With the exception of bacteria, living cells contain most of their DNA inside a structure called the nucleus. The membranes of the nucleus form a protective wall around the DNA, while pores within this wall act as entry check-points, controlling what can and cannot get inside. Maintaining the structure of this wall is critical for cell survival. Problems can occur if the nuclear wall or its pores become disrupted, as in the case of cancer and neurodegenerative diseases. Thankfully cells have developed a protective surveillance system that can rapidly identify and repair any damage made to the nuclear wall. However, how this damage is found and what activates its repair is poorly understood.

Now, Thaller et al. have investigated two key proteins that they suspected were involved in the surveillance of the nuclear border in budding yeast: Chm7 and Heh1. Chm7 is part of a complex group of proteins that can cut and sculpt the shape of membranes, while Heh1 is normally embedded on the inside of the nucleus. Thaller et al. discovered that, when the nuclear wall is disrupted, Heh1 recruits Chm7 to the site of damage and activates it. Once activated Chm7 can repair the damage to the nuclear wall, by sealing over defective nuclear pores and closing gaps caused by breakages.

Thaller et al. showed that the transport system that normally moves molecules into and out of the nucleus also imports Heh1 and actively excludes Chm7, physically segregating them to opposite sides of the nuclear border. If the nuclear wall becomes damaged this leads to the local meeting of Heh1 and Chm7 at these sites. Heh1 will then activate the membrane shaping mechanisms of Chm7, rapidly repairing the nuclear border in response to the damage.

It is possible cell structures other than the nucleus use a similar surveillance system to protect their borders. Manipulating the border surveillance system of the nucleus could be used to treat the detrimental impacts of damage caused to the nuclear wall by disease.

The molecular machinery that biochemically segregates the nucleus and the cytoplasm has been extensively investigated. The foundational components of this selective barrier include the double-membrane nuclear envelope with embedded nuclear pore complexes (NPCs). NPCs impose a ‘soft’ diffusion barrier to macromolecules larger than ~40 kD (Popken et al., 2015; Timney et al., 2016) while providing binding sites for the rapid and selective transport of signal-bearing (nuclear localization and nuclear export signals; NLSs and NESs) macromolecules, which are ferried through the NPC by shuttling nuclear transport receptors (NTRs; a.k.a. karyopherins/importins/exportins; Schmidt and Görlich, 2016). Directionality and energy for NTR-selective transport is imparted by the spatial segregation of the Ran-GTPase whose nuclear GTP-bound form destabilizes and stabilizes import and export complexes, respectively (Floch et al., 2014).

Interestingly, the robustness of the nuclear envelope barrier has been shown to be compromised in several different contexts, including in diverse human diseases (Hatch and Hetzer, 2014; Lusk and King, 2017). For example, there is an emerging body of work linking the function of NTRs and NPCs with neurodegenerative diseases like ALS and FTD (Nousiainen et al., 2008; Freibaum et al., 2015; Jovičić et al., 2015; Kaneb et al., 2015; Zhang et al., 2015; Kim and Taylor, 2017; Shi et al., 2017). These studies, coupled to the observations of age-related declines in NPC function in both post-mitotic multicellular systems (D'Angelo et al., 2009; Savas et al., 2012; Toyama et al., 2013) and also in replicative aging models like budding yeast (Janssens et al., 2015; Lord et al., 2015), support a theme in which the function of the nuclear envelope could be mitigatory of age-related disease progression (Schreiber and Kennedy, 2013; Jevtić et al., 2014; Serebryannyy and Misteli, 2018). Of similar interest, the hallmark cellular pathophysiology of early-onset dystonia are nuclear envelope herniations (Goodchild et al., 2005) that emanate from NPC-like structures (Laudermilch et al., 2016). As analogous herniations have been observed in many genetic backgrounds associated with defects in NPC biogenesis in yeast over several decades (Thaller and Lusk, 2018), this has contributed to the idea that the herniations might be the result of either defective NPC assembly events (Scarcelli et al., 2007; Onischenko et al., 2017; Zhang et al., 2018) and/or the triggering of a NPC (Wente and Blobel, 1993) or NPC assembly quality control pathway (Webster et al., 2014; Webster et al., 2016). The latter could depend on the function of the endosomal sorting complexes required for transport (ESCRT), a membrane scission machinery that has been proposed to seal-off malforming NPCs (Webster et al., 2016).

That there could be mechanisms to surveil the assembly of NPCs makes considerable sense as there are hundreds of NPCs, each containing hundreds of nucleoporins/nups (Kosinski et al., 2016; Kim et al., 2018), that are assembled during interphase in mammalian cells (Maul et al., 1972; Doucet et al., 2010; Dultz and Ellenberg, 2010). There are approximately 100 NPCs formed during a budding yeast cell cycle, which includes a closed mitosis (Winey et al., 1997). As interphase NPC assembly likely occurs through an inside-out evagination of the inner nuclear membrane (INM) followed by membrane fusion with the outer nuclear membrane (ONM) (Otsuka et al., 2016), holes are constantly being formed in the nuclear envelope. Without mechanisms to surveil this process, de novo NPC biogenesis might pose a threat to nuclear-cytoplasmic compartmentalization (Webster et al., 2014). Consistent with this idea, malformed or damaged NPCs are not passed on to daughter cells in budding yeast (Colombi et al., 2013; Makio et al., 2013; Webster et al., 2014). Further, deletion of the ESCRT machinery in the context of genetic backgrounds where nuclear envelope herniations have been observed for example nup116Δ (Wente and Blobel, 1993) or apq12Δ (Scarcelli et al., 2007) cells require a nuclear envelope-specific ESCRT, Chm7 (the orthologue of mammalian CHMP7), for viability (Bauer et al., 2015; Webster et al., 2016). While we have previously proposed that a biochemical signature of malforming NPCs is surveilled by integral inner nuclear membrane proteins of the Lap2-emerin-MAN1 (LEM) domain family, specifically Heh2, it remains to be formally established what the signal that leads to ESCRT recruitment to the nuclear envelope actually comprises (Webster et al., 2014).

Evidence that the ESCRT machinery acts at holes in the nuclear envelope is further exemplified by their critical role in performing annular fusion events during the final stages of nuclear envelope reformation at the end of mitosis in mammalian cells (Olmos et al., 2015; Olmos et al., 2016; Vietri et al., 2015; Gu et al., 2017; Ventimiglia et al., 2018). Moreover, ESCRTs are also required for the efficient repair of nuclear ruptures that arise during the migration of cells through tight constrictions (Denais et al., 2016; Raab et al., 2016). And, it is most likely that they also act to repair nuclear envelope ruptures that are induced by intracellular mechanical stresses from either the actin cytoskeleton (Hatch and Hetzer, 2016; Robijns et al., 2016), or from those observed during telomere crisis (Maciejowski et al., 2015). Lastly, recent work also suggests a role for ESCRTs in the context of turning over NPCs in quiescent cells (Toyama et al., 2019). It remains an open question, however, whether the mechanisms that repair nuclear ruptures, seal the nuclear envelope at the end of mitosis, and protect against defective NPC assembly respond to an identical upstream signal and proceed through the same membrane-sealing mechanism.

Clues to what might constitute the upstream signal that leads to nuclear envelope-recruitment of ESCRTs could be drawn from other contexts where ESCRTs protect membrane compartments including endolysosomes (Radulovic et al., 2018; Skowyra et al., 2018) and the plasma membrane (Jimenez et al., 2014; Scheffer et al., 2014; Gong et al., 2017). In both of these cases, there is evidence to suggest that the local release of Ca²⁺ is a trigger for ESCRT recruitment, through (at least at the plasma membrane) a Ca²⁺ binding protein, ALG-2 (Jimenez et al., 2014; Gong et al., 2017). Whether Ca²⁺ plays a role at the nuclear envelope remains unaddressed. More generally, there are two, often redundant, recruitment mechanisms seeded by either an ESCRT-I, II complex and/or ESCRT-II and ALIX (Bro1 in yeast) that bind and activate ESCRT-III subunit polymerization (Wemmer et al., 2011; Henne et al., 2012; Tang et al., 2015; Tang et al., 2016; Christ et al., 2016) on specific membranes throughout the cell (reviewed in Schöneberg et al., 2017; McCullough et al., 2018).

ESCRT-III polymers predominantly made up of the most abundant ESCRT-III (Snf7/CHMP4B) scaffold negative but also in at least one case, positive membrane curvature (McCullough et al., 2015), and directly contribute to membrane scission (Adell et al., 2014; Adell et al., 2017; Schöneberg et al., 2018). The AAA +ATPase Vps4 disassembles ESCRT-III filaments by directly interacting with MIM (MIT interacting motif) domains present on a subset of ESCRT-III subunits (Obita et al., 2007; Stuchell-Brereton et al., 2007; Agromayor et al., 2009; Xiao et al., 2009; Han et al., 2015) by threading the ESCRT-III filaments through the central cavity of a hexameric ring (Yang et al., 2015; Han et al., 2017; Monroe et al., 2017; Su et al., 2017). It is likely that ESCRT-III disassembly by Vps4 directly contributes force to promote membrane scission (Schöneberg et al., 2018). Whether the membrane scission reaction is different in distinct subcellular contexts like at the nuclear envelope remains to be understood.

Consistent with the idea that there might be unique ESCRT membrane remodeling mechanisms at play in distinct compartments, a step-wise recruitment and activation mechanism requiring the ESCRT-II Vps25 and the ESCRT-III Vps20 is thought to be required at budding yeast endosomes (Saksena et al., 2009; Teis et al., 2010; Tang et al., 2015; Tang et al., 2016), but both of these proteins are absent from the genetic and biochemical analyses of the nuclear envelope arm of the ESCRT pathway (Webster et al., 2014; Webster et al., 2016). These data suggest that other proteins likely contribute to ESCRT-III activation at the nuclear envelope. Key candidates are Chm7 and the inner nuclear membrane (INM) proteins, Heh1/Src1 (orthologue of LEM2) and Heh2 (orthologue of MAN1 or other LEM-domain proteins). These proteins have collectively been shown to interact biochemically and genetically with Snf7 (Webster et al., 2014; Webster et al., 2016) and Heh1 is required for the focal accumulation of Chm7 at the nuclear envelope in genetic backgrounds where NPC assembly is inhibited (Webster et al., 2016). Remarkably, the interactions between Heh1 and Chm7 are well conserved in both fission yeast (Gu et al., 2017) but also in mammalian cells, where LEM2 is required to recruit CHMP7 to the reforming nuclear envelope at the end of mitosis (Gu et al., 2017). It remains to be understood, however, whether LEM proteins (or Chm7) directly contribute to ESCRT-III activation at the nuclear envelope or whether additional proteins are involved.

Heh1 and Heh2 contain an N-terminal helix-extension-helix (heh) motif (the LEM domain), followed by an INM targeting sequence that (at least in the case of Heh2 but likely also Heh1 [King et al., 2006; Lokareddy et al., 2015]) includes an NLS and a ~ 200 amino acid unstructured region that are both required for INM targeting (Meinema et al., 2011). They both also contain a second nuclear-oriented domain, which likely folds into a winged helix (WH) (also called MAN1/Src1-C-terminal homology domain or MSC; Caputo et al., 2006); this domain is also well conserved through evolution (Mans et al., 2004; Mekhail et al., 2008). The LEM domain proteins as a family have been ascribed diverse roles in gene expression either through binding to transcription factors, BAF, or the lamins (Barton et al., 2015). While yeasts lack BAF and lamins, the LEM domain proteins nonetheless directly interface with chromatin (Grund et al., 2008; Barton et al., 2015), contribute to rDNA repeat stability (Mekhail et al., 2008) and the mechanical robustness of the nucleus (Schreiner et al., 2015). This latter function is likely revealed by the observation in many diverse yeast species that loss of Heh1 leads to nuclear envelope disruption (Yewdell et al., 2011; Gonzalez et al., 2012; Yam et al., 2013), however, it might also reflect Heh1’s role in recruiting ESCRTs to the nuclear envelope. Thus, the relationship between how the LEM proteins contribute to nuclear integrity and ESCRT-mediated surveillance remains to be clearly defined.

In the following, we further explore the molecular determinants of Chm7 recruitment to the budding yeast nuclear envelope by Heh1. We determine that the spatial segregation of Chm7 and Heh1 on either side of the nuclear envelope is driven by NTRs and the robustness of the nuclear transport system. Perturbations of this system, or the exposure of Heh1 to the cytosol leads to the local recruitment and Heh1 WH-dependent activation of Chm7. At sites of Chm7 hyperactivation, we observe remarkable alterations to nuclear envelope morphology including nuclear envelope herniations and intranuclear INM invaginations suggesting a role for membrane expansion and remodeling during nuclear envelope repair.

While there has been considerable focus over the last few decades on mechanisms that control the targeting of proteins and lipids to distinct intracellular compartments, it is equally important to understand the protective mechanisms that maintain this compartmentalization in the face of challenges to membrane integrity and/or the specific biochemical identity of organelles. Here, we further explore the mechanism of ESCRT surveillance of the nuclear envelope. We interpret our data in a model where the nuclear envelope is surveilled by two principle components, the ESCRT Chm7 and the integral INM protein, Heh1. This surveillance system appears to be set up to respond directly to perturbations in the nuclear envelope barrier in a way that we suggest is agnostic as to whether the perturbation is a result of defectively formed NPCs or a mechanical (or other) disruption of the nuclear membranes.

The rationale behind this assertion is that the nuclear envelope surveillance system is itself directly established by a functioning nuclear transport system, which physically segregates Chm7 and Heh1 on either side of the nuclear envelope. For example, prior work has shown that Heh1 requires the function of the NTRs Kap-α and Kap-β1 in addition to the Ran-GTPase in order to be actively targeted to the INM through NPCs (King et al., 2006). Here, we establish that Chm7, while small enough to passively leak through the NPC diffusion barrier, is actively exported by the major export NTR, Xpo1 (Figure 1). Therefore, any perturbations that impact active nuclear transport or the diffusion barrier across the nuclear envelope would lead to increased Chm7 diffusion into the nucleus and/or a deficit in its nuclear export increasing the likelihood that it meets Heh1. While similar perturbations could also lead to Heh1 mistargeting and/or its diffusion into the ONM, we suspect that this would be kinetically slower than Chm7 diffusion into the nucleus as Heh1 is bound to chromatin at the INM (Grund et al., 2008; Mekhail et al., 2008; Gonzalez et al., 2012; Yam et al., 2013; Barton et al., 2015; Schreiner et al., 2015). Indeed, it is probable that Heh1 functions in two major roles with respect to nuclear integrity: first, it provides mechanical stability to the nucleus by binding chromatin, and second, provides a binding site for Chm7 through its C-terminal WH domain. These two inter-related roles could also help to explain why both the N- and C-terminal domains of Heh1 are required to maintain viability of apq12Δ cells (Figure 2C), and why HEH1 is generally more essential in budding and fission yeasts compared to CHM7. This likely holds true in mammalian models as well as LEM2 is essential whereas CHMP7 is dispensable for viability (in cell culture) (Hart et al., 2015).

Once a perturbation occurs to the nuclear envelope barrier through defects in NPC assembly, loss of function of NPCs, or mechanical disruption of the nuclear membranes, Chm7 and Heh1 are able to come together. While we have so far been unsuccessful in reconstituting a direct biochemical interaction between Chm7 and Heh1 (although others have between LEM2 and CHMP7 [Gu et al., 2017]), we have nonetheless provided evidence that this interaction likely leads to Chm7 activation, thus tightly coupling recruitment and activation (Figure 4). Our data support a model in which the C-terminal WH domain of Heh1 in addition to a membrane anchor are necessary and sufficient for this activation event. As our prior work (Webster et al., 2016) and that from Olmos et al. (2016), support that it is the N-terminal ESCRT-II-like domain of Chm7 (which, interestingly, is also predicted to be made up of tandem WH domains [Horii et al., 2006; Bauer et al., 2015; Figure 1A]) that is necessary for recruitment to the nuclear envelope, it seems likely that the binding between the Heh1-WH domain and the N-terminus of Chm7 could trigger activation by removing some form of autoinhibition, which is a common theme among ESCRT-III proteins (Zamborlini et al., 2006; Shim et al., 2007; Lata et al., 2008; Bajorek et al., 2009; Henne et al., 2012; Tang et al., 2015; Tang et al., 2016) that ensures polymerization in the correct compartment. Clearly, the precise molecular mechanism of Chm7 activation by Heh1 will require structural insight, which is no doubt on the horizon.

Even with structural information, there are many additional factors that need to incorporated into a nuclear envelope surveillance mechanism. For example, future work must directly address how Heh2 fits into this pathway. While Heh2 has a similar domain architecture as Heh1 including a WH domain (King et al., 2006), it does not appear to impact Chm7 activation within the FETA assay (Figure 5—figure supplement 1D), nor was it detected in any of the affinity purifications (Figure 3). This was surprising, as we had previously established that the N-terminus of Heh2 (that lacks the WH domain), can directly bind to both Chm7 and Snf7, at least in their ‘open’ forms (Webster et al., 2016). Moreover, we reported physical interactions between Chm7 and Snf7 with Heh2 in vivo (Webster et al., 2014; Webster et al., 2016). While there are many possible explanations for these results, we favor models that consider Heh2 in a regulatory role that might modulate Chm7 and/or Snf7 function at the nuclear envelope, although this remains speculative and awaits direct experimentation.

Additional downstream (of Chm7 and Heh1) components also need to be figured into any nuclear envelope surveillance mechanism including Snf7, but also Did2, Vps24 and Vps2 (Figure 3B,C). That these ESCRT-IIIs impact the recruitment of Vps4 to the nuclear envelope (Figure 3E) raises the possibility that the sequence of their recruitment might be similar to that found at ILVs during MVB formation, which begins with Snf7 and ends with Did2, Vps24, and Vps2 (Babst et al., 2002; Teis et al., 2008), the latter of which play important roles in Vps4 recruitment and ATPase activation required for membrane scission (Azmi et al., 2008; Shestakova et al., 2010; Schöneberg et al., 2018). Interestingly, however, while we observe Vps4 recruitment to sites of chm7_OPEN at the INM (Figure 3E), we do not have any explicit evidence for membrane scission per se. Indeed, the INM evaginations and nuclear envelope herniations appear to be stabilized by ~45 nm diameter necks (Figures 6, 7 and 8B). That these necks have similar diameters to those observed at the plasma membrane or endosomes when HIV budding (von Schwedler et al., 2003; Morita et al., 2011; Cashikar et al., 2014; Jackson et al., 2017) or ILV formation (Adell et al., 2014; Buono et al., 2017; Frankel et al., 2017; Wenzel et al., 2018), respectively, are stalled, is suggestive that they are stabilized by a similar ESCRT-III-like polymer. We suggest that the most likely explanation is that Chm7 itself might play a unique role in a nuclear membrane-scission step, perhaps through its potential MIM1 domain that might directly recruit Vps4 but is absent from the chm7_OPEN construct. Such a conclusion is supported by the presence of similar INM evaginations and nuclear envelope herniations observed in the presence of full length Chm7 but lacking Vps4 (Figure 7).

Any scission mechanism that requires a direct interaction between the MIM1 domain of Chm7 and Vps4 will also need to consider the overlap of this sequence with NES2 (Figure 1). This clearly predicts a competition between Xpo1 and Vps4 for Chm7, which provides a potential entryway for an additional level of regulation for any polymer formation or scission reaction by this NTR (that would also be modulated by Ran-GTP). Indeed, NTRs might regulate this pathway at the level of Heh1 as well. Heh1 is synthesized and inserted into ER membranes so is exposed to the cytosol and would thus be capable of binding to Chm7 before it is targeted to the INM. Such an assumption is supported by the observation that the exposure of the WH domain of Heh1 is sufficient to recruit Chm7 to ER membranes (Figure 2). Does NTR-binding to the NLSs in the N-terminus of Heh1 inhibit Chm7 recruitment in this compartment? Answering this question will also be relevant in mammalian systems where LEM2 and CHMP7 are found in the same compartment during mitotic nuclear envelope breakdown suggesting the need for mechanisms to prevent their binding in this phase of the cell cycle. Interestingly, recent work is suggesting that proteins like Lgd/CC2D1B help control the spatiotemporal timing of CHMP4B and CHMP7 activity in mammalian cells (Ventimiglia et al., 2018), supporting the existence of regulatory mechanisms of this pathway.

Lgd/CC2D18 acts during nuclear envelope reformation when ESCRTs play critical roles in sealing the nuclear envelope at the end of mitosis, often at sites where spindle microtubules perforate the nascent nuclear envelope (Olmos et al., 2015; Olmos et al., 2016; Vietri et al., 2015). It will be interesting to understand the similarities and differences between how ESCRTs function at the end of mitosis (in an open mitosis) versus events that are triggered upon acute disruption of nuclear envelope integrity or NPC function during interphase. For example, two of the more interesting observations of the EM tomographic analyses are the generation of intranuclear membrane invaginations and the proximity of ER sheets and vesicles at the ONM sites of nuclear envelope herniations (Figure 6, Figure 6—figure supplement 1, Figure 7). Both of these observations suggest that nuclear envelope surveillance might be coupled to the delivery of new membrane at sites of rupture. It seems plausible that sealing holes in the nuclear envelope (particularly those larger than a nuclear pore) would require the local delivery of membranes. Similarly, NPC quality control mechanisms that suggest the sealing of defective NPCs would likely require some form of expansion of the pore membrane, as has been proposed (Wente and Blobel, 1993). Whether such membrane is derived from new synthesis or from the mobilization of existing stores remains to be explored. Of note, recent work supports that even the INM may be metabolically active and have the capacity to generate new lipid locally (Romanauska and Köhler, 2018). Further, close examination of sites of chm7_OPEN accumulation reveals that the morphology of the INM extensions are both sheet-like and tubular in nature and resemble the ‘normal’ continuities between the ONM and the broader ER network (West et al., 2011); together these data suggest that ER-shaping proteins and lipid synthesis pathways might also play an important role in contributing to nuclear envelope sealing - topics for the future.

Both ER remodeling proteins and lipid synthesis pathways are required for de novo NPC biogenesis (Schneiter et al., 1996; Dawson et al., 2009; Hodge et al., 2010). As NPC biogenesis proceeds through an INM evagination step, we also must consider whether the INM evaginations observed in the electron tomograms reflect a function for Chm7 in this process. However, while this remains a compelling hypothesis, our data do not yet support this idea. For example, the herniations observed in the context of inhibiting NPC assembly are morphologically distinct from those found in the chm7_OPEN or vps4Δpom152Δ scenarios with wider necks that contain nups (Figure 8A,B). Further, they arise in the absence of CHM7 and are thus more likely to be formed due to a defect in NPC biogenesis (perhaps because of an inhibition of INM-ONM fusion), or, through the triggering of a NPC assembly surveillance mechanism (Webster et al., 2016; Thaller and Lusk, 2018). Should Chm7 be a critical component of the latter mechanism, it would be predicted that the nuclear envelope herniations associated with nups would be unsealed without CHM7. Indeed, that we observe often-dramatic openings in the nuclear envelope in apq12Δchm7Δ cells (Figure 8D and Figure 8—figure supplement 2), suggest that the herniations themselves might be prone to rupture with Chm7 required for their repair. It follows then that like in mammalian cells where much larger nuclear envelope herniations are precursors to nuclear rupture (De Vos et al., 2011; Vargas et al., 2012; Hatch et al., 2013; Denais et al., 2016; Hatch and Hetzer, 2016; Raab et al., 2016), these smaller NPC-assembly associated herniations might also impact nuclear envelope integrity through mechanisms that remain to be fully defined. In either case, it reinforces the concept that the assembly of NPCs can be perilous, and it will be important to consider this possibility when interpreting the underlying pathology of human diseases that are associated with defects in NPC function or assembly, for example, DYT1 early-onset dystonia (Laudermilch et al., 2016; Pappas et al., 2018) or Steroid Resistant Nephrotic Syndrome (Miyake et al., 2015; Braun et al., 2016; Braun et al., 2018).

Lastly, while we acknowledge then that the INM evaginations that we observe in cases of Chm7 hyperactivation might not necessarily be a physiological event in a nuclear envelope sealing process, it remains tempting to speculate that they might be in the context of proposed mechanisms of nuclear egress be it Mega-RNPs (Speese et al., 2012; Jokhi et al., 2013), viruses (Lee et al., 2012; Lee et al., 2016; Arii et al., 2018), or nucleophagy (Roberts et al., 2003; Dou et al., 2015; Mochida et al., 2015; Mostofa et al., 2018) that so-far remain obscure but would nonetheless require a membrane scission step. Interestingly, recent work suggests that herpes virus nuclear egress requires ESCRTs (Arii et al., 2018), including a role in controlling INM extensions into the nucleus; such intranuclear membrane might also be relevant in cell types that have so-called ‘nucleoplasmic reticulum’ (Malhas et al., 2011). The biogenesis and function of nucleoplasmic reticulum remain enigmatic but our observations of intranuclear fenestrated membrane emanating from the INM might suggest a yet-to-be discovered role for Chm7 and Heh1 in forming such structures as well.

All data generated and analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figure 3.

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Author details

1. David J Thaller

   Department of Cell Biology, Yale School of Medicine, New Haven, United States

   Contribution

   Conceptualization, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3577-5562

2. Matteo Allegretti

   Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse, Germany

   Contribution

   Formal analysis, Validation, Investigation, Visualization, Writing—review and editing

   Competing interests

   No competing interests declared

3. Sapan Borah

   Department of Cell Biology, Yale School of Medicine, New Haven, United States

   Contribution

   Investigation, Visualization, Writing—review and editing

   Competing interests

   No competing interests declared

4. Paolo Ronchi

   Electron Microscopy Core Facility, European Molecular Biology Laboratory, Meyerhofstrasse, Germany

   Contribution

   Resources, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

5. Martin Beck

   Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse, Germany

   Contribution

   Resources, Supervision, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7397-1321

6. C Patrick Lusk

   Department of Cell Biology, Yale School of Medicine, New Haven, United States

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   patrick.lusk@yale.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4703-0533