenced flies surviving to adulthood. This study expanded the spectrum of NUP93 variants detected in paediatric onset SRNS and demonstrate its incidence within a national cohort. Silencing of either D. melanogaster Nup93 ortholog caused a severe nephrocyte phenotype, signaling an important role for the nucleoporin complex in podocyte biology (Bierzynska, 2022).

A missense mutation in Nucleoporin107 (Nup107; D447N) underlies XX-ovarian-dysgenesis, a rare disorder characterized by underdeveloped and dysfunctional ovaries. Modeling of the human mutation in Drosophila or specific knockdown of Nup107 in the gonadal soma resulted in ovarian-dysgenesis-like phenotypes. Transcriptomic analysis identified the somatic sex-determination gene doublesex (dsx) as a target of Nup107. Establishing Dsx as a primary relevant target of Nup107, either loss or gain of Dsx in the gonadal soma is sufficient to mimic or rescue the phenotypes induced by Nup107 loss. Importantly, the aberrant phenotypes induced by compromising either Nup107 or dsx are reminiscent of bone morphogenetic protein (BMP signaling hyperactivation). Remarkably, in this context, the metalloprotease AdamTS-A, a transcriptional target of both Dsx and Nup107, is necessary for the calibration of BMP signaling. As modulation of BMP signaling is a conserved critical determinant of soma-germline interaction, the sex- and tissue-specific deployment of Dsx-F by Nup107 seems crucial for the maintenance of the homeostatic balance between the germ cells and somatic gonadal cells (Shore, 2022).

This study has shown that Nup107 activity in the somatic component of the gonad is necessary for the proper development and function of the ovaries. In which somatic cell type, the activity of Nup107 is necessary? The fact that KD of Nup107 using the tj-Gal4 driver resulted in larval and adult aberrant phenotypes indistinguishable from those induced by the Nup107 loss of function mutation, indicates that the ovarian function of Nup107 is primarily required in the Tj-expressing cells. Notably, the tj-Gal4 driver is not expressed in somatic terminal filament (TF) cells either at larval or adult stages. TF cells together with the CCs constitute the ovarian stem cell niche. During larval ovarian development, Tj is expressed in the somatic support cells, known as intermingled cells (ICs), Cap cells (CCs), and follicle stem cells (FSC) progenitors. CCs, which are derived from the ICs, are formed at the base of fully formed TFs at the transition from the third larval instar to prepupal stage. Therefore, the aberrant primordial germ cells (PGCs) and ICs observed in the larval gonads are not due to impairment of Nup107 or Dsx activities in CCs. Likewise, FSC progenitors are also not the candidate cells for the site of action of Nup107 activity as they reside posterior to ICs with a minimal physical contact with only a few posteriorly located PGCs. Together these data imply that Nup107 acts specifically in ICs enabling them to effectively interact with the PGCs. Consistent with this notion, loss of Nup107 affected the behavior of ICs such that these cells showed varying degrees of failure to mingle with the PGCs. A severe failure of ICs and PGCs to interact in the larval gonad is expected to cause an ovarian-dysgenesis-like phenotype as it is essential for the germarium development and ovariole formation. Likewise, a milder failure of intermingling in the larval gonad would allow for the formation of adult ovaries. However, in the adult ovary Nup107 activity is further required in ECs for the formation of their cellular extensions and regulation of differentiation of the GSCs. Thus, it will be important to determine in future studies the functional relationship between Nup107 and the signaling pathways which previously were shown to regulate the formation of these cellular extensions (Shore, 2022).

These studies have revealed that Nup107, a ubiquitously expressed nuclear envelope protein, is a crucial player during female gonad formation. How does an essential housekeeping protein critical for nuclear transport, perform such a sex- and tissue-specific function(s)? Two possible scenarios, not necessarily mutually exclusive, are envisioned to explain how the specific mutation in Nup107 results in ovary-specific aberrant phenotypes. In the first scenario, Nup107 would specifically mediate nucleocytoplasmic translocation of factor(s) or downstream effector(s) required for ovarian development. Indeed, recent studies have demonstrated that Nup107 is involved in translocation of specific factors. For instance, in the event of DNA damage, Nup107 directly interacts with the apoptotic protease activating factor 1 (Apaf-1 also known as Drosophila ARK) and mediates its transport into the nucleus to elicit cell-cycle arrest. Furthermore, it has been shown in tissue culture that specific Nucleoporins, including Nup107, are required for nuclear translocation of SMAD1, an important downstream effector of the Dpp/BMP pathway (Shore, 2022).

Alternatively, accumulating evidence has documented that in addition to their primary function in regulating the exchange of molecules between the nucleus and cytoplasm, NPC components may contribute to genome organization and tissue-specific regulation of gene expression in a nuclear transport-independent manner. Consequently, such moonlighting activities may not be confined to the nuclear envelope which is the primary native location of these proteins. For instance, mammalian Nup107-160 complex (a subcomplex of the NPC of which Nup107 is a key component) has recently been shown to shuttle in and out of glycine-leucine-phenylalanine-glycine (GLFG) nuclear bodies containing Nup98, a nucleoporin that regulates multiple aspects of gene regulation. Consistently, Nup107 was shown to regulate levels of specific RNAs through gene imprinting. Furthermore, using an RNAi-based assay, Nup107 was identified as a positive regulator of OCT4 and NANOG expression in human ESCs. In this regard, it is noteworthy that the recently published chromatin-binding profile of Nup107 suggested that Nup107 specifically targets active genes (Gozalo, 2020). Altogether these data support the possibility that Nup107 affects transcription of specific target genes in a tissue- and sex-specific manner either directly or indirectly (Shore, 2022)

In Drosophila melanogaster, Sex-lethal (Sxl) is the master determinant of somatic sexual identity, regulating a splicing dependent regulatory cascade resulting in the presence of alternatively spliced sex-specific isoforms of Dsx protein, Dsx-F and Dsx-M, in females and males, respectively. Subsequent dimorphic sexual development including sex-specific gonad morphogenesis is under the control of these Dsx isoforms. Consistently, Dsx proteins deploy components of the housekeeping machinery to achieve sex-specific development of the gonads. Thus, such 'maintenance' factors are unlikely to be involved in any regulatory capacity. These data challenge this notion and demonstrate the presence of sexually dimorphic circuitry downstream of a 'housekeeping' nuclear envelope protein, Nup107, which regulates the expression the female form of dsx (Shore, 2022)

The similar sex-specific and ovary-restricted phenotype associated with compromised Nup107 activity in both humans and flies implies common underlying molecular mechanisms. This study has identified Dsx as the primary target acting downstream of Nup107 in Drosophila ovarian development. The mammalian homologues of Dsx, the Dmrt family of transcription factors, also function during sex-specific gonad development. However, in mammals the main function of Dmrt genes in the gonad is to promote male-specific differentiation. While detailed functional analysis is not available, it is plausible that in mammals, another key female-specific transcription factor, like Foxl2 (female-specific forkhead box L2) may act downstream of Nup107 to substitute for DsxF in flies (Shore, 2022)

The key components of the stem cell niche, that is the hub in males and the TFs in the case of females, have been shown to still be formed in the absence of dsx, but this happens in a stochastic manner in both XX and XY dsx null mutant individuals. These results indicate that in the context of the developing stem cell niche, Dsx may not act in an instructive manner, but is instead required to ensure that the proper program (male or female) is selected, which does not require Dsx activity for the execution of subsequent sex-specific development. Nevertheless, these findings clearly demonstrated that the resulting adult ovaries and testes are improperly formed, consist of aberrant structures, arguing that Dsx activity, is critical for proper gonad development outside of the stem cell niche. The observations of this study are consistent with this suggestion. Moreover, the current and previous experimental strategies and results differ in two important ways. First, the current experiments have relied on reduction of only the female form of dsx that is dsx-F which allowed for sex determination and thus ni 'male' structures or cellular identity transformations were observed. Second, in the curren experiments tj-Gal4 driver was used, which is not expressed in the stem cell niche (TF cells) but in other somatic gonadal cells. This experimental design enabled uncovering of a novel developmental function of Dsx-F in ICs and their adult descendants, ECs. Supporting this notion other studies showed that RNAi KD of dsx also resulted in small ovaries (Shore, 2022)

This study has found that the secreted metalloprotease AdamTS-A is an important downstream component in the Nup107-Dsx axis, as KD of AdamTS-A results in phenotypes similar to those elicited by loss of either Nup107 or dsx. In the adult ovary, these aberrant phenotypes include loss of EC membrane protrusions and expanded BMP signaling. This raised the question of how AdamTS-A regulates the range of BMP signaling. The ECM, which is produced and secreted by cells, has the structure of a complex fibrillar meshwork and provides structural support and tissue integrity, playing an active role in regulating cell behavior. ECM proteoglycans sequester and modulate chemical signals, including growth factors and guidance molecules. Furthermore, type IV collagens, major components of the ECM, were shown to restrict Dpp signaling in the ovary. This is particularly intriguing, since in C. elegans Gon-1, the homolog of AdamTS-A, was shown to genetically interact with a type IV collagen (EMB-9) in the regulation of gonadogenesis. This raised the possibility that AdamTS-A, secreted by ECs, restricts Dpp movement in the germarium through cleavage of ECM components. However, by knocking down coracle in ECs, this study has shown that disruption of their cellular protrusions, which encapsulate the germ cells, leads to expansion of BMP signaling. This implies that the activity provided by these cellular extensions is necessary and sufficient for restricting the BMP signal (Shore, 2022)

Thus, Nup107, Dsx, and AdamTS-A all function in escort cells (ECs) and are necessary for the formation and maintenance of the cellular protrusions which are required for restricting the BMP signal emanating from the GSC's niche. Further, it appears that Adam-TS-A activity in the ECM is required for the formation and/or maintenance of these cellular protrusions. The results indicate that in this context AdamTS-A regulates BMP signal distribution indirectly via regulation of the cellular protrusion maintenance. It is also possible that AdamTS-A utilizes these cellular extensions in order to reach the ECM away from the ECs in the GSC region, where it acts to restrict Dpp trafficking (Shore, 2022)

Overall, these results support a model where Nup107 regulates the expression of dsx, either directly or indirectly, while Dsx directly regulates the transcription of multiple target genes including AdamTS-A. These observations have also uncovered that DsxF controls somatic niche function by calibrating the range and/or strength of Dpp/BMP signaling, possibly via modulation of the level and/or activity of the ECM components. Thus, it will be critical to elucidate how activities of nonsex-specific components such as Nup107 are coordinated with sex-specific regulation to achieve the precise specification and patterning underlying gonad development. This is of particular significance since modulation of BMP signaling circuitry is inextricably linked with the establishment and maintenance of stem cell fate. Importantly, as in the case of Nup107, BMP signaling is also required in a nonsex-specific manner in a variety of developmental contexts.These observations therefore open new avenues toward the critical examination of how a productive molecular dialog is established between nonsex-specific housekeeping machinery and versatile intersecting developmental pathways, in order to ultimately achieve proper sex-specific gonadogenesis crucial for fertility, and transmission of genetic information (Shore, 2022)

For proper function of proteins, their subcellular localization needs to be monitored and regulated in response to the changes in cellular demands. In this regard, dysregulation in the nucleocytoplasmic transport (NCT) of proteins is closely associated with the pathogenesis of various neurodegenerative diseases. However, it remains unclear whether there exists an intrinsic regulatory pathway(s) that controls NCT of proteins either in a commonly shared manner or in a target-selectively different manner. To dissect between these possibilities, this study investigated the molecular mechanism regulating NCT of truncated ataxin-3 (ATXN3) proteins of which genetic mutation leads to a type of polyglutamine (polyQ) diseases, in comparison with that of TDP-43. In Drosophila dendritic arborization (da) neurons, dynamic changes were observed in the subcellular localization of truncated ATXN3 proteins between the nucleus and the cytosol during development. Moreover, ectopic neuronal toxicity was induced by truncated ATXN3 proteins upon their nuclear accumulation. Consistent with a previous study showing intracellular calcium-dependent NCT of TDP-43, NCT of ATXN3 was also regulated by intracellular calcium level and involves Importin α3 (Imp α3). Interestingly, NCT of ATXN3, but not TDP-43, was primarily mediated by CBP. It was further shown that acetyltransferase activity of CBP is important for NCT of ATXN3, which may acetylate Imp α3 to regulate NCT of ATXN3. These findings demonstrate that CBP-dependent acetylation of Imp α3 is crucial for intracellular calcium-dependent NCT of ATXN3 proteins, different from that of TDP-43, in Drosophila neurons (Cho, 2022).

Disruption of the nuclear pore complex (NPC) and nucleocytoplasmic transport (NCT) have been implicated in the pathogenesis of neurodegenerative diseases. A GGGGCC hexanucleotide repeat expansion (HRE) in an intron of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia, but the mechanism by which the HRE disrupts NCT is incompletely understood. This study found that expression of GGGGCC repeats in Drosophila neurons induces proteasome-mediated degradation of select nucleoporins of the NPC. This process requires the Vps4 ATPase and the endosomal-sorting complex required for transport complex-III (ESCRT-III), as knockdown of ESCRT-III/Vps4 genes rescues nucleoporin levels, normalizes NCT, and suppresses GGGGCC-mediated neurodegeneration. GGGGCC expression upregulates nuclear ESCRT-III/Vps4 expression, and expansion microscopy demonstrates that the nucleoporins are translocated into the cytoplasm before undergoing proteasome-mediated degradation. These findings demonstrate a mechanism for nucleoporin degradation and NPC dysfunction in neurodegenerative disease (Dubey, 2022).

Nuclear pore complexes have emerged in recent years as chromatin-binding nuclear scaffolds, able to influence target gene expression. However, how nucleoporins (Nups) exert this control remains poorly understood. This study shows that ectopically tethering Drosophila Nups, especially Sec13, to chromatin is sufficient to induce chromatin decondensation. This decondensation is mediated through chromatin-remodeling complex PBAP (see polybromo), as PBAP is both robustly recruited by Sec13 and required for Sec13-induced decondensation. This phenomenon is not correlated with localization of the target locus to the nuclear periphery, but is correlated with robust recruitment of Nup Elys. Furthermore, this study identified a biochemical interaction between endogenous Sec13 and Elys with PBAP, and a role for endogenous Elys in global as well as gene-specific chromatin decompaction. Together, these findings reveal a functional role and mechanism for specific nuclear pore components in promoting an open chromatin state (Kuhn, 2019).

Functionally, several Nups were found to be required for the transcriptional output and regulation of at least a subset of their target genes. In metazoans, Nup targets include genes important for tissue-specific development, regulation of the cell cycle, and antiviral responses. One conserved regulatory mechanism that requires Nups is transcriptional memory, a process by which genes are marked as recently transcribed to allow more robust transcriptional responses to future activation. Loss-of-function studies have demonstrated that specific Nups are required for multiple molecular steps involved in transcription and transcriptional memory, including binding of poised RNA polymerase (RNAP) II, H3K4 methylation, chromatin remodeling, and formation of activation-induced genomic loops. However, while Nups have been shown to be required for these molecular events, it remains unclear which specific steps of the transcriptional or epigenetic processes Nups are sufficient to induce (Kuhn, 2019).

In Drosophila melanogaster, Nups such as Nup98, Sec13, and Nup62 have been detected at a large number of active genes via DNA adenine methyltransferase identification, chromatin immunoprecipitation (ChIP), and imaging studies. Depletion of Sec13 or Nup98 in fly culture cells or in salivary gland tissues has been shown to lead to more compact chromatin, decreased levels of active RNAP II, and reduced mRNA production at select target genes. Nup98 has been extensively implicated in maintaining transcriptional memory of its target genes in yeast, fly, and mammalian cells, and it has been recently reported that Nup98 is involved in stabilization of enhancer-promoter contacts of ecdysone-inducible genes. However, the molecular functions performed by other transcription-associated Nups such as Sec13 and Nup62 at Nup-chromatin contacts remain unknown. Additionally, many of the Nup-chromatin contacts can occur off-pore in the nuclear interior, as these Nups have been found to shuttle NPCs on and off and/or have distinct intranuclear pools. However, it remains unclear if gene regulatory functions of Nups are independent of nuclear localization (Kuhn, 2019).

To examine these functions and to identify which chromatin- or transcription-associated changes Nups are sufficient to induce, a gain-of-function approach was used. A tethering system was generated to create ectopic chromatin-binding sites of Sec13 and Nup62 in the genome of transgenic Drosophila strains. Using this system, it was observed that NPC component Sec13 consistently induces robust chromatin decondensation at multiple genomic locations. In dissecting the mechanism of this phenomenon, Nup Elys was implicated as the primary mediator of chromatin decompaction, and a robust interaction was identified of Sec13 and Elys with the ATP-dependent chromatin-remodeling complex polybromo-containing Brahma (Brm)-associated proteins (PBAP), as well as a role of Elys in endogenous chromatin decondensation. These findings suggest that promoting chromatin decondensation is a critical and previously underappreciated molecular function of specific Nups in the process of gene regulation (Kuhn, 2019).

The presented findings, combined with previous findings in the field demonstrating functional roles for Nups in regulating gene expression, lead to a model whereby certain Nups primarily influence chromatin state, which in turn can affect downstream gene expression. It is proposed that chromatin-bound Nups, such as Elys and Sec13, recruit factors associated with formation of open chromatin, specifically GAF and components of PBAP. This results in a permissive, open-chromatin state, which, in the right cellular contexts, may allow for binding of appropriate transcription factors, for RNAP II recruitment, and subsequently in an increase in downstream gene expression. Together, these results and model suggest a specific chromatin-decondensing function of certain Nups, particularly Elys, as an early step in the process of gene activation (Kuhn, 2019).

Evidence is provided that Nups facilitate chromatin decondensation. The resulting 'holes' in DNA stain that appear upon Nup tethering and the associated loss of IF signal from antibodies against both core histone H3 and histone modifications favor this notion. This interpretation is further supported by the observed recruitment and functional involvement of the chromatin-remodeling PBAP complex and by additional biochemical data showing global and gene-specific defects in nucleosome occupancy upon Elys depletion. Furthermore, the robust biochemical interaction between Nups and components of PBAP and the correlation between the amount of Brm recruitment by Nups and the level of observed decondensation at lacO 96C further suggest that Nups have the capacity to promote chromatin decondensation. One interesting outstanding question is whether these Nup-induced changes in chromatin structure can occur entirely de novo (or rapidly after Nup binding) or require the process of chromatin assembly during replication to take effect. The current experiments have not differentiated between these possibilities. Further experiments, perhaps in blocking replication and assaying for similar Nup functions, could differentiate between these mechanisms further. Regardless, the findings strongly support the function of Nups in regulating compaction states of chromatin, while the particular cell cycle stage and the dynamic time frame at which this process takes place remain to be elucidated (Kuhn, 2019).

As transcription and chromatin decompaction are intimately intertwined, it was of interest to know if the Nup-induced changes in chromatin were primary or secondary to transcriptional regulation. Increased transcription of the gene directly downstream from the lacO 96C integration, dan, was observed upon tethering of Sec13, which also promoted decondensation here. However, since Brm recruitment and chromatin decondensation appear to be much more robustly detected than the presence of RNAP II upon Sec13 tethering, it is believed that decondensation is likely the primary effect of Sec13 tethering, and increased gene expression a secondary consequence. This is supported by the fact that Nup62 is able to induce a small amount of detectable decondensation at 96C lacO, associated with low-level recruitment of Elys and Brm, but does not result in significant levels of RNAP II recruitment. Further evidence that the primary effect of Nup targeting is decondensation rather than transcriptional activation is the increased nucleosome occupancy at both Hph and B52 genes upon Elys knockdown, but a transcriptional change is detected only at Hph. The differential effect of Elys depletion on Hph and B52 transcription again suggests that the primary chromatin-associated role of certain Nups is to facilitate the step of chromatin decondensation (Kuhn, 2019).

Although this study found tethering of Sec13 to elicit chromatin decondensation in the ectopic context, the data suggest that Elys may be the Nup primarily responsible for facilitating decondensation. As discussed above, there is a striking correlation between levels of Elys recruitment and level of decondensation at multiple lacO loci, and endogenous Elys appears to interact much more robustly with components of PBAP in S2 cells than Sec13. Significantly, Elys depletion shows a defect in global genomic MNase digestion, whereas Sec13 depletion does not. The latter experiment also suggests that the role of Elys in chromatin decondensation is independent of NPC integrity, as both Elys and Sec13 (which is a core component of the Nup107-Nup160 complex) are required for nuclear pore assembly. Therefore, a lack of phenotype of Sec13 RNAi in the MNase assay suggests that the observed reduction in nucleosomal accessibility in Elys RNAi conditions does not stem from a defect in NPC assembly. This conclusion is supported by the previously published observation that inhibiting transport capabilities of the NPC with WGA treatment does not lead to chromatin decondensation defects. It is further hypothesize that since Elys exhibits a particularly robust genome-wide binding while Sec13 appears to bind fewer loci, Elys has a stronger and more detectable effect on global chromatin decompaction. It remains to be determined whether Sec13 and Elys share a subset of target genes, and whether chromatin-bound Sec13 co-functions with Elys in chromatin decompaction of such targets (Kuhn, 2019).

The data presented in this study provide functional and mechanistic evidence for the long-standing visual correlation between NPCs and open chromatin and validates the hypothesized relationship between them. Interestingly, previous genetic and proteomic experiments have reported interactions between the Caenorhabditis elegans homologue of Elys, MEL-28, and chromatin-remodeling complexes, including the SWI/SNF complex subunit SWSN-2.2, suggesting an evolutionarily conserved role for Elys in regulating chromatin state. Furthermore, genetic and physical interactions between yeast NPC components and the chromatin-remodeling RSC complex have also been reported. Elys is known to bind condensed postmitotic chromatin to nucleate NPC assembly during nuclear envelope reformation, and recent work has reported a defect in global postmitotic chromatin decompaction associated with depletion of Elys from chromatin (Aze, 2017). Thus, an intriguing possibility is that in addition to NPC assembly, postmitotic chromatin binding of Elys may also play a role in postmitotic chromatin decompaction through mechanisms similar to those described in this study. A role for Nups in facilitating the formation or maintenance of open chromatin is also consistent with the evolutionarily conserved phenomenon of viral genome integration into open/active chromatin regions that are associated with NPCs. Finally, the interaction of Nups with developmentally critical GAF and PBAP suggests that this relationship may be relevant to the establishment of tissue-specific open chromatin regions or the global genome decompaction during organismal development. It is possible that the potential role of Elys and possibly other Nups in postmitotic chromatin decondensation has extended to regulation of chromatin structure in the context of interphase transcription, thus contributing to regulation of developmental transcriptional programs (Kuhn, 2019).

Mitotic spindles are composed of microtubules (MTs) that must nucleate at the right place and time. Ran regulates this process by directly controlling the release of spindle assembly factors (SAFs) from nucleocytoplasmic shuttle proteins importin-αβ and subsequently forms a biochemical gradient of SAFs localized around chromosomes. The majority of spindle MTs are generated by branching MT nucleation, which has been shown to require an eight-subunit protein complex known as augmin. In Xenopus laevis, Ran can control branching through a canonical SAF, TPX2, which is non-essential in Drosophila melanogaster embryos and HeLa cells. Thus, how Ran regulates branching MT nucleation when TPX2 is not required remains unknown. This study used in vitro pulldowns and TIRF microscopy to show that augmin is a Ran-regulated SAF. Augmin was shown to directly interact with both importin-α and importin-&beta; through two nuclear localization sequences on the Haus8 subunit, which overlap with the MT binding site. Moreover, Ran was shown to control localization of augmin to MTs in both Xenopus egg extract and in vitro. These results demonstrate that RanGTP directly regulates augmin, which establishes a new way by which Ran controls branching MT nucleation and spindle assembly both in the absence and presence of TPX2 (Kraus, 2023).

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