Coupling lipid synthesis with nuclear envelope remodeling

Abstract

The nuclear envelope (NE) is a protective barrier to the genome, yet its membranes undergo highly dynamic remodeling processes that are necessary for cell growth and maintenance. While mechanisms by which proteins promote NE remodeling are emerging, the types of bilayer lipids and the lipid-protein interactions that define and sculpt nuclear membranes remain elusive. The NE is continuous with the endoplasmic reticulum (ER) and recent evidence suggests that lipids produced in the ER are harnessed to remodel nuclear membranes. In this review, we examine new roles for lipid species made proximally within the ER and locally at the NE to control NE dynamics. We further explore how the biosynthesis of lipids coordinates NE remodeling to ensure genome protection.

The dynamics and biogenesis of nuclear membranes

The nucleus is the distinguishing feature of eukaryotic cells that houses the genome and is defined by the nuclear envelope (NE; see Glossary) [1, 2]. The NE is a double membrane sheet that is physically continuous with the endoplasmic reticulum (ER), a large membrane system essential for protein and lipid synthesis (Figure 1).The ER extends from the NE to the cell periphery in the form of flat membrane sheets and tubules [1]. Tubule-to-sheet junctions connect the ER to the outer nuclear membrane (ONM), which is contiguous with the inner nuclear membrane (INM) via curved membranes surrounding 100 nm holes containing nuclear pore complexes (NPCs) (Figure 1A) [3]. Unlike ER sheets that can be highly fenestrated [4], the NE is sealed except for at pores occupied by NPCs [2]. Beyond NPCs, the NE, specifically the INM, harbors a unique set of proteins, including LEM-domain proteins that interact with chromatin and lamins, a dense meshwork of nuclear intermediate filaments that provide structural support to the NE, and linker of nucleoskeleton and cytoskeleton (LINC) complex proteins that traverse the INM/ONM to connect the NE with the cytoskeleton (Figure 1A) [5, 6]. INM proteins are associated with a striking diversity of human diseases that range from progeria to lipodystrophy, and emerging studies suggest that disease mutations in INM proteins disrupt NE integrity [7, 8], thus highlighting the importance of understanding how the NE is generated and maintained as a selective permeability barrier to the genome.

Figure 1. The composition and dynamics of the nuclear envelope (NE) during cell division and nuclear expansion.

(A) Schematic of a region fo the NE and endoplasmic reticulum (ER) that enclose a shared lumen (light blue). Shown is the distinct protein composition of the inner nuclear membrane (INM) that faces chromatin. A representative LAP2β - Emerin- MAN1 (LEM) domain protein (dark orange) that binds to the the DNA crosslinker Barrier to autointegration factor (BAF) (orange) and links chromatin to the nuclear periphery is shown. Nuclear lamins (gray) form filaments underneath the INM and associate with INM proteins (such as LBR, shown in green). Also shown is the Linker of the nucleoskeleton and cytoskeleton (LINC) complex proteins (purple) that traverse the INM, lumen, and outer nuclear membrane (ONM) to connect the NE to the cytoskeleton. A nuclear pore complex (NPC) (light green) is shown inserted at a fusion point between the INM/ONM. (B) A schematic of the continuous ER formed as sheets in proximity to the NE and tubules extending to the cell periphery in an interphase cell. NE breakdown and ER reorganization upon entry into mitosis occur as chromosomes (gray) condense and the spindle assembles (light grey circles are spindle poles, light grey lines are microtubules, MTs). During chromosome segregation, the ER/NE network is restricted to the cell periphery. NE reformation follows chromatin partitioning whereby nuclear membranes re-associate with decondensing chromatin and holes left by spindle MTs are sealed to re-establish the nuclear barrier. Core and non-core regions at the NE (inset) are shown relative to spindle MTs, and the peripheral ER and ESCRT membrane remodeling proteins in royal blue. (C–E) Schematic of NE expansion accomplished by incorporation of nearby ER lipids that may flow to expand the ONM/INM. Expansion of both the NE and ER result from increased glycerolipid (GPL) biosynthesis as shown in (D) and protein or lipid barriers (magenta squares) positioned at ER tubule-sheet junctions or the pore membrane may restrict the diffuison of lipids to limit expansion as shown in (E).

Establishment of NE integrity for genome protection involves remodeling of the NE during the cell division cycle (breakdown, reformation, sealing, and growth). Metazoans undergo an open mitosis and so NE breakdown initiates at the onset of mitosis and [2, 9, 10] (Figure 1B), directly after chromosome segregation, ER-derived membranes re-associate with chromatin to reform the NE [11] (Figure 1B). NE reformation, the second phase of NE remodeling, is guided by LEM-domain proteins that bind barrier to autointegration factor (BAF), a soluble DNA-binding protein that crosslinks DNA to restrict membranes to the surface of the chromatin [12] (Figure 1B). ER-derived membranes that enclose chromatin first form the outer “non-core” region, which is exposed and not occluded by spindle microtubules (MTs), and extend into the inner “core” regions as spindle MTs begin to disassemble (Figure 1B, inset). NPCs assemble at the outer core to initiate nuclear transport [13], and the Endosomal Sorting Complex Required for Transport (ESCRT) remodeling machinery is recruited to any remaining NE holes at the inner core region by NE adaptors for ESCRTs, the INM protein LEMD2 and the ESCRT-II/III protein CHMP7 [14] (Figure 1B, inset). These holes contain persisting spindle MTs, and ESCRTs coordinate the severing of these MTs with membrane sealing to establish nuclear compartmentalization [15, 16]. In addition to NE remodeling in mitosis, the NE can also undergo rupture and repair [17] (see Box 1), fusion [18], and rapid expansion during interphase [19]. The nucleus grows two-fold in volume during interphase (Figure 1C) and this is coupled to chromatin decondensation, as well as the insertion of new NPCs into a sealed NE [20, 21]. Similar proteins and mechanisms contribute to NE remodeling in interphase and mitosis [22] (see Box 1), although different regulatory pathways likely signal and coordinate NE dynamics during these distinct cell cycle stages.

Box 1. Nuclear envelope (NE) rupture and repair.

In the past decade, transient ruptures of the NE have been observed in cultured cells under mechanical stress and when lamins, the structural component of the NE, are depleted or mutated [7]. The cause and significance of transient nuclear ruptures to cell physiology has been an intense area of research, especially given the relationship between human disease and loss of NE integrity [13, 17, 53, 78–80]. In new work on mouse models of lamin-related muscular dystrophy, migrating nuclei of muscle cells that suffer repeated NE rupture lead to increased DNA damage, reduced cell viability, and increased disease severity [81]. Using an in vitro and mouse xenograft model of breast cancer progression, Nader et al. (2020) [82] showed that continued DNA damage in cells in the constricted environment of breast cancer tumor fronts leads to increased invasion and metastasis.Given these severe consequences of NE breaches on genome integrity and cellular behavior, current work aims to identify mechanisms of repairing NE holes caused by rupture. Repair of NE holes during interphase and NE closure following chromosome segregation in mitosis both involve the ESCRT membrane remodeling machinery and BAF [13, 14, 22]. However, the presence of the mitotic spindle is a major difference between mitotic NE remodeling and interphase NE repair. While NE sealing after mitosis occurs in a synchronous and rapid fashion, the timing of NE repair in interphase can vary from minutes to hours depending on the context [13, 22, 83]. Several papers have proposed that breaches in the NE are detected by 1) cytoplasmic exposure of DNA or 2) access to repair proteins (e.g. Chmp7) to the inner nuclear membrane [14, 22]. Although there are hints that lipid synthesis through the CTDNEP1/lipin pathway coordinates with sealing of NE holes following rupture [61], future work is needed to understand how lipid signals and regulation of membrane biogenesis regulates NE repair and relates to the diseases characterized by repeated loss of nuclear integrity.

Bilayer lipids synthesized on the cytoplasmic leaflet of the ER produce the membranes that form the NE. After NE reformation, the ER and NE maintain their continuity, indicating that lipids can diffuse from the ER to the ONM via ER-ONM junctions and to the INM via the nuclear pore membrane [23] (Figure 1A, C). This is unlike non-contiguous organelles in which the ER controls lipid composition via vesicular trafficking or direct lipid transfer at membrane contact sites [24]. At steady state, the ER makes up 51% of total membranes, whereas the NE only makes up <1% [25], so there is an overabundance of available ER lipids to form nuclear membranes. What mechanisms then control lipid flow between the ER and NE? While the answer to this question is not entirely clear, it is well established from yeast to humans that the biosynthesis of bilayer glycerophospholipids (GPLs) at the ER is tightly coupled to the generation of nuclear membranes – shifting the flux of the lipid synthesis pathway towards these major membrane lipids causes expansion of ER membranes and increases the surface area of the NE [26–31] (Figure 1D). However, the fact that the ER retains the majority of the lipids in the continuous ER/NE network indicates that potential barriers regulate the content and abundance of lipids exchanged between these domains [23] (Figure 1E). Thus, the biogenesis of ER and NE involves crosstalk and coordination.

Here, we explore these new areas and discuss evidence for regulation of bilayer lipid composition and ER/NE lipid production during NE breakdown, formation, sealing, and growth. We mainly focus on work done in metazoans and explore lessons from yeast that have provided critical genetic and biochemical insight into the regulation of lipid metabolism in relation to NE remodeling.

Compartmentalizing lipid synthesis at the nuclear envelope

The ER and NE are mainly made up of the major membrane GPLs, phosphatidylcholine (PC), and phosphatidylethanolamine (PE), as well as the low abundant lipid phosphatidylinositol (PI) [32, 33]. The de novo synthesis of GPLs that comprise the NE and ER occurs in the cytoplasmic leaflet of the ER. Unidentified flippases and scramblases maintain lipid content between the lumenal and outer leaflet of the ER [23]. The first steps in the de novo lipid synthesis pathway require the sequential addition of fatty acids (FAs) to glycerol-3-phosphate (G3P) to form phosphatidic acid (PA) [23] (Figure 2A, B). These FAs have short and desaturated chains (see examples of fatty acid tails in Figure 2B) that are more flexible than the saturated acyl chains that pack tightly with cholesterol to generate the impermeable barrier of the plasma membrane [34, 35]. PA is then converted to diacylglycerol (DAG), which serves as a precursor to PC and PE (Figure 2A). Conversion of PA to cytidine diphosphate (CDP)-DAG produces phosphatidylinositol (PI) (Figure 2A). Steps in the sphingolipid and cholesterol synthesis pathway also occur within ER membrane bilayers [23]; however, these lipids and lipid intermediates enrich in ER microdomains destined for the membrane trafficking pathway and thus make up a very small percentage of total steady state ER/NE lipids [33].

Figure 2. Glycerolipid synthesis and fatty acid desaturation in relation to regulation of nuclear envelope (NE) lipids.

(A) A simplified representation of the glycerolipid (GPL) synthesis pathway with the major bilayer lipids that make up the NE/endoplasmic reticulum (ER) highlighted in green (PC, phosphatidylcholine; PE, phosphatidylethanolamine; and PI, phosphatidylinositol) and the reaction catalyzed by lipin shown with generic structures of phosphatidic acid (PA) and diacylglycerol (DAG). Inset shows opposing kinase and phosphatase activities that regulate lipin with the CTDNEP1 complex highlighted. In addition to its catalytic activity, lipin regulates transcription through an unknown mechanism. (B) Representative structure of a GPL containing a glycerol backbone and two fatty acid tails. Fatty acids differ in chain length, saturation level, and positioning of double bonds. Examples of fatty acid chains are shown to represent the differences in their structure that imposes flexibility and thus may impact membrane fluidity and lipid packing. (C) Schematic representation of peripheral membrane binding enzymes, lipin and CCTα, associated with a single leaflet of a membrane bilayer through their amphipathic helices.

A key enzyme in GPL synthesis is the peripheral membrane binding phosphatidic acid phosphatase (PAP) lipin (Saccharomyces cerevisiae Pah1, S. pombe Ned1, Caenorhabditis elegans LPIN-1, mammalian lipin 1, 2, and 3) that catalyzes the dephosphorylation of PA to yield DAG [23] (Figure 2A, C). The levels of PA and DAG make up <1% of total ER lipids [33] and lipin itself is not rate-limiting for lipid synthesis [36]. Instead, modulating the activity and membrane association of lipin influences signaling and transcriptional pathways that can have profound effects on lipid homeostasis [36]. Lipins bind to membranes through multiple regions and their membrane binding and catalytic activity is regulated by post-translational modifications, as well as the concentration of PA and composition of FAs within membrane bilayers [37–41]. Mammalian lipin 1, which has the highest PAP activity of the three mammalian lipins, has at least 20 predicted and mapped phosphorylation sites [42]. In vitro based activity assays show that dephosphorylated lipin 1 is more active towards PA [37], which would be predicted to produce more DAG for bulk membrane synthesis. However, an overabundance of ER membranes results from expression of a constitutively phosphorylated (less active) lipin, suggesting that the phosphorylation state of lipin may either directly or indirectly serve as a signal to change the levels of ER lipid synthesis in vivo – how this relates to changes in its enzyme activity as measured in vitro is not clear [26, 29, 31, 43, 44]. Shuttling of lipin 1 between the nucleus and cytoplasm, which also depends on its phosphorylation state, influences the transcription of genes necessary for FA synthesis and thus suggests a potential feedback mechanism for regulation of bulk membrane synthesis [45] (Figure 2A). A major phosphatase for lipin is the integral protein phosphatase CTDNEP1 (budding and fission yeasts Nem1, C. elegans CNEP-1, mammalian CTDNEP1) [31, 46] (Figure 2A). Importantly, in C. elegans embryos, which are transcriptionally quiescent, CTDNEP1 (C. elegans CNEP-1) is enriched at the NE and regulates NE dynamics through lipin [26]. Recent work has also shown that CTDNEP1 can concentrate at the NE in fly and mouse cells [47], further suggesting that CTDNEP1 may have a conserved role in locally regulating a pool of lipin. Although there is still much to learn about how CTDNEP1 modulates lipin’s phosphorylation state from the NE to influence lipid synthesis and NE dynamics, evidence exits that lipin 1 is active in the production of triglycerides (TAG) from DAG at the INM when excess FAs are present [48] (see Box 2), indicating that lipin 1 PAP activity is at least in part regulated at the INM. Thus, through regulation of its phosphorylation state, lipin is poised to both locally control NE lipid composition and globally control ER lipid homeostasis.

Box 2. Nuclear Lipid Droplets.

Lipid droplets (LDs) are multifunctional organelles composed of a membrane monolayer that encass neutral lipids. The accumulation of triglycerides (TAG) and sterols in the endoplasmic reticulum (ER) membrane initiates LD formation and budding from the outer leaflet of the ER [84]. Observations of LDs inside the nucleus raised many interesting questions about their formation (derived from the inner nuclear membrane (INM) or traveled from the ER) and whether they have a distinct composition and functions than those out in the cytoplasm. Romanaska & Kohler (2018) provided definitive evidence that nuclear LDs form from the INM [51]. Another study in budding yeast showed that Lro1, a fungal specific enzyme that transfers a fatty acid from glycerophospholipids (GPLs) to diacylglycerol (DAG) to form TAG, localizes to and generates TAG at the INM [85]. Nuclear LDs have also been observed in mammalian cells, although different mechanisms are required depending on the cell type [48, 86–88]. A recent study in an osteosarcoma cell line (U2OS) showed that LDs form directly from the INM in human cells and that enzymes required for TAG synthesis are active and able to reach the INM [48]. While the discovery of the INM as an active platform for lipid metabolism is exciting, questions still remain on the functional significance of nuclear LDs. Do they serve to actively regulate the transcription of lipid synthesis genes through protein sequestration or do they direct storage for membrane biogenesis during episodes of nuclear expansion [89]?

The role of PA and DAG as biochemical precursors for membrane synthesis through lipin’s catalytic activity is relatively well understood (Figure 2A). PA and DAG are also well known for their signaling roles at other membranous organelles [49, 50]. One possibility is that PA/DAG serve as signals for feedback regulatory pathways that control overall lipid homeostasis in response to changes in lipin activity. Alternativley or in addition, PA/DAG may directly modulate the dynamics of the NE. In budding yeast, DAG is enriched at the INM and PA accumulates in NE herniations that form from hyperactivation of the ESCRT-III NE remodeling machinery [51, 52]. In mammalian cells, DAG activates PKC to disassemble the nuclear lamina [53]. A future challenging goal will be understand potential signaling roles of PA and DAG in controlling NE dynamics.

A similar role in regulation of lipid homeostasis from the INM has been suggested for the key peripheral membrane binding enzyme in PC synthesis, CTP:Phosphocholine Cytidylyltransferase (CCT) [54] (Figure 2A). PC is the most abundant lipid in intracellular membranes and accounts for >60% of total lipids in ER membranes [32]. Under steady state conditions, CCTα is enriched in the nucleus where it is retained in its autoinhibited, inactive form. Lipid packing defects resulting from low PC levels are favorable for the association of CCTα to the INM and relieve its autoinhibition, leading to restoration of PC levels (Figure 2C). A form of CCTα that is targeted to the ER also restores PC levels, suggesting that CCTα does not have to localize to the INM to control PC homeostasis [54]. The formation of intranuclear membranes (see Box 3) results from overexpression of either wild type or inactive forms of CCTα and so whether CCTα assists in local production of PC for NE biogenesis remains unclear [55].

Box 3. Lipid signaling and nuclear mechanics.

Two recent studies reveal an important role for lipid signaling at the nuclear envelope (NE) in response to compression of the nucleus [90, 91]. These studies show that natural folds of the NE are important for sensing cellular compression to facilitate cell migration in confinement. The authors show that when cells are confined, the nucleus flattens and the normally wrinkled nuclear membrane becomes unfolded. This triggers calcium release and subsequent activation of cytosolic phospholipase A2 (cPLA2) that localizes to the NE and generates arachidonic acid (AA), which promotes myosin-II-mediated contractility at the cell cortex. Heightened actomyosin contractility is necessary to squeeze the cell out of the confined environment and survive physical compression. The NE folds described in these studies may be a form of intranuclear membranes that have been described in both healthy and diseased states [92]. There are two types of intranuclear membranes (termed nucleoplasmic reticulum) that have been classified: (1) Type I invaginations occur when the INM alone extends into the nucleoplasm (2) Type II invaginations occur when both INM and ONM invade the nuclear interior [93]. Intranuclear membranes have been shown to result from overproduction of membranes and defects in NE remodeling [61] and while still highly speculative it would be interesting to understand whether their presence disrupts lipid signaling from the NE in response to tension. Further elucidating connections between mechanical forces and lipid signaling and synthesis in will be an interesting area of future research that might open doors to our understanding of pathologies related to the nuclear envelope.

Thus, lipin and CCT are two key enzymes in the GPL pathway that may sense INM lipids as a direct read out of changes in the composition of the ER and may also locally regulate lipid composition and lipid synthesis at the INM. While lipin and CCT have the unique property of binding on and off membranes depending on the local lipid composition (Figure 2C), there is evidence for the integral membrane protein enriched at the INM, Lamin B receptor (LBR), in regulation of cholesterol synthesis [56]. The separation of the INM from other membrane bound organelles in the cytoplasm may provide a sheltered environment for distinct regulation of lipid metabolizing enzymes and allow access to a subset of lipids, without exposure to lipids of other membrane bound compartments where these enzymes may inappropriately act. The proximity of the INM to the genome may also be important for coupling the local sensing of lipids to regulation of transcriptional networks to maintain lipid homeostasis, which has been proposed as a potential role for nuclear lipid droplets (LDs) [51] (see Box 2). Defining the specific roles of these lipid metabolizing enzymes in NE remodeling will be essential to understanding how lipid composition and lipid synthesis is coupled to NE dynamics.

Coordinating lipid synthesis with nuclear envelope breakdown

Several lines of evidence suggest a role for lipid synthesis in coordinating NE remodeling during cell division through regulation of lipin activity. This coordination of membrane synthesis with early events in mitosis has been shown using multiple model organisms that differ in the degree of NE breakdown during mitosis (Figure 3A). In budding and fission yeasts, for example, the mitotic spindle forms inside the nucleus, requiring the NE to expand during mitosis to accommodate the elongating mitotic spindle [57]. Unlike metazoans, yeast can convert PA to major membrane GPLs and so a reduction in lipin^Pah1 activity is thought to increase the flux of PA towards GPL synthesis [43]. In line with this, in S. pombe, lipin hyperphosphorylation in mitosis by the mitotic kinase Cdk1 leads to the increased production of membranes that expand the NE as the spindle elongates [58]. In S. japonicus, the expansion of the NE is uncoupled from spindle elongation, resulting in partial rupture of the NE [57]. Lipin’s hyperphosphorylation during mitosis in S. pombe produces nuclear membranes to support their expansion, however the phosphorylation status of lipin in S. japonicus remains unchanged during cell division, leading to reduced membrane availability and transient rupture of the NE (termed “semi-closed” mitosis) [58].

Figure 3. Nuclear envelope (NE) remodeling in mitosis and membrane flow.

(A) Schmatic of nuclear membranes in different organisms that undergo “closed” and “open” mitosis. In closed mitoses of fission yeasts, the NE breaks down in local area (between the membrane bridge in S. pombe and tears in S. japonicus). In C. elegans, the NE undergoes permeabilization, but membrane remnants remain in the vicinity, but excluded from, the mitotic spindle and segregating chromosomes. In open mitosis, the NE and its associated proteins retract into the endoplasmic reticulum (ER) and reorganize to the cell periphery. (B) Schematic of pronuclear fusion in C. elegans embryos. Successful pronuclear fusion and retraction allows mixing of chromosomes and formation of a single nucleus containing both the oocyte- and sperm-derived chromosomes. “Twinned” nuclei form from failure to fuse pronuclear membranes, such as in cnep-1 mutant embryos (see [26]). (C) Schematic representation of stacked membrane sheets undergoing fusion. A “tubule-to-sheet junction” may transition into a “sheet-to-sheet junction” that then allows feeding of the membrane into an opening. Membrane remodeling of the junction may be required to properly feed in the membrane. Adapted from [18].

Lipin regulation has also been shown to promote the removal of nuclear membranes during pronuclear fusion in C. elegans embryos by limiting membrane availability [26, 28, 59]. In the C. elegans zygote, mitotic spindle MTs penetrate the poles of the permeabilized NEs of the oocyte and sperm-derived pronuclei (Figure 3A). Membranes between metaphase chromosomes must retract prior to anaphase onset to allow genome mixing such that a single nucleus forms in each daughter cell that contains the full complement of parental chromosomes (Figure 3B) [60]. In C. elegans embryos deleted for the NE localized protein phosphatase cnep-1 (CTDNEP1 in mammals) that regulates lipin, an excess layer of ER membrane sheets enwrap the existing NEs of pronuclei [26]. This results in 8 membrane bilayers (instead of 4) persisting between pronuclei and delaying membrane retraction between the two pronuclei, resulting in the formation of mostly oblong and twinned nuclear phenotypes in daughter cells [26, 28, 59] (Figure 3B). Interestingly, the pathway that produces major membrane lipids from PA in fungi does not exist in metazoans and so how excess ER membranes are generated in this genetic background remains unclear. The depletion of enzymes that synthesize PI from PA (Figure 2A) suppresses the cnep-1 mutant phenotypes of C. elegans embryos, which are transcriptionally silent, and so excess membranes may result from signaling (or lackthereof) by an intermediate lipid product that is more readily produced (or depleted) because of the shift in the flux in the lipid synthesis pathway towards PI synthesis (see Figure 2A).

Recent work used focused-ion beam scanning electron microscopy to study how the membranes between C. elegans pronuclei are remodeled to promote mixing of oocyte and sperm-derived chromosomes [18]. This work revealed that membrane fusion, rather than (or in addition to) NE breakdown, is necessary for the timely retraction of membranes between pronuclei. The two outer membrane bilayers of the juxtaposed oocyte- and sperm-derived pronucleus form “outer-outer junctions” as well as unique “three way sheet junctions” in which an inner and outer membrane from one pronucleus forms a fused junction with the outer membrane of the other pronucleus (Figure 3B, C) [18]. These fusions resolve at enlarged holes that likely result from the coalescence of smaller holes generated from the removal of NPCs, to form a single membrane sheet [18]. Thus, preventing an additional membrane sheet from enwrapping the NE by restricting membrane production through CNEP-1 regulation of lipin is likely important to ensure timely fusion of membranes between pronuclei. Interestingly, loss of the NE adaptor for ESCRT machinery CHMP7 (C. elegans chmp-7) dramatically increases the propensity to form twinned nuclei when membranes are in excess in cnep-1 mutants, suggesting a role for ESCRT-mediated NE remodeling in the pronuclear fusion process [61].

In mammalian cells, NE and ER membranes are cleared from the spindle region to the cell periphery to promote mitotic progression and proper chromosome segregation [31, 62]. Persistent association of membranes with chromatin results in severe defects in mitosis [5, 63–65]. Excess ER membrane production resulting from loss of CTDNEP1 regulation of lipin 1 causes delayed clearance of ER/NE membranes from the spindle region [31]. While in C. elegans excessive ER/NE membranes enwrap the existing NEs, in mammalian cells ectopic ER membranes invade the spindle region and surround mitotic chromosomes [31]. Inhibition of the rate limiting step in FA synthesis restores ER expansion in CTDNEP1 knockout cells, supporting the idea that there is a potential feedback regulation that increases FA synthesis, and thus bulk ER membrane synthesis, in response to loss of a CTDNEP1-dependent dephosphroylated pool of lipin 1 [31].

Thus, regulating ER lipid synthesis through phospho-regulation of lipin is a conserved feature across phylogeny critical for mitotic membrane reorganization and remodeling.

Nuclear envelope closure: coupling membrane sealing and biogenesis

Following mitosis (or interphase NE rupture, see Box 1), the holes in the NE must seal so that the NE can perform its function as a selective permeability barrier (Figure 1B). Recent studies aimed at elucidating the roles of proteins in NE sealing that are associated with ESCRT membrane remodeling machinery have also revealed roles for lipid synthesis and lipid composition in the closure of NE holes, further discussed below [61]. Studies in mammalian cells find that spindle MTs that persist at the end of mitosis are surrounded by nuclear membranes forming 50–100 nm holes in the reforming NE (Figure 1B) [66]. The INM protein LEMD2 (S. cerevisiae Heh1, S. pombe Lem2, C. elegans LEM-2) associates with persistent MTs through a MT-binding region that may function through phase separation [67]. The C-terminal winged helix domain of LEMD2 binds and activates ESCRTII/III protein CHMP7 that in turn recruits downstream ESCRT components and the MT severing protein spastin to coordinate MT disassembly with membrane sealing [15, 66, 67]. Coupling of ESCRT-III filament polymerization and MT dissasembly at NE holes is mediated by the coiled-coil- and C2 domain-containing protein B (CC2D1B) that interacts with the membrane phospholipid phosphatidylinositol 4,5 bi-sphosphate (PIP₂) on the NE, suggesting that the production of specific lipid species on the reforming NE recruits membrane remodeling machinery for closure of NE holes following open mitosis [16].

While ESCRTs execute fission of membrane bilayers that are ~50–100 nm apart [66], studies show that ESCRT-remodeling machinery is not required for sealing of larger holes in the NE [61, 68, 69]. In C. elegans embryos, regulation of lipid synthesis by the CNEP-1 (CTDNEP1)/lipin pathway is important for closure of the large 1 μm hole of the reforming NE surrounding the meiotic spindle [61]. Excess ER membrane sheets in cnep-1 mutants disrupt NE sealing by overflowing into the large hole, causing the formation of intranuclear membranes. 3D rendering of electron microsopy (EM) tomograms of wild type embryos revealed ER sheets feeding into NE holes that are sometimes occupied by MTs, suggesting membrane feeding narrows and closes holes in a coordinated fashion (see Figure 3C). The loss of C. elegans CHMP7 (chmp-7) or LEMD2 (lem-2) in cnep-1 mutants or parital RNA interference (RNAi)-depletion of lipin exacerbates defects in NE sealing and increases the severity of intranuclear membranes, suggesting that CHMP7/LEMD2 may be important for remodeling membranes as they feed into the NE to close holes. In support of this, in vitro purification of the C-terminal winged helix domain of LEMD2 together with CHMP7 activates the formation of oligomeric rings that resemble membrane fission spiral component of the ESCRT machinery, suggesting that these proteins may have membrane remodeling activity on their own independent of other ESCRT components [67]. Taken together, this work revealed that membrane synthesis cooperates with membrane remodeling machinery to ensure closure of large holes in the NE, thus driving new hypotheses for how bilayer lipids and NE-associated membrane remodeling proteins might facilitate hole closure.

Evidence also exists for the composition of FAs in membrane bilayers impacting NE hole closure. In S. japonicus, overexpression of the conserved FA desaturase (Ole1 is fission yeasts, SCD1 in humans), which adds a double bond to FA tails that incorporate into GPLs (Figure 2A), partially rescues defects in NE sealing in chmp7 mutants, suggesting that regulation of FA composition may act independently from ESCRT-III remodeling to close holes [70]. One idea that remains speculative is that defects in hole closure in cnep-1 mutants may result directly from the overrepresentation of PI in ER/NE membranes, which has a large headgroup and is more likely to have polyunsaturated fatty acids that increase membrane fluidity [34]. The kinked FA tails of desaturated lipids may help create a more disordered bilayer at the curved edges of the NE hole, making membrane fusion more energetically favorable [71]; however, one possibility is that the ratio of saturated to unsaturated FAs may be key to promoting or impairing NE closure. In S. pombe, increasing the production of lipids with long saturated FA tails rescues loss of NE integrity in a sensitized lem2 (LEMD2 in humans) mutant background, supporting the idea that hole closure in the NE involves regulation of the length and degree of saturation in membrane lipids [72].

Overall these studies provide evidence for a conserved role in the regulation of lipid synthesis in NE closure. It will be interesting to determine whether regulation of lipid composition or restricting ER/NE biogenesis, independent of lipid bilayer composition, is required to seal the NE. The lipin activation pathway appears central to coordinating the production and ratio of appropriate lipids for sealing holes during NE closure and the degree of FA saturation may also play an important role in establishing and maintaining NE integrity. A close analysis of FA profiles and the impact of acute and local depletion of lipid metabolism enzymes will be necessary to understand the role of lipid diversity on NE closure.

Expanding the nuclear envelope by membrane insertion and flow

After NE formation, the NE expands as chromatin decondenses. The expansion of the NE is limited by the overall levels of GPL production in the ER [26, 31, 73]. In line with this, the membrane pool of the perinuclear ER has recently been shown to control the rate of expansion and steady state size of the NE in both sea urchin and Xenopus laevis eggs [74], further suggesting that ER lipids and their flow into the NE control NE expansion and size (see Figure 1C). Disrupting the perinuclear ER through inhibition of MTs/dynein or overexpression of reticulon 4b, an ER tubule-shaping protein, reduces nuclear growth [74]. These experiments suggest that 1) ER lipids proximal to the NE feed into the expanding NE to control nuclear size and 2) the topology of nearby ER membranes plays a role in NE growth. ER membrane sheets containing NPCs (termed annulate lamellae) have been shown to incorporate into the NE to rapidly expand the NE in rapidly growing nuclei of Drosophila oocytes [19]. The underlying topology faciliating membrane incorporation during nuclear growth may resemble the sheet-to-sheet junctions observed during pronuclear fusion in C. elegans embryos given that the fusion of two membrane sheets would substantially increase the surface area of the single, fused membrane product (Figure 3C) [18].

Transmembrane proteins at NE-ER junctions, at the nuclear pore membrane, and at the INM may regulate NE growth by controlling the flow of lipids between the ER and NE (Figure 1E). In S. pombe, the INM protein Lem2 (LEMD2 in humans) restricts changes in nuclear size resulting from inhibition of nuclear export or excess lipid synthesis [75]. Lem2 also prevents shrinkage of the NE when available ER lipids are reduced by inhibition of FA synthesis. Lem2 cooperates with Lunapark, an ER transmembrane protein that preferentially associates with membrane structures at three-way ER tubule junctions, to regulate membrane flow. In fission yeast lacking both Lem2 and Lunapark, partitioning of the ER and NE membranes is dramatically disrupted, explaining previously reported growth defects upon loss of the two proteins [76]. The fact that overproduction of GPLs by disrupting the lipin activation pathway in budding yeast causes extrusions of the NE at selective regions further suggests that barriers to lipid flow and membrane insertion exist at or near the NE [27]. Given that INM stretching can trigger mechanochemical reponses (see Box 3), it is also possible NE folding or tension could contribute to the regulation of lipid flow between the NE and ER either by affecting barrier proteins stability and localization or through mechanochemical signaling. Furthermore, evidence in yeast suggests that lipids in LDs can be utilized for membrane GPLs (see Box 2), providing a route to directly expand the NE. In sum, little is known about the existence of potential protein barriers or valves that regulate lipid flow in metazoans but there is a clear need for coordinating lipid synthesis with NE growth since the overproduction of ER lipids results in nuclear expansion, abnormal nuclear structures, and internal nuclear membranes.

Concluding remarks

Similar themes from multiple model organisms are emerging in which regulation of lipid synthesis coordinates membrane feeding, remodeling, and fusion events that facilitate the breakdown, sealing, and growth of the NE. 3D tomography and high resolution imaging reveal similarities in membrane topologies that facilitate these dynamic events, and genetic and cell biological studies suggest that similar pathways and proteins are involved. While the functions and dynamics of proteins during NE remodeling are well-studied, the roles for lipids within the ER/NE membranes are far less understood. Moreover, regulated changes in nuclear membrane lipids likely in turn affect the functions of ONM/INM proteins. Future work involving the development and use of reliable lipid biosensors, as well as in vitro studies to complement in vivo studies on lipid metabolism, will be critical to learn more about lipid functions during NE remodeling (see Outstanding Questions).

Outstanding Questions.

• What regulatory pathways and signals coordinate the production of lipids with nuclear envelope (NE) dynamics?

• How do lipid metabolism enzymes that are spatially restricted at the NE sense and respond to changes in the lipid content of the endoplasmic reticulum (ER)/NE membrane system?

• How do signals for cellular growth and maintenance impact the lipid environment of the NE and do changes in the lipid content induce downstream regulation of the genome?

• Does the inner nuclear membrane (INM) contain a unique lipid composition relative to the rest of the ER and if so how is this lipid asymmetry achieved within a continuous membrane system?

• What are the protein barriers that regulate the flow of lipids between the NE and ER?

• How is lipid flow coupled to nuclear growth and dynamic membrane remodeling events?

• Do specific lipids at the inner nuclear membrane confer important biophysical properties to control its remodeling?

• How does mechanical strain on the nucleus affect lipid content and protein organization of the NE?

• Do specific lipid-protein interactions or lipid microdomains sculpt nuclear membranes during NE remodeling?

Highlights.

• The genome is surrounded by the nuclear envelope (NE), a sealed double membrane sheet that is contiguous with the endoplasmic reticulum (ER).

• The NE is highly dynamic and undergoes membrane remodeling such as breakdown, reformation, sealing and growth.

• New data suggests that remodeling of the NE involves crosstalk and coordination with lipid synthesis, which occurs primarily within the ER.

• Some lipid synthesis enzymes also act at the NE and may locally regulate the biogenesis and remodeling of nuclear membranes.

• Insights into the specific chemistry and types of lipids required to remodel nuclear membranes are also emerging.

• Research into how the regulation of bilayer lipid production and composition is coupled to NE remodeling is key to understanding mechanisms underlying diseases related to loss of NE integrity.

Glossary

Arachidonic acid

polyunsaturated fatty acid present in cell membranes; confers membrane flexibility and serves as a signaling lipid

CTP:Phosphocholine Cytidylyltransferase (CCT/PCYT1)

the rate-limiting enzyme in the de novo synthesis of phospho-choline. CCT catalyzes the formation of CDP-choline from phosphocholine and CTP, which is combined with DAG to produce phosphatidylcholine (PC)

CTDNEP1

C-terminal domain nuclear envelope phosphatase 1 is a serine/threonine protein phosphatase of the haloacid dehalogenase family that has a DXDT/V as its active site [77]. (S. cerevisiae Nem1; C. elegans CNEP-1)

Diacylglycerol (DAG)

glycerolipid consisting of two fatty acid chains attached to the central glycerol backbone; functions as a lipid intermediate in lipid metabolism and the cone shaped lipid and the headgroup of DAG confer specificity for protein interactions allowing for downstream activation of signaling pathways

ER sheets

uniform sacs of membranes that are separated by an inner space (the ER lumen). Large ER sheets are usually located in close vicinity to the nuclear envelope and are associated with ribosomes

ER tubules

curved membrane tubes that are stabilized by curvature-stabilizing proteins. ER tubules are highly dynamic and associate with microtubules and form contacts with nearly every membrane bound organelle to facilitate the transfer of lipids or regulate their morphology

ESCRT membrane remodeling machinery

the Endosomal Sorting Complexes Required for Transport (ESCRT), a multisubunit complex that assembles on the cytoplasmic side of membrane to execute membrane scission

Glycerophospholipids (GPLs)

glycerol based backbone attached to two fatty acid chains. The glycerol backbone is linked to a polar headgroup by a phosphodiester bond; main constituent of biological membranes

LEM (Lap2-emerin-MAN1) domain

a domain in a family of conserved inner nuclear membrane proteins defined by a 40 amino acids that in metazoans binds to the DNA crosslinking protein Barrier to Autointegration Factor (BAF or BANF1)

Lipin

a Mg²⁺⁻ dependent peripheral membrane binding protein phosphatidic acid phosphatase that dephosphorylates phosphatidic acid to yield diacylglycerol and inorganic phosphate; lipin proteins play major roles in lipid synthesis both through their enzymatic activity and through regulation of transcription (S. cerevisiae Pah1; S. pombe Ned1, C. elegans LPIN-1)

NE breakdown

specific to open mitosis of metazoans, NE breakdown involves permeabilization of the nuclear envelope by removal of nuclear pore complexes, detachment of nuclear membranes from chromatin, diffusion of integral NE proteins and retraction of associated membranes into ER

Nuclear envelope (NE)

a polarized double lipid membrane separated by a lumen in eukaryotic cells that protects, shapes and bounds the genome; it is composed of the inner nuclear membrane (INM) and outer nuclear membrane (ONM) and is contiguous with the ER. Nuclear pore complexes are embedded within the NE to facilitate transport between the nucleus and cytoplasm. The NE is structurally supported by the nuclear lamina, a meshwork of intermediate filaments (lamins), assembled on the INM and a unique set of inner nuclear membrane proteins that are stably enriched at the INM

Nuclear pore complexes (NPCs)

large, ring-shaped aqueous channels in the NE that regulate the selective transport of macromolecules between the nucleus and cytoplasm

Phosphatidic acid phosphatase (PAP)

catalytic activity of enzymes that dephosphorylate phosphatidic acid to produce diacylglycerol

Phosphatidic acid (PA)

small, low-abundant glycerolipid with a phosphate headgroup connected to two fatty acid chains by a glycerol backbone. Serves as the precursor for other lipids; also functions in signaling similar to DAG by recruiting proteins to the membrane

Phosphatidylcholine (PC)

cylindrical shaped glycerolipid that is the major structural component of eukaryotic membranes

Phosphatidylinositol 4,5 bisphosphate (PIP2)

most-abundant of the phosphorylated states of phosphatidylinositol (inositol ring phosphorylated on positions 4 and 5); enriched at the cytoplasmic leaflet of the plasma membrane and acts as a substrate for multiple signaling pathways; recruits proteins to the plasma membrane via specific domains or electrostatic interactions with basic amino acid residues

Tubule-to-sheet ER junctions

fusion points between ER tubules and sheets. This would include the connection between the points of fusion between the NE (a large sheet) and ER tubules