Mechanisms for assembly of the nucleoplasmic reticulum

Abstract

The nuclear envelope consists of an outer membrane connected to the endoplasmic reticulum, an inner membrane facing the nucleoplasm and a perinuclear space separating the two bilayers. The inner and outer nuclear membranes are physically connected at nuclear pore complexes that mediate selective communication and transfer of materials between the cytoplasm and nucleus. The spherical shape of the nuclear envelope is maintained by counterbalancing internal and external forces applied by cyto- and nucleo-skeletal networks, and the nuclear lamina and chromatin that underly the inner nuclear membrane. Despite its apparent rigidity, the nuclear envelope can invaginate to form an intranuclear membrane network termed the nucleoplasmic reticulum (NR) consisting of Type-I NR contiguous with the inner nuclear membrane and Type-II NR containing both the inner and outer nuclear membranes. The NR extends deep into the nuclear interior potentially facilitating communication and exchanges between the nuclear interior and the cytoplasm. This review details the evidence that NR intrusions that regulate cytoplasmic communication and genome maintenance are the result of a dynamic interplay between membrane biogenesis and remodelling, and physical forces exerted on the nuclear lamina derived from the cyto- and nucleo-skeletal networks.

Introduction

The nuclear envelope (NE) is composed of an outer nuclear membrane (ONM) contacting the cytoplasm and an inner nuclear membrane (INM) that faces the nucleoplasm (Fig. 1). Narrow membrane junctions physically connect the ONM and endoplasmic reticulum (ER), while connections between the ONM and INM occur at the perimeter of nuclear pore complexes (NPCs), the selective portals that facilitate protein and nucleic acid transport between the cytoplasm and nucleoplasm [1]. The INM has a unique repertoire of proteins involved in nuclear function, notably an underlying lamina network composed of Lamin A and B intermediate filament proteins and their interacting partners that regulate genomic organization and expression [2] (Fig. 1). The perinuclear space between the ONM and INM is a continuum of the ER lumen and thus contains chaperones, calcium regulatory proteins and secretory factors. In addition, the INM and ONM are bridged by linkers of the nucleoskeleton to the cytoskeleton (LINC) complexes composed of Sad1 and UNC84 Domain containing (SUN) proteins in the INM that interact with the lamina and KASH domain-containing nesprins in the ONM [3]. The LINC complex is central to mechano-transduction; the process by which signals related to extracellular matrix stiffness and cell adhesion induce changes in gene expression and NE morphology to respond appropriately to mechanical cues from the surrounding environment [4].

Fig. 1.

Type-I and Type-II nucleoplasmic reticulum. The Type-I NR are formed by invaginations of the INM at sites that are relatively lamina deficient, include the perinuclear space and are devoid of NPCs. The Type-II NR are invaginations of both the INM and ONM, have a cytoplasmic core as well as proteins associated with the nuclear envelope, such as lamins, LINC complexes and NPCs. Figure created using BioRender.com

The NE has a relatively planar surface but with regions of high membrane curvature at NE-ER junctions [5], and at the perimeter of NPCs where the ONM and INM fuse [6] (Fig. 1). These highly curved membrane contacts between the ER, ONM and INM are constriction points that regulate the exchange of proteins and lipids that define the unique composition and function of the three compartments (reviewed in [7, 8]). In addition to these membrane connections within the NE, ultrastructure analysis using 3D confocal, 2-photon and transmission electron microscopy revealed the presence of NE invaginations that form an extensive tubule membrane network throughout the interior of the nucleus of many cells and tissues [9–13]. These nuclear membrane invaginations, termed the nucleoplasmic reticulum (NR) due to similarities to the tubular ER [9], are subdivided into Type-II NR formed by invagination of both nuclear membranes and the Type-I NR invaginations of the INM [14] (Fig. 1). Type-II NR includes the perinuclear space, NPCs that connect the INM and ONM, nuclear lamina, and a cytoplasmic core that is frequently large enough to accommodate organelles, including mitochondria, vesicles, lipid droplets and cytoskeletal filaments, [10, 15]. Type-I NR is relatively lamin-deficient, lacks a cytoplasmic core and NPCs, and extends both the INM and perinuclear space into the nuclear interior [10, 14]. The NR has a high degree of structural heterogeneity, exhibiting Type-I and Type-II branched structures, Type-I branches emanating from the Type-II NR, and Type-II NR that terminate in or completely transverses the nucleus (reviewed in [14, 16]). Depending on the cell type, Type-II NR tubules average 0.1-0.2 μm in diameter but can be as large as 1 μm [10, 14, 16, 17].

The NR has been observed in numerous normal and transformed cultured cells and tissues from different metazoan species, forms in interphase cells independent of NE breakdown, is remodelled and modified on a time scale of minutes, and dynamically responds to cytoplasmic and nuclear stimuli [14, 16, 18]. Due to its ubiquity and potential to bring a cytoplasmic and ER luminal interface to the interior of the nucleus, the NR is proposed to facilitate nuclear-cytoplasmic signalling and the transfer of solutes, nucleic acid and proteins [10]. Indeed, Lamin A- and B-positive NR are frequently found in close proximity to the nucleoli, promyelocytic leukemia nuclear bodies (PML NBs), heterochromatin and sites of DNA damage, and are hot-spots for calcium signalling [9, 19–21]. These activities are contingent on the dynamic extension of the NR to subdomains within the interior of the nucleus, which would obviate reliance on diffusion or transport to the nuclear periphery. Live-cell imaging showing changes in the organization and location of the Type-II NR [10] and frequent proximity to nuclear substructures suggested a de novo mechanism for NR assembly that is under regulatory control. Phospholipid synthesis, lamin intermediate filaments, and microtubule and actin networks have been identified as key factors in NR assembly, but how they are integrated to respond to nuclear stimuli is poorly understood. This review will focus on our current understanding of the mechanism(s) for NR biogenesis and remodelling, and its role as conduit and interface for signaling and transport between the nuclear and cytoplasmic compartments.

Membrane biogenesis and remodelling promote NR assembly

Since the NR is composed of single or double membranes, its extension into the nuclear interior is dependent on the acquisition of phospholipids from the ER and/or phospholipid synthesis in the NE. Biogenesis of Type-II NR requires the coordinated expansion of both the ONM and INM. Phosphatidylcholine (PC) species comprise 65% of NE phospholipids, with phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine (PE) each contributing 5–10% [22, 23]. Since the bulk synthesis of these phospholipids occurs in in the ER and ONM [24, 25], extension of the ONM into Type-II NR tubules could involve the lateral transport of pre-existing phospholipids from the ER through membrane junctions with the ONM (Fig. 1) and/or de novo synthesis in the ONM. Phospholipids could be transported to the ONM from other organelles at membrane contact sites [26]. The expansion of the INM into Type-I or Type-II NR could also involve acquisition of phospholipids from the ONM and ER. However, this is potentially restricted by high membrane curvature where the ONM and INM fuse at NPCs (Fig. 1) that could prevent the lateral bilayer diffusion of specific phospholipids between the two membranes. There is also increasing evidence that the INM of eukaryotic cells is a site of active lipid synthesis [27, 28]. The INM of U2OS and hepatoma cells harbour most enzymes required for the synthesis of the phospholipid precursors phosphatic acid (PA) and diacylglycerol (DAG), including glycerophosphate acyltransferases, lysophosphatidic acid acyltransferase, and the phosphatidic acid phosphatase Lipin1 [28–30]. Fluorescent biosensors also revealed that the products of this pathway, DAG and PA, are present in the INM [28, 29]. PA and DAG are precursors for the assembly of the major phospholipid classes via the CDP-DAG and CDP-choline/ethanolamine pathways, respectively. These phospholipid precursors also directly affect nuclear functions, such as NPC assembly and NE repair, and are used to make triacylglycerol (TAG) that is stored in lipid droplets (reviewed in [7, 31, 32]).

While the INM has the capacity to synthesize the phospholipid precursors DAG and PA, whether these precursors are used for the in situ synthesis of phospholipids in the INM is less certain. Isolated mammalian cell nuclei have the capacity to synthesize PC [33], the major membrane constituent of the NE [22], but this could occur in the ONM or be the result of ER contamination. The rate-limiting enzyme in the CDP-choline pathway for synthesis of PC (Fig. 2A), CTP: phosphocholine cytidylyltransferase (CCT), is in the nucleoplasm and INM of most eukaryotic cells [34]. The terminal enzyme in the CDP-choline pathway that synthesizes PC and PE, choline/ethanolamine phosphotransferase 1 (CEPT1), was localized to the NE and NR but its presence on the INM has not been confirmed [35, 36]. The INM of S. cerevisiae also has the enzymes for synthesis of DAG and PA [27], and a split GFP screen localized the terminal enzyme for PC (Cpt1) and PE (Ept1) biosynthesis on the INM [37]. These studies infer that expansion of the INM and ONM during NR formation is facilitated by lateral flow of phospholipids from neighbouring membranes and/or phospholipid synthesis at sites proximal to membrane extension.

Fig. 2.

CCT promotes membrane curvature and phosphatidylcholine synthesis to promote NR formation. (A) Phosphatidylcholine (PC) and phosphatidylethanolamine (PE), the predominant phospholipids of nuclear membranes, are synthesized by the CDP-choline and CDP-ethanolamine pathways, respectively. (B) The catalytic domain of CCTα (green) is activated by insertion of the Domain M amphipathic helix (blue) into the INM when enriched in anionic lipids and non-bilayer lipids or deficient in PC. CCTα translocation and activation is also accompanied by dephosphorylation of the C-terminus. (C) CCTα induces positive curvature in membrane tubules by insertion of Domain M helix (blue, shown in cross-section) into the lipid bilayer. (D) Cooperation between CCT insertion into the INM, the nuclear lamina and coupling of the ONM and INM via LINC complexes drives the extension of a Type-II NR tubule. Abbreviations: CK, choline kinase, CEPT1, choline/ethanolamine phosphotransferase 1; CHPT1, choline phosphotransferase 1; EK, ethanolamine kinase; ECT, phosphoethanolamine cytidylyltransferase; EPT1, ethanolamine phosphotransferase. Figure created using BioRender.com

Studies of cultured cells treated with the mono-unsaturated fatty acid oleate support a model wherein NR formation involves a complex interplay between de novo PC synthesis, membrane deformation and nuclear lamins. Oleate activates PC synthesis by stimulating the translocation of nucleoplasmic CCT onto the INM by insertion of its C-terminal 50 amino acid amphipathic helix (termed Domain M) into the membrane [34] (Fig. 2B). The Domain M helix has positively charged residues at the interface between its polar and non-polar surfaces that charge-pair with lipid head groups, and a large non-polar surface that wedges into the interior of the membrane [34]. Charge-pairing of Domain M with anionic lipids, such as fatty acids and PA [38–40], facilitates its insertion into membranes. As well, membrane elastic curvature stress due to PC depletion and enrichment in non-bilayer lipids, such as DAG and PE, creates gaps in the membrane bilayer that favour association of the non-polar surface of Domain M [41–43]. These properties define Domain M as a membrane sensing module that regulates the rate of PC synthesis at the INM by responding to a deficiency in the end product and the availability of pathway precursors, such as fatty acids and DAG.

In addition to translocation to the INM, CCT in oleate-treated CHO-K1, fibroblasts and NIH-3T3 cells was localized on Type-II NR that contained Lamin A and B, NPCs and ER markers [17, 44]. A doubling of NR tubules after exposure to oleate for 4 h, and a paucity of NR in CHO-K1 cells with a temperature sensitive CCT mutation, pointed to CCT activation and PC synthesis as a mechanism for NR proliferation. However, the Type-II NR network in CCT-deficient CHO-K1 cells was restored by expression of either catalytically-active or -inactive CCT, but not by CCT with Domain M mutations that prevented membrane binding [44]. The conclusion that CCT promoted the proliferation of the NR network independent of PC synthesis and by a membrane remodelling mechanism is supported by the observation that incubation of liposomes with recombinant CCT resulted in formation of 50 nm membrane tubules that emanated from the membrane surface [17]. Cornell and co-workers later showed that (1) in vitro tubule formation from liposomes was proportional to CCT or domain M peptide density on the membrane, (2) CCT induced tubulation of planar membranes but showed a preference for positively curved membranes, and (3) tubule formation was favoured by a heterogenous phospholipid composition [45]. Mechanistically, insertion of Domain M into the leaflet of a membrane creates bilayer mismatch and positive curvature, a common feature of proteins that either sense or generate membrane curvature in organelle membranes [46] (Fig. 2C). Type-II NR tubules are on average 0.1–0.2 μm in diameter and have a high degree of positive curvature [10, 17]. Assuming that positive curvature induction at the INM by CCT is a driving force in NR formation, positive and negative curvature would also occur on the luminal and cytoplasmic leaflets, respectively, of the ONM (Fig. 2D). Curvature of the ONM could be driven by physical coupling of INM and ONM by NPCs and LINC complexes, and by the presence of specific lipids that favour positive or negative curvature. For example, negative curvature at the neck of NE invaginations in zeocin-treated yeast was facilitated by DAG and/or PA [47].

It is important to highlight that the expression in cells of a constitutively active, membrane-binding deficient CCT mutant increased both PC synthesis and NR formation without oleate treatment [17]. However, membrane-binding defective CCT formed 50% fewer NR tubules compared to the wild-type enzyme, indicating that PC synthesis promotes NR formation but ineffectively unless coupled with membrane curvature induction by CCT. The possibility of localized PC synthesis and incorporation into nascent NR was addressed using nanoscale secondary ion mass spectrometry of deuterated choline-labeled cells [48, 49]. Although there was incorporation of labelled choline into PC at specific sites in NR tubules, the pulse-labeling period of 12 h does not allow discrimination between PC synthesized in the NR versus PC imported from other membranes. Without solid evidence that CCT provides CDP-choline directly to CEPT1 in the INM [35], one is left to conclude that NR biogenesis likely proceeds by acquisition of PC and other phospholipids from preexisting membranes.

Nuclear lamins and NR formation

Lamin A and B have established roles in NE structure, chromatin organization and mechano-sensing through their interaction with a large network of protein partners [50, 51]. Studies employing mammalian cell models have shown that the lamina underlying the Type-II NR, and to a lesser extent the Type-I NR, is important for stability and expansion. Expression of A- and B-type Lamins in mammalian, D. melanogaster and X. laevis cells resulted in NE proliferation and appearance of a NR tubular network [52, 53]. Overexpression of GFP-Lamin A in CHO-K1 cells afforded a 2.5-fold increase in NR tubules while RNA silencing of LMNA inhibited NR formation under basal and oleate-stimulated conditions [44]. In contrast, co-expression of Lamin B1, but not Lamin A, with CCT enhanced oleate-induced NR proliferation, and LMNB silencing resulted in comparable reduction in NR formation to LMNA/C silencing in the absence of oleate, but the effect was less in the presence of oleate. The concept that coordinated activity of CCT and lamins promotes NR proliferation was supported by the observation that a CCT domain M mutant that constitutively associated with membranes caused accumulation of unusual bundles of membrane tubules within the nucleus that were devoid of lamins [44]. Thus, a mismatch between lamin and CCT expression and activity caused the proliferation of an abnormal tubule network. A similar relationship was identified in a study showing that overexpression of the constitutively farnesylated lamin A mutant progerin resulted in an extensive NR network that was dependent on CCT expression [54].

Collectively, these studies highlight the importance of coordinated assembly of the membrane and lamina during proliferation of the Type-II NR. Both nascent farnesylated Lamin A as well as Lamin B1 are rapidly incorporated into the lamina underlying the NR [48] where it provides a structural framework on which to extend NR tubules in response curvature induction by CCT or external forces supplied by the cytoskeleton (see below). The membrane deforming activity of CCT would be enhanced by the phospholipid composition of the NE, which is rich in unsaturated acyl-chain species of PC and PI that would favour a more elastic, malleable membrane [22, 23]. In oleate-treated cells, de novo synthesis of PC was dispensable for NR proliferation implying that the NE has sufficient membrane mass for NR assembly. This conclusion is supported by the observation that NR tubule proliferation in response to overexpression of Lamin A or B did not increase de novo PC synthesis [35]. However, it is possible that phospholipids synthesis replenishes the ONM and INM over a longer time scale than employed in these studies.

The cytoskeleton and NR formation

EM and confocal imaging of different cell types identified cytosolic microtubules, intermediate filaments and actin in Type-II NR [10, 15, 55–57]. These cytoskeletal elements may form or extend the NR by ‘poking’ or ‘pushing’ the NE into the nuclear interior. Evidence summarized below recognizes the cytoskeleton as a driving force behind Type-II NR biogenesis involved in DNA repair and genome surveillance.

Nuclear shape is modified in response to DNA and telomere damage by mechanical forces exerted by microtubules and actin connected to the NE via the LINC complex [50]. DNA damage repair occurs at the NE and NPCs [58, 59] but most damage sites are scattered throughout the nucleus. In D. melanogaster and S. cerevisiae, DNA strand breaks (DSB) move to the peripheral NE on damage-induced nuclear microtubules and actin filaments [60, 61]. In mammalian cells, NE invagination occurs rapidly upon the induction of DNA DSBs in concert with rapid decondensation and ultrastructural changes in chromatin [62, 63] and, in contrast to yeast, persistent DSBs become immobilized at both PML nuclear bodies and the NE [62, 64, 65]. However, many DSBs do not appear to directly access the peripheral NE. The association of the NR with sites of DNA damaged marked by H2AX and 53BP1 suggested that the NR could be a repair scaffold [21], and in particular may represent a site where DNA DSBs in heterochromatin are repaired [66, 67]. In support, fibroblasts treated with etoposide for as briefly as 6 min caused the appearance of vertical and horizontal Lamin B1-positive invaginations that resolved almost completely by 30 min [62]. The appearance of the NR coincided with decreased chromatin density and H2AX and MRE11 repair foci. The observation that 53BP1 promotes increased mobility of DSBs and dysfunctional, uncapped telomeres in a LINC- and microtubule-dependent manner further implicated the NR in DNA damage repair under normal and pathological conditions [68].

A recent study using etoposide-treated U2OS cells revealed that a Type-II NR containing Lamin B, NPCs, microtubules and the LINC complex was associated with intranuclear DSBs [20]. The mechanism for NR extension involved DNA damage repair kinases ataxia telangiectasia and Rad3-related (ATR), ataxia telangiectasia mutated (ATM) and DNA dependent protein kinases, which activated tubulin acetyltransferase 1 to increase kinesin KIF5B-dependent extension of acetylated microtubules at the tips of the NR (Fig. 3A). The reversal of NR extensions upon etoposide removal required the minus-end kinesin KIF3C [20]. Damage at a single genomic locus by restriction enzyme-induced ribosomal DSBs at the nucleolar periphery was associated with NR invaginations and dependent on LINC complex and the actin cytoskeleton [69]. Thus, microtubule- and actin-induced invaginations driven by DNA repair kinases bring homologous repair machinery on the NE and NPCs in close proximity to DSBs, reducing the requirement for migration to the nuclear periphery.

Fig. 3.

Mechanisms for Type-II NR formation and function. (A) DNA damage signaling stimulates the formation of Type-II NR through the activation of DNA repair kinases ATM, ATR, and DNA dependent protein kinase, leading to ATAT1-KIF5B-LINC mediated extension of cytoplasmic microtubules into the nuclear envelope. (B) Extracellular vesicles (EVs) taken up by cells in late endosomes are escorted into Type-II NR by a Rab7, ORP3, and VAPA-dependent mechanism. Fusion of the EVs with late endosome membrane releases cargo in Type-II NR that are imported by NPC into nucleoplasm to affect transcription. (C) Calcium release through inositiol-3-phosphate receptors (IP3R) and ryanodine receptors (RyR) promotes calcium enrichment in the Type-II NR lumen. Calcium released into Type-II NR can diffuse through NPCs and, along with possible calcium release from Type-I NR, activate PKCγ and promote its translocation to the INM. Calcium uptake into the perinuclear space occurs by the channel proteins SERCA or ORA1-STIM1 and is regulated by the INM protein emerin. Figure created using BioRender.com

Since all cells experience replicative and genotoxic DNA damage events, DNA damage repair could be a primary function of the NR, and the prevalence of NR intrusions should correlate with levels of genome instability. This supposition aligns with the general observation that transformed and cancerous cells, which suffer from high rates of genome instability, also have an abundant and elaborate NR compared to normal tissue [14, 16]. DNA damage induced by bacterial genotoxins such as cytolethal distending toxin from H. pylori also induced a Type-II NR in intestinal cells that initiated DNA repair and autophagy activation to promote survival of infected cells [70, 71]. Cells from individuals with Hutchinson-Gilford progeria syndrome (HGPS) have an abnormal lamina, lobulated NE and increased Type-1 and -II NR [35, 54]. HGPS fibroblasts are also defective in DNA repair responses, showing delayed recruitment of 53BP1 to DSBs [72], telomere attrition [73, 74], heterochromatin loss [75] and increased non-homologous end joining (NHEJ) and genomic amplification events [76]. Relative to wild-type Lamin A, progerin is strongly associated with the NE, aligns in lamina microdomains and is resistant to, but less able to dissipate mechanical stress [77]. Since the NE in HGPS cells is relatively rigid and resistant to invaginations unless acted on by external forces, the extensive NR in progeroid cells could result from a defective lamina that is unable to repair and resolve DNA damage.

Extracellular vesicle uptake at the Type-II NR

Extracellular vesicles (EVs) encompass a large class of membrane structures released into the extracellular milieu from multivesicular bodies, plasma membrane budding and apoptotic bodies. EVs contain a variety of protein, lipid and nucleic acid cargoes that mediate intercellular communication under normal and pathological conditions. The contents of EVs are delivered to cells by fusion with the plasma membrane or following uptake by endocytic, pinocytic or phagocytic mechanisms [78]. How EV cargoes reach cellular organelles to exert their biological activity is poorly understood, particularly the mechanism for accessing the nucleus.

Recently, a complex of oxysterol binding protein-related protein 3 (ORP3), vesicle-associated membrane protein-associated protein (VAP) A and Rab7 in the Type-II NR were identified as key factors in the transfer of EVs packaged in late endosomes to the nucleoplasm [79, 80]. RNA interference silencing of VAPA, but not its closely related orthologue VAPB, prevented the recruitment of ORP3 and Rab7-late endosomal EVs into Type-II NR and the release of EV contents into the nucleoplasm [80] (Fig. 3B). These results are consistent with the known role of VAPA-ORP complexes to form molecular bridges at organelle membrane contact sites [81]. Interestingly, the VAPA-ORP1L complex, which specifically tethers Rab7-positive late endosomes to the ER [82], was not implicated in EV uptake into the nucleus [80]. ORP3 lacks a canonical Rab7 interaction motif suggesting that other unidentified factors are involved in Rab7 recognition. The release of EV contents into the nucleoplasm was prevented by an inhibitor of importin-1 implicating transit via the NPC [79]. A model was proposed wherein the ORP3/VAPA/Rab7 complex promoted the tethering and fusion of EVs with the late endosome membrane with subsequent release of EV contents into the Type-II NR for import through NPCs (Fig. 3B). The confined space within the Type-II NR lumen could facilitate tethering of EVs to an NPC and more efficient uptake of EV contents. An important feature of this transfer pathway is that CD9 + EVs increased the frequency of nuclear envelope invaginations in breast cancer cell lines [79]. Whether EVs or their contents stimulate NR formation via a membrane, lamina or cytoskeletal mechanism is not known.

The NR and nuclear calcium signaling

Nuclear functions, including protein kinase activity, transcription and chromatin structure, are regulated by calcium released from the NE perinuclear space or cytoplasm at the nuclear periphery. Initial studies by Lui and co-workers demonstrated a calcium store in Type-II NR that was released by phorbol esters [83, 84]. Later, studies using caged inositol 3-phosphate (IP3) and growth factor stimulation of epithelial and hepatoma cells implicated IP3 receptors in the release of calcium stores from the NR into the interior of the nucleus [9, 85]. This release of calcium specifically activated protein kinase C γ translocation to the NE but not the plasma membrane [9]. NR-specific calcium release also involves ryanodine receptors capable of calcium-induced calcium release [86], as well as stromal interaction molecule 1 (STIM1) and ORAI1 that mediate store operated calcium entry [87]. Sarco-endoplasmic reticulum calcium ATPase channels, which restore resting calcium levels in the nucleus [88], were also identified in the NR of mouse C2C12 myoblasts and neonatal cardiomyocytes [87].

Calcium signalling in the nucleus of mouse cardiomyocytes also specifically alters NR structure. In this case, hypertrophic stimuli reduced NR tubules, increased nuclear volume, and increased the half-decay time of calcium transients [89]. This effect on calcium transients and the NR was reproduced by silencing of emerin, and in cells from individuals with Emery-Dreyfuss Muscular Dystrophy (EDMD), implicating emerin as a specific regulator of NR calcium uptake. Since EDMD is also caused by mutations in the genes for Lamin A, SUN1, SUN2 and nesprins, the NR could have a general role in calcium homeostasis and transcriptional responses in muscle [90]. Additionally, the NR could provide structural stiffness and support to the myocyte nucleus under conditions of contraction-induced mechanical stress by enhancing LINC mechano-transduction and sensing pathways [50]. Whether NR abnormalities are specifically involved in muscle dysfunction or reflective of a more generalized defect in the NE is unclear. However, the established role of nuclear calcium signaling in transcription, DNA repair, and apoptosis [91], and the collective evidence that the NR has most of the major calcium regulatory channels implies a critical role in regulation of these signalling events in the nuclear interior.

Nuclear egress of DNA viruses via the Type-I NR

Type-I NR are observed as INM extensions from both the Type-II NR and the NE, lack NPCs and LINC complexes and are relatively deficient in lamins [10, 14]. A well-documented role for the Type-I NR is the membrane encapsulation and nuclear egress of the Herpesviridae family, including herpes simplex virus (HSV), cytomegalovirus and pseudorabies virus. Mature viral capsids are enveloped by the lamina-free INM of Type-I NR, migrating through tubules to eventually fuse with the ONM and release the naked virions [92, 93] or proceed through the perinuclear space and ER-Golgi secretory pathway [94]. Several conserved viral proteins in Herpesviridae have been demonstrated to participate in this pathway, notably the UL31 and UL34 membrane proteins of the herpesvirus nuclear egress complex (NEC). UL34 is a transmembrane protein that recruits soluble UL31 to the INM where the heterodimers form a lattice of hexameric complexes that promote positive membrane curvature, inducing vesicular formation from the INM and Type-I NR invaginations [95, 96]. In a study modeling the effects of these proteins on artificial membranes, purified pseudorabies virus pUL34 reconstituted in proteolipid giant unilamellar vesicles interacted with pUL31 to induce invaginations that developed into intralumenal vesicles [97]. Targeting of UL34 and UL31 in HSV-1-infected cells to the INM promoted Type-I NR invaginations by a mechanism that required the kinase activity of the HSV-1 protein US3 to disrupt the nuclear lamina [98–101]. The loss of US3 during pseudorabies virus infection resulted in accumulation of enveloped virions within the perinuclear space [98]. The ability of UL31 and UL34 to bind Lamin A/C and modify its conformation and localization would suggest that these proteins expose the surface of the INM, allowing for the assembly of the curvature-promoting NEC to facilitate the formation of Type-I NR. However, it is unclear whether the formation of Type-I NR is a consequence of or required for viral egress. In this context, mutant pseudorabies virus lacking pUL34 promoted viral egress but with deterioration of the NE and release of immature capsids lacking viral DNA. Thus, NEC-facilitated virion egress serves as a quality control mechanism by preventing premature NE breakdown to maximize virion progeny [102].

Type -I NR proliferation and nuclear lipid droplet biogenesis

The Type-I NR has a crucial role in biogenesis of nuclear lipid droplets (LDs) in hepatocytes (20, 22). Nuclear LDs are structurally related to the more abundant cytoplasmic LDs but are restricted to specific mammalian cells, such as hepatocytes, intestinal cells and U2OS cells, and are found at relatively low frequency in non-transformed cells [31, 103]. In hepatoma cells, the biogenesis of nuclear LDs involves Type-I NR proliferation under the control of SUN1/2 LINC complex proteins, receptor expression enhancing proteins (REEP) 3 and 4 and PML protein isoform II (Fig. 4) [104]. SUN1/2 are integral membrane proteins that span the INM and attach to nesprins 1–4 that span the ONM. SUN1/2 and nesprins make up the LINC complex, with SUN1/2 adhering to the nuclear lamina and nesprins interacting directly with actin filaments or indirectly with microtubules and intermediate filaments. Nuclear LD biogenesis and Type-I NR is constrained by SUN2 (and to a lesser extent SUN1) expression in Huh7 cells [30], indicating that formation of nuclear LDs and Type-I NR results from INM expansion of the NE or Type-II NR where the INM and ONM are uncoupled due to a deficiency in LINC complexes [104]. REEP3 and 4, which are responsible for tubulating ER membranes and excluding the ER from the metaphase plate during mitosis [105], were similarly found to inhibit nuclear LD biogenesis and proliferation of Type-I NR [104].

Fig. 4.

Type-I NR formation and nuclear lipid droplet biogenesis in hepatocytes. Promyelocytic leukemia protein (PML) isoform II localizes to the INM and excludes Lamin A and B, SUN1/2, and REEP3/4 to promote Type-I NR formation. In the case of SUN1/2, the absence of these proteins allows for uncoupling of the INM and ONM. ApoB-free ER luminal lipids droplets (eLDs) positive for apolipoprotein E (ApoE) and C3 (ApoC3) are precursors for very low-density lipoproteins (VLDL). ER stress causes these precursors to accumulate and migrate through the ER lumen into the perinuclear space and eventually into Type-I NR. There, PML II mediates the emergence of eLDs into the nucleoplasm to form a nuclear LDs, which retain PML structures to their surface and recruit lipid biosynthetic enzymes such as CCTα. Figure created using BioRender.com

PML protein isoform II enhances both nuclear LD biogenesis and Type-I NR formation in hepatoma cells upstream of SUN1/2 and REEP3/4 (Fig. 4). RNAi-mediated PML II silencing reduced nuclear LD biogenesis and prevented the proliferation of Type-I NR induced by RNAi-mediated silencing of SUN1/2 or REEP3/4. PML II harbours a membrane-targeting motif that is essential for its role in promoting nuclear LD biogenesis by accumulating at sites of the INM deficient in Lamin B receptor, Lamin B, Lamin A/C and SUN1 [30, 106] (Fig. 4). Whether PMLII is promoting the degradation or excluding these proteins from membrane sites is unknown, as is the mechanism responsible for recruiting PML II to the INM.

The nuclear LDs that enter the nucleus via the Type-I NR are derived from ER luminal lipoprotein precursors (Fig. 4) [104]. The frequency of nuclear LDs and Type-I NR was increased in Huh7 cells treated with tunicamycin and oleate, indicating involvement of ER stress and glycerolipid synthesis. Transmission electron microscopy revealed that cells treated with tunicamycin and oleate accumulated ER luminal lipoprotein precursors that migrated through the ER lumen into Type-I NR and there appeared to rupture the INM and enter into the nucleoplasm (Fig. 4). The formation of nuclear LDs was dependent on the activity of microsomal triglyceride transfer protein (MTP), which transfers lipids to lipoprotein precursors and nascent very low-density lipoproteins in the ER lumen. Type-I NR proliferation was not dependent on MTP activity but was nonetheless upregulated in response to ER stress and further exacerbated by oleate treatment. The Type-I NR pathway for nuclear LD biogenesis in response to excess fatty acids was not observed in intestinal cell lines that have NR, MTP and secrete chylomicrons [107]. Thus, the Type-I NR pathway could be specific for hepatocytes exposed to combined ER and fatty acid stress.

Future perspectives

This review has highlighted how lipid metabolism, DNA repair, calcium signaling and EV uptake either modify or expand the NR network by affecting membrane biogenesis and remodeling, the nuclear lamina and/or cyto/nucleoskeletal networks. However, it remains to be established whether these mechanisms for NR expansion work cooperatively or in isolation to promote metabolic and signaling activities. In the case of cells treated with oleate to activate lipid synthesis, evidence points to a cooperative mechanism to extend NR tubules involving CCT, PC synthesis and nuclear lamins. The cytoskeleton could provide a mechanical force to extend the NR under these conditions but which networks are involved is unknown. Similarly, the membranes required for NR extension to DNA damage sites could come from increased phospholipid synthesis or recruitment from the INM, ONM or ER. However, the rapid extension and contraction of the NR during the DNA damage response [20, 62] suggests that phospholipids are recruited from surrounding membranes rather than local synthesis. Enhanced NR formation due to constitutive DNA damage, such as in cancer cells, would necessitate the synthesis and assembly of new membranes via sustained activation of CCT and PC synthesis. For example, ras-transformed intestinal epithelial cells have an extensive Lamin A-positive NR with constitutively associated CCT and increased PC synthesis [108].

Another potential link between NR extension and membrane organization are the LINC proteins SUN1 and SUN2. Both are essential for NR extensions during the DNA damage response, with the SUN1 N-terminal domain required to form lamina- and chromatin-dependent interactions [20]. In addition to these domains, SUN2 has an amphipathic helix facing the nucleoplasm that associates with lipid-induced packing defects in the INM caused by increased DAG, the product of the PA phosphatase Lipin1 [109]. Dissociation of the SUN2 helix from the INM results in increased proteolytic degradation, potentially linking INM protein composition to mechanical forces exerted by the cytoskeleton.

Studies linking NR dynamics to calcium transients and EV uptake have not explored mechanisms for tubule extension. On a speculative note, calcium release at the INM of the NR could activate PKCs, leading to phosphorylation and disassembly of the nuclear lamina [2]. Localized delamination at the INM could allow access to other proteins that promote tubule extension. Calcium signaling could have a role in Type-II NR in DNA damage repair and extracellular vesicle trafficking by promoting microtubule disassembly and preventing nucleation in vitro [110]. Similarly, calcium activation of calpains that modify the stability of microtubules could negatively impact Type-II NR formation [111]. The well characterized role of calcium in promoting actin polymerization and crosslinking could also have a role in NR tubule extension.

The NR navigates the nucleoplasm in response to internal and external cues that effect different structural elements comprising its tubular membrane network. A systematic approach should be applied to understand the contribution of membrane synthesis and remodelling, lamina and nucleo- and cyto-skeleton, to NR formation. As well, the application of unbiased approaches, such as biotin proximity labelling or genetic screens, could be employed to identify other components of the NR assembly apparatus.

Abbreviations

CCTα CTP

Phosphocholine Cytidylyltransferase α

CEPT1

Choline/Ethanolamine Phosphotransferase 1

DAG

Diacylglycerol

DSB

DNA Strand Break

EDMD

Emery-Dreyfuss Muscular Dystrophy

EV

Extracellular Vesicle

HGPS

Hutchinson-Gilford Progeria Syndrome

HSV

Herpes Simplex Virus

IP3

Inositol 3-Phosphate

LAPS

Lipid-Associated PML Structures

LINC

Linkers of the Nucleoskeleton to the Cytoskeleton

LD

Lipid Droplet

MTP

Microsomal Triglyceride Transfer Protein

NEC

Nuclear Egress Complex

NR

Nucleoplasmic Reticulum

ONM

Outer Nuclear Membrane

INM

Inner Nuclear Membrane

NE

Nuclear Envelope

ORP

Oxysterol Binding Protein-Related Protein

PA

Phosphatidic Acid

PC

Phosphatidylcholine

PE

Phosphatidylethanolamine

PML NB

Promyelocytic Leukemia Nuclear Bodies

NPC

Nuclear Pore Complex

REEP

Receptor Expression Enhancing Proteins

SUN

Sad1 and UNC84 Domain containing

TAG

Triacylglycerol

VAP

Vesicle-Associated Membrane Protein-Associated Protein

Author contributions

All authours contributed equally to the preparation of this manuscript.

Funding

Not applicable.

Data availability

Not applicable.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authours have no financial or non-financial interests to disclose.