POM121 and Sun1 play a role in early steps of interphase NPC assembly

Nuclear pore complexes (NPCs) assemble at the end of mitosis during nuclear envelope (NE) reformation and into an intact NE as cells progress through interphase. Although recent studies have shown that NPC formation occurs by two different molecular mechanisms at two distinct cell cycle stages, little is known about the molecular players that mediate the fusion of the outer and inner nuclear membranes to form pores. In this paper, we provide evidence that the transmembrane nucleoporin (Nup), POM121, but not the Nup107-160 complex, is present at new pore assembly sites at a time that coincides with inner nuclear membrane (INM) and outer nuclear membrane (ONM) fusion. Overexpression of POM121 resulted in juxtaposition of the INM and ONM. Additionally, Sun1, an INM protein that is known to interact with the cytoskeleton, was specifically required for interphase assembly and localized with POM121 at forming pores. We propose a model in which POM121 and Sun1 interact transiently to promote early steps of interphase NPC assembly.     

MEL-28, a novel nuclear-envelope and kinetochore protein essential for zygotic nuclear-envelope assembly in C. elegans

The nuclear envelope (NE) of eukaryotic cells separates nucleoplasm from cytoplasm, mediates nucleo-cytoplasmic transport, and contributes to the control of gene expression. The NE consists of three major components: the nuclear membranes, the nuclear pore complexes (NPCs), and the nuclear lamina. The list of identified NE proteins has increased considerably during recent years but is most likely not complete. In most eukaryotes, the NE breaks down and is then reassembled during mitosis. The assembly of NPCs and the association and fusion of nuclear membranes around decondensing chromosomes are tightly coordinated processes. Here, we report the identification and characterization of MEL-28, a large protein essential for the assembly of a functional NE in C. elegans embryos. RNAi depletion or genetic mutation of mel-28 severely impairs nuclear morphology and leads to abnormal distribution of both integral NE proteins and NPCs. The structural defects of the NE were associated with functional defects and lack of nuclear exclusion of soluble proteins. MEL-28 localizes to NPCs during interphase, to kinetochores in early to middle mitosis then is widely distributed on chromatin late in mitosis. We show that MEL-28 is an early-assembling, stable NE component required for all aspects of NE assembly.     

PLCgamma is enriched on poly-phosphoinositide-rich vesicles to control nuclear envelope assembly

Nuclear envelope assembly is an essential event in each cell cycle but the proteins and lipids involved in its regulation remain mostly unknown. Assembly involves membrane fusions but neither specific SNAREs nor Rab GTPases have been identified in its control. We report that a precursor membrane population (MV1) required for NE assembly has a unique lipid composition consisting prominently of poly-phosphatidylinositides. The lipid composition was determined by adapting HPLC electrospray ionisation tandem mass spectrometry to phosphoinositide analysis, revealing the capacity of this technique to document dynamic lipid transitions of functional importance in natural membrane populations. MV1 is >100-fold enriched in endogenous PLCgamma and >25-fold enriched in the PLC substrate phosphatidylinositol bisphosphate (PtdInsP2) compared to the second membrane population, derived largely from endoplasmic reticulum (ER), that contributes most of the NE. During NE formation PLCgamma becomes transiently phosphorylated at the tyrosine 783 site indicative of its activation. In addition specific inhibition of PLCgamma blocks nuclear envelope formation. In vivo, PLCgamma is concentrated on vesicles of similar size to purified MV1. These associate with nuclei during the period of NE formation and are distinct from ER membranes. The unprecedented concentration of PLCgamma and its substrate PtdInsP2 in a subset of membranes that binds to only two regions of the nucleus, and activation of PLCgamma by GTP during initial stages of NE formation provide a mechanism for temporal control of NE assembly and offer an explanation for how such a process of membrane fusion can be spatially regulated.     

Requirement for Lamin B Receptor and Its Regulation by Importin β and Phosphorylation in Nuclear Envelope Assembly during Mitotic Exit

Lamin B receptor (LBR), a chromatin and lamin B-binding protein in the inner nuclear membrane, has been proposed to target the membrane precursor vesicles to chromatin mediated by importin β during the nuclear envelope (NE) assembly. However, the mechanisms for the binding of LBR with importin β and the membrane targeting by LBR in NE assembly remain largely unknown. In this report, we show that the amino acids (aa) 69–90 of LBR sequences are required to bind with importin β at aa 45–462, and the binding is essential for the NE membrane precursor vesicle targeting to the chromatin during the NE assembly at the end of mitosis. We also show that this binding is cell cycle-regulated and dependent on the phosphorylation of LBR Ser-71 by p34^cdc2 kinase. RNAi knockdown of LBR causes the NE assembly failure and abnormal chromatin decondensation of the daughter cell nuclei, leading to the daughter cell death at early G₁ phase by apoptosis. Perturbation of the interaction of LBR with importin β by deleting the LBR N-terminal spanning region or aa 69–73 also induces the NE assembly failure, the abnormal chromatin decondensation, and the daughter cell death. The first transmembrane domain of LBR promotes the NE production and expansion, because overexpressing this domain is sufficient to induce membrane overproduction of the NE. Thus, these results demonstrate that LBR targets the membrane precursor vesicles to chromatin by interacting with importin β in a LBR phosphorylation-dependent manner during the NE assembly at the end of mitosis and that the first transmembrane domain of LBR promotes the LBR-bearing membrane production and the NE expansion in interphase.     

Direct membrane protein-DNA interactions required early in nuclear envelope assembly

Among the earliest events in postmitotic nuclear envelope (NE) assembly are the interactions between chromatin and the membranes that will fuse to form the NE. It has been proposed that interactions between integral NE proteins and chromatin proteins mediate initial membrane recruitment to chromatin. We show that several transmembrane NE proteins bind to DNA directly and that NE membrane proteins as a class are enriched in long, basic domains that potentially bind DNA. Membrane fractions that are essential for NE formation are shown to bind directly to protein-free DNA, and our data suggest that these interactions are critical for early steps in NE assembly.     

The integral membrane nucleoporin pom121 functionally links nuclear pore complex assembly and nuclear envelope formation

The metazoan nuclear envelope (NE) breaks down and reforms at each mitosis. Nuclear pore complexes (NPCs), which allow nucleocytoplasmic transport during interphase, assemble into the reforming NE at the end of mitosis. Using in vitro NE assembly assays, we show that one of the two transmembrane nucleoporins, pom121, is essential for NE formation, whereas the second, gp210, is dispensable. Depletion of either pom121-containing membrane vesicles or the protein alone does not affect vesicle binding to chromatin but prevents their fusion to form a closed NE. When the Nup107-160 complex, which is essential for integration of NPCs into the NE, is also depleted, pom121 becomes dispensable for NE formation, suggesting a close functional link between NPC and NE formation and the existence of a checkpoint that monitors NPC assembly state.     

SUN proteins facilitate the removal of membranes from chromatin during nuclear envelope breakdown

SUN proteins reside in the inner nuclear membrane and form complexes with KASH proteins of the outer nuclear membrane that connect the nuclear envelope (NE) to the cytoskeleton. These complexes have well-established functions in nuclear anchorage and migration in interphase, but little is known about their involvement in mitotic processes. Our analysis demonstrates that simultaneous depletion of human SUN1 and SUN2 delayed removal of membranes from chromatin during NE breakdown (NEBD) and impaired the formation of prophase NE invaginations (PNEIs), similar to microtubule depolymerization or down-regulation of the dynein cofactors NudE/EL. In addition, overexpression of dominant-negative SUN and KASH constructs reduced the occurrence of PNEI, indicating a requirement for functional SUN–KASH complexes in NE remodeling. Codepletion of SUN1/2 slowed cell proliferation and resulted in an accumulation of morphologically defective and disoriented mitotic spindles. Quantification of mitotic timing revealed a delay between NEBD and chromatin separation, indicating a role of SUN proteins in bipolar spindle assembly and mitotic progression.     

Nuclear assembly with lambda DNA in fractionated Xenopus egg extracts: an unexpected role for glycogen in formation of a higher order chromatin intermediate

Crude extracts of Xenopus eggs are capable of nuclear assembly around chromatin templates or even around protein-free, naked DNA templates. Here the requirements for nuclear assembly around a naked DNA template were investigated. Extracts were separated by ultracentrifugation into cytosol, membrane, and gelatinous pellet fractions. It was found that, in addition to the cytosolic and membrane fractions, a component of the gelatinous pellet fraction was required for the assembly of functional nuclei around a naked DNA template. In the absence of this component, membrane-bound but functionally inert spheres of lambda DNA were formed. Purification of the active pellet factor unexpectedly demonstrated the component to be glycogen. The assembly of functionally active nuclei, as assayed by DNA replication and nuclear transport, required that glycogen be pre-incubated with the lambda DNA and cytosol during the period of chromatin and higher order intermediate formation, before the addition of membranes. Hydrolysis of glycogen with alpha- amylase in the extract blocked nuclear formation. Upon analysis, chromatin formed in the presence of cytosol and glycogen alone appeared highly condensed, reminiscent of the nuclear assembly intermediate described by Newport in crude extracts (Newport, J. 1987. Cell. 48:205- 217). In contrast, chromatin formed from phage lambda DNA in cytosol lacking glycogen formed "fluffy chromatin-like" structures. Using sucrose gradient centrifugation, the highly condensed intermediates formed in the presence of glycogen could be isolated and were now able to serve as nuclear assembly templates in extracts lacking glycogen, arguing that the requirement for glycogen is temporally restricted to the time of intermediate formation and function. Glycogen does not act simply by inducing condensation of the chromatin, since similarly isolated mitotically condensed chromatin intermediates do not form functional nuclei. However, both mitotic and fluffy interphase chromatin intermediates formed in the absence of glycogen can be rescued to form functional nuclei when added to a second extract which contains glycogen. This study presents a novel role for a carbohydrate in nuclear assembly, a role which involves the formation of a particular chromatin intermediate. Potential models for the role of glycogen are discussed.     

Reshaping of the endoplasmic reticulum limits the rate for nuclear envelope formation

During mitosis in metazoans, segregated chromosomes become enclosed by the nuclear envelope (NE), a double membrane that is continuous with the endoplasmic reticulum (ER). Recent in vitro data suggest that NE formation occurs by chromatin-mediated reorganization of the tubular ER; however, the basic principles of such a membrane-reshaping process remain uncharacterized. Here, we present a quantitative analysis of nuclear membrane assembly in mammalian cells using time-lapse microscopy. From the initial recruitment of ER tubules to chromatin, the formation of a membrane-enclosed, transport-competent nucleus occurs within ~12 min. Overexpression of the ER tubule-forming proteins reticulon 3, reticulon 4, and DP1 inhibits NE formation and nuclear expansion, whereas their knockdown accelerates nuclear assembly. This suggests that the transition from membrane tubules to sheets is rate-limiting for nuclear assembly. Our results provide evidence that ER-shaping proteins are directly involved in the reconstruction of the nuclear compartment and that morphological restructuring of the ER is the principal mechanism of NE formation in vivo.     

Chromatin-bound NLS proteins recruit membrane vesicles and nucleoporins for nuclear envelope assembly via importin-α/β

The mechanism for nuclear envelope (NE) assembly is not fully understood. Importin-β and the small GTPase Ran have been implicated in the spatial regulation of NE assembly process. Here we report that chromatin-bound NLS (nuclear localization sequence) proteins provide docking sites for the NE precursor membrane vesicles and nucleoporins via importin-α and -β during NE assembly in Xenopus egg extracts. We show that along with the fast recruitment of the abundant NLS proteins such as nucleoplasmin and histones to the demembranated sperm chromatin in the extracts, importin-α binds the chromatin NLS proteins rapidly. Meanwhile, importin-β binds cytoplasmic NE precursor membrane vesicles and nucleoporins. Through interacting with importin-α on the chromatin NLS proteins, importin-β targets the membrane vesicles and nucleoporins to the chromatin surface. Once encountering Ran-GTP on the chromatin generated by RCC1, importin-β preferentially binds Ran-GTP and releases the membrane vesicles and nucleoporins for NE assembly. NE assembly is disrupted by blocking the interaction between importin-α and NLS proteins with excess soluble NLS proteins or by depletion of importin-β from the extract. Our findings reveal a novel molecular mechanism for NE assembly in Xenopus egg extracts.     

Participation of a fusogenic protein,glyceraldehyde-3-phosphate dehydrogenase,in nuclear membrane assembly

We found an autoimmune serum, K199, that strongly suppresses nuclear membrane assembly in a cell-free system involving a Xenopus egg extract. Four different antibodies that suppress nuclear assembly were affinity-purified from the serum using Xenopus egg cytosol proteins. Three proteins recognized by these antibodies were identified by partial amino acid sequencing to be glyceraldehyde-3-phosphate dehydrogenase (GAPDH), fructose-1,6-bisphosphate aldolase, and the regulator of chromatin condensation 1. GAPDH is known to be a fusogenic protein. To verify the participation of GAPDH in nuclear membrane fusion, authentic antibodies against human and rat GAPDH were applied, and strong suppression of nuclear assembly at the nuclear membrane fusion step was observed. The nuclear assembly activity suppressed by antibodies was recovered on the addition of purified chicken GAPDH. A peptide with the sequence of amino acid residues 70-94 of GAPDH, which inhibits GAPDH-induced phospholipid vesicle fusion, inhibited nuclear assembly at the nuclear membrane fusion step. We propose that GAPDH plays a crucial role in the membrane fusion step in nuclear assembly in a Xenopus egg extract cell-free system.     

Concentration of Ran on chromatin induces decondensation,nuclear envelope formation and nuclear pore complex assembly

Nuclear envelope (NE) formation can be studied in a cell-free system made from Xenopus eggs. In this system, NE formation involves the small GTPase Ran. Ran associates with chromatin early in nuclear assembly and concentration of Ran on inert beads is sufficient to induce NE formation. Here, we show that Ran binds to chromatin prior to NE formation and recruits RCC1, the nucleotide exchange factor that generates Ran-GTP. In extracts prepared by high-speed centrifugation, increased concentrations of Ran are sufficient to induce chromatin decondensation and NE assembly. Using field emission in-lens scanning electron microscopy (FEISEM), we show that Ran promotes the formation of smoothed membranes and the assembly of nuclear pore complexes (NPCs). In contrast, RanT24N, a mutant that fails to bind GTP and inhibits RCC1, does not support efficient NE assembly, whereas RanQ69L, a mutant locked in a GTP-bound state, permits some membrane vesicle recruitment to chromatin, but inhibits vesicle fusion and NPC assembly. Thus, binding of Ran to chromatin, followed by local generation of Ran-GTP and GTP hydrolysis by Ran, induces chromatin decondensation, membrane vesicle recruitment, membrane formation and NPC assembly. We propose that the biological activity of Ran is determined by its targeting to structures such as chromatin as well as its guanine nucleotide bound state.     

Remodeling the sperm nucleus into a male pronucleus at fertilization

After fertilization, the dormant sperm nucleus undergoes morphological and biochemical transformations leading to the development of a functional nucleus, the male pronucleus. We have investigated the formation of the male pronucleus in a cell-free system consisting of permeabilized sea urchin sperm nuclei incubated in fertilized sea urchin egg extract containing membrane vesicles. The first sperm nuclear alteration in vitro is the disassembly of the sperm nuclear lamina as a result of lamin phosphorylation mediated by egg protein kinase C. The conical sperm nucleus decondenses into a spherical pronucleus in an ATP-dependent manner. The new nuclear envelope (NE) forms by ATP-dependent binding of vesicles to chromatin and GTP-dependent fusion of vesicles to each other. Three cytoplasmic membrane vesicle fractions with distinct biochemical, chromatin-binding and fusion properties, are required for pronuclear envelope assembly. Binding of each fraction to chromatin requires two detergent-resistant lipophilic structures at each pole of the sperm nucleus, which are incorporated into the NE by membrane fusion. Targeting of the bulk of NE vesicles to chromatin is mediated by a lamin B receptor (LBR)-like integral membrane protein. The last step of male pronuclear formation involves nuclear swelling. Nuclear swelling is associated with import of soluble lamin B into the nucleus and growth of the nuclear envelope by fusion of additional vesicles. In the nucleus, lamin B associates with LBR, which apparently tethers the NE to the lamina. Thus male pronuclear envelope assembly in vitro involves a highly ordered series of events. These events are similar to those characterizing the remodeling of somatic and embryonic nuclei transplanted into oocytes. The relationship between sperm nuclear remodeling at fertilization and nuclear remodeling after nuclear transplantation is discussed.     

Nuclear pore biogenesis into an intact nuclear envelope

Nuclear pore complexes (NPCs) serve as transport channels across the nuclear membrane, a double lipid bilayer that physically separates the nucleoplasm and cytoplasm of eukaryotic cells. New evidence suggests that the multiprotein nuclear pores also play a role in chromatin organization and gene expression. Given the importance of NPC function, it is not surprising that a growing list of human diseases and developmental defects have been linked to its malfunction. In order to fully understand the functional repertoire of NPCs and their essential role for nuclear organization, it is critical to determine the sequence of events that lead to the formation of nuclear pores. This is particularly relevant since NPC number, and possibly composition, are tightly linked to metabolic activity. Most of our knowledge is derived from NPC formation that occurs in dividing cells at the end of mitosis when the nuclear envelope (NE) and NPCs reform from disassembled precursors. However, NPC assembly also takes place during interphase into an intact NE. Importantly, this process is not restricted to dividing cells but also occurs during cell differentiation. Here, we will review aspects unique to this process, namely the regulation of nuclear expansion and the mechanisms of fusion between the outer and inner nuclear membranes. We will then discuss conserved and diverging mechanisms between post-mitotic and interphase assembly of the proteinaceous structure in light of recently published data.     

Spatial and temporal control of nuclear envelope assembly by Ran GTPase

Using evidence derived primarily from studies using Xenopus egg extracts, a model for the role of Ran in multiple stages during NE assembly can be proposed (Figure 2). Ran is concentrated on chromatin prior to NE assembly and recruits RCC1 that generates Ran-GTP locally. Recruitment of RCC1 to chromatin may be a specialized mechanism to initiate NE assembly following fertilization of the egg, whereas in somatic cells, RCC1 may be present on chromatin throughout mitosis. Ran-GTP recruits vesicles to the surface of chromatin, and promotes vesicle fusion to form the double membrane of the NE. Ran-GTP may recruit membrane vesicles to chromatin through binding to integral membrane proteins through importin-beta. A transient complex would be formed between Ran-GTP, importin-beta and the target protein, which would be released locally to promote assembly of a precursor complex. GTP hydrolysis by Ran would release importin-beta, but may also play a role in vesicle fusion. Ran-GTP also promotes NPC assembly by releasing nucleoporins such as Nup107 from inhibitory complexes with importin-beta. In vertebrate cells undergoing mitosis, the majority of Ran molecules are excluded from the chromosomes and dispersed into the cytoplasm. Relocalization of Ran to chromatin at the end of mitosis may co-ordinate the initiation of NE assembly with disassembly of the mitotic spindle. The function of Ran in this transition is likely to be coupled to changes in the activity of cyclin-dependent protein kinases and other activities that control the progression of the cell cycle. Thus, changes in the localization of Ran and its regulators provide temporal and spatial control of NE assembly at the end of mitosis.     

A role for nuclear lamins in nuclear envelope assembly.

The molecular interactions responsible for nuclear envelope assembly after mitosis are not well understood. In this study, we demonstrate that a peptide consisting of the COOH-terminal domain of Xenopus lamin B3 (LB3T) prevents nuclear envelope assembly in Xenopus interphase extracts. Specifically, LB3T inhibits chromatin decondensation and blocks the formation of both the nuclear lamina-pore complex and nuclear membranes. Under these conditions, some vesicles bind to the peripheral regions of the chromatin. These "nonfusogenic" vesicles lack lamin B3 (LB3) and do not bind LB3T; however, "fusogenic" vesicles containing LB3 can bind LB3T, which blocks their association with chromatin and, subsequently, nuclear membrane assembly. LB3T also binds to chromatin in the absence of interphase extract, but only in the presence of purified LB3. Additionally, we show that LB3T inhibits normal lamin polymerization in vitro. These findings suggest that lamin polymerization is required for both chromatin decondensation and the binding of nuclear membrane precursors during the early stages of normal nuclear envelope assembly.     

Nuclear envelope dynamics.

The nuclear envelope (NE) provides a semi permeable barrier between the nucleus and cytoplasm and plays a central role in the regulation of macromolecular trafficking between these two compartments. In addition to this transport function, the NE is a key determinant of interphase nuclear architecture. Defects in NE proteins such as A-type lamins and the inner nuclear membrane protein, emerin, result in several human diseases that include cardiac and skeletal myopathies as well as lipodystrophy. Certain disease-linked A-type lamin defects cause profound changes in nuclear organization such as loss of peripheral heterochromatin and redistribution of other nuclear envelope components. While clearly essential in maintenance of nuclear integrity, the NE is a highly dynamic organelle. In interphase it is constantly remodeled to accommodate nuclear growth. During mitosis it must be completely dispersed so that the condensed chromosomes may gain access to the mitotic spindle. Upon completion of mitosis, dispersed NE components are reutilized in the assembly of nuclei within each daughter cell. These complex NE rearrangements are under precise temporal and spatial control and involve interactions with microtubules, chromatin, and a variety of cell-cycle regulatory molecules.     

Role of importin-beta in the control of nuclear envelope assembly by Ran

Compartmentalization of the genetic material into a nucleus bounded by a nuclear envelope (NE) is the hallmark of a eukaryotic cell. The control of NE assembly is poorly understood, but in a cell-free system made from Xenopus eggs, NE assembly involves the small GTPase Ran. In this system, Sepharose beads coated with Ran induce the formation of functional NEs in the absence of chromatin. Here, we show that importin-beta, an effector of Ran involved in nucleocytoplasmic transport and mitotic spindle assembly, is required for NE assembly induced by Ran. Concentration of importin-beta on beads is sufficient to induce NE assembly in Xenopus egg extracts. The function of importin-beta in NE assembly is disrupted by a mutation that decreases affinity for nucleoporins containing FxFG repeats. By contrast, a truncated protein that cannot interact with importin-alpha is functional. Thus, importin-beta functions in NE assembly by recruiting FxFG nucleoporins rather than by interaction through importin-alpha with karyophilic proteins carrying classical nuclear localization signals. Importin-beta links NE assembly, mitotic spindle assembly, and nucleocytoplasmic transport to regulation by Ran and may coordinate these processes during cell division.     

Reticulon 4a/NogoA locates to regions of high membrane curvature and may have a role in nuclear envelope growth

Reticulon 4a (Rtn4a) is a membrane protein that shapes tubules of the endoplasmic reticulum (ER). The ER is attached to the nuclear envelope (NE) during interphase and has a role in post mitotic/meiotic NE reassembly. We speculated that Rtn4a has a role in NE dynamics. Using immuno-electron microscopy we found that Rtn4a is located at junctions between membranes in the cytoplasm, and between cytoplasmic membranes and the outer nuclear membrane in growing Xenopus oocyte nuclei. We found that during NE assembly in Xenopus egg extracts, Rtn4a localises to the edges of membranes that are flattening onto the chromatin. These results demonstrate that Rtn4a locates to regions of high membrane curvature in the ER and the assembling NE. Previously it was shown that incubation of egg extracts with antibodies against Rtn4a caused ER to form into large vesicles instead of tubules. To test whether Rtn4a contributes to NE assembly, we added the same Rtn4a antibody to nuclear assembly reactions. Chromatin was enclosed by membranes containing nuclear pore complexes, but nuclei did not grow. Instead large sacs of ER membranes attached to, but did not integrate into the NE. It is possible therefore that Rtn4a may have a role in NE assembly.