Nuclear Envelope In Interphase Research Articles

An ESCRT grommet cooperates with a diffusion barrier to maintain nuclear integrity.

The molecular mechanisms by which the endosomal sorting complexes required for transport (ESCRT) proteins contribute to the integrity of the nuclear envelope (NE) barrier are not fully defined. We leveraged the single NE hole generated by mitotic extrusion of the Schizosaccharomyces pombe spindle pole body to reveal two modes of ESCRT function executed by distinct complements of ESCRT-III proteins, both dependent on CHMP7/Cmp7. A grommet-like function is required to restrict the NE hole in anaphase B, whereas replacement of Cmp7 by a sealing module ultimately closes the NE in interphase. Without Cmp7, nucleocytoplasmic compartmentalization remains intact despite NE discontinuities of up to 540 nm, suggesting mechanisms to limit diffusion through these holes. We implicate spindle pole body proteins as key components of a diffusion barrier acting with Cmp7 in anaphase B. Thus, NE remodelling mechanisms cooperate with proteinaceous diffusion barriers beyond nuclear pore complexes to maintain the nuclear compartment.

A membrane reticulum, the centriculum, affects centrosome size and function in Caenorhabditis elegans

Centrosomes are cellular structures that nucleate microtubules. At their core is a pair of centrioles that recruit pericentriolar material (PCM). Although centrosomes are considered membraneless organelles, in many cell types, including human cells, centrosomes are surrounded by endoplasmic reticulum (ER)-derived membranes of unknown structure and function. Using volume electron microscopy (vEM), we show that centrosomes in the Caenorhabditis elegans (C.elegans) early embryo are surrounded by a three-dimensional (3D), ER-derived membrane reticulum that we call the centriculum, for centrosome-associated membrane reticulum. The centriculum is adjacent to the nuclear envelope in interphase and early mitosis and fuses with the fenestrated nuclear membrane at metaphase. Centriculum formation is dependent on the presence of an underlying centrosome and on microtubules. Conversely, increasing centriculum size by genetic means led to the expansion of the PCM, increased microtubule nucleation capacity, and altered spindle width. The effect of the centriculum on centrosome function suggests that in the C.elegans early embryo, the centrosome is not membraneless. Rather, it is encased in a membrane meshwork that affects its properties. We provide evidence that the centriculum serves as a microtubule "filter," preventing the elongation of a subset of microtubules past the centriculum. Finally, we propose that the fusion between the centriculum and the nuclear membrane contributes to nuclear envelope breakdown by coupling spindle elongation to nuclear membrane fenestration.

Torsin and NEP1R1‐CTDNEP1 phosphatase affect interphase nuclear pore complex insertion by lipid‐dependent and lipid‐independent mechanisms

The interphase nuclear envelope (NE) is extensively remodeled during nuclear pore complex (NPC) insertion. How this remodeling occurs and why it requires Torsin ATPases, which also regulate lipid metabolism, remains poorly understood. Here, we show that Drosophila Torsin (dTorsin) affects lipid metabolism via the NEP1R1‐CTDNEP1 phosphatase and the Lipin phosphatidic acid (PA) phosphatase. This includes that Torsins remove NEP1R1‐CTDNEP1 from the NE in fly and mouse cells, leading to subsequent Lipin exclusion from the nucleus. NEP1R1‐CTDNEP1 downregulation also restores nuclear pore membrane fusion in post‐mitotic dTorsinKO fat body cells. However, dTorsin‐associated nuclear pore defects do not correlate with lipidomic abnormalities and are not resolved by silencing of Lipin. Further testing confirmed that membrane fusion continues in cells with hyperactivated Lipin. It also led to the surprising finding that excessive PA metabolism inhibits recruitment of the inner ring complex Nup35 subunit, resulting in elongated channel‐like structures in place of mature nuclear pores. We conclude that the NEP1R1‐CTDNEP1 phosphatase affects interphase NPC biogenesis by lipid‐dependent and lipid‐independent mechanisms, explaining some of the pleiotropic effects of Torsins.

Interphase epichromatin: last refuge for the 30-nm chromatin fiber?

"Interphase epichromatin" describes the surface of chromatin located adjacent to the interphase nuclear envelope. It was discovered in 2011 using a bivalent anti-nucleosome antibody (mAb PL2-6), now known to be directed against the nucleosome acidic patch. The molecular structure of interphase epichromatin is unknown, but is thought to be heterochromatic with a high density of "exposed" acidic patches. In the 1960s, transmission electron microscopy of fixed, dehydrated, sectioned, and stained inactive chromatin revealed "unit threads," frequently organized into parallel arrays at the nuclear envelope, which were interpreted as regular helices with ~ 30-nm center-to-center distance. Also observed in certain cell types, the nuclear envelope forms a "sandwich" around a layer of closely packed unit threads (ELCS, envelope-limited chromatin sheets). Discovery of the nucleosome in 1974 led to revised helical models of chromatin. But these models became very controversial and the existence of in situ 30-nm chromatin fibers has been challenged. Development of cryo-electron microscopy (Cryo-EM) gave hope that in situ chromatin fibers, devoid of artifacts, could be structurally defined. Combining a contrast-enhancing phase plate and cryo-electron tomography (Cryo-ET), it is now possible to visualize chromatin in a "close-to-native" situation. ELCS are particularly interesting to study by Cryo-ET. The chromatin sheet appears to have two layers of ~ 30-nm chromatin fibers arranged in a criss-crossed pattern. The chromatin in ELCS is continuous with adjacent interphase epichromatin. It appears that hydrated ~ 30-nm chromatin fibers are quite rare in most cells, possibly confined to interphase epichromatin at the nuclear envelope.

Diverse cellular functions of barrier-to-autointegration factor and its roles in disease.

Barrier-to-autointegration factor (BAF; encoded by BANF1) is a small highly conserved, ubiquitous and self-associating protein that coordinates with numerous binding partners to accomplish several key cellular processes. By interacting with double-stranded DNA, histones and various other nuclear proteins, including those enriched at the nuclear envelope, BAF appears to be essential for replicating cells to protect the genome and enable cell division. Cellular processes, such as innate immunity, post-mitotic nuclear reformation, repair of interphase nuclear envelope rupture, genomic regulation, and the DNA damage and repair response have all been shown to depend on BAF. This Review focuses on the regulation of the numerous interactions of BAF, which underlie the mechanisms by which BAF accomplishes its essential cellular functions. We will also discuss how perturbation of BAF function may contribute to human disease.

Epichromatin and chromomeres: a 'fuzzy' perspective.

‘Epichromatin’, the surface of chromatin beneath the interphase nuclear envelope (NE) or at the surface of mitotic chromosomes, was discovered by immunostaining with a specific bivalent mouse monoclonal anti-nucleosome antibody (mAb PL2-6). ‘Chromomeres’, punctate chromatin particles approximately 200–300 nm in diameter, identified throughout the interphase chromatin and along mitotic chromosomes, were observed by immunostaining with the monovalent papain-derived Fab fragments of bivalent PL2-6. The specific target for PL2-6 appears to include the nucleosome acidic patch. Thus, within the epichromatin and chromomeric regions, this epitope is ‘exposed’. Considering that histones possess unstructured ‘tails’ (i.e. intrinsically disordered peptide regions, IDPR), our perception of these chromatin regions becomes more ‘fuzzy’ (less defined). We suggest that epichromatin cationic tails facilitate interactions with anionic components of NE membranes. We also suggest that the unstructured histone tails (especially, histone H1 tails), with their presumed promiscuous binding, establish multivalent binding that stabilizes each chromomere as a unit of chromatin higher order structure. We propose an ‘unstructured stability’ hypothesis, which postulates that the stability of epichromatin and chromomeres (as well as other nuclear chromatin structures) is a consequence of the collective contributions of numerous weak histone IDPR binding interactions arising from the multivalent nucleosome, analogous to antibody avidity.

Function of nuclear membrane proteins in shaping the nuclear envelope integrity during closed mitosis.

The nuclear envelope (NE) not only protects the genome from being directly accessed by detrimental agents but also regulates genome organization. Breaches in NE integrity threaten genome stability and impede cellular function. Nonetheless, the NE constantly remodels, and NE integrity is endangered in dividing or differentiating cells. Specifically, in unicellular eukaryotes undergoing closed mitosis, the NE expands instead of breaking down during chromosome segregation. The newly assembling nuclear pore complexes (NPCs) penetrate the existing NE in interphase. A peculiar example of NE remodelling during nuclear differentiation in Tetrahymena involves formation of the redundant NE and clustered NPCs. Even under these conditions, the NE remains intact. Many recent studies on unicellular organisms have revealed that nuclear membrane proteins, such as LEM-domain proteins, play a role in maintaining NE integrity. This review summarizes and discusses how nuclear membrane proteins participate in NE integrity.

Lamins position the nuclear pores and centrosomes by modulating dynein.

Lamins, the type V nuclear intermediate filament proteins, are reported to function in both interphase and mitosis. For example, lamin deletion in various cell types can lead to an uneven distribution of the nuclear pore complexes (NPCs) in the interphase nuclear envelope, whereas deletion of B-type lamins results in spindle orientation defects in mitotic neural progenitor cells. How lamins regulate these functions is unknown. Using mouse cells deleted of different combinations or all lamins, we show that lamins are required to prevent the aggregation of NPCs in the nuclear envelope near centrosomes in late G2 and prophase. This asymmetric NPC distribution in the absence of lamins is caused by dynein forces acting on NPCs via the dynein adaptor BICD2. We further show that asymmetric NPC distribution upon lamin depletion disrupts the distribution of BICD2 and p150 dynactin on the nuclear envelope at prophase, which results in inefficient dynein-driven centrosome separation during prophase. Therefore lamins regulate microtubule-based motor forces in vivo to ensure proper NPC distribution in interphase and centrosome separation in the mitotic prophase.

Structure and biological functions of mammalian LEM-domain proteins

During the process of open mitosis in higher eukaryotic cells, the nuclear envelope (NE) is disassembled and reassembled with highly organized and periodical dynamic morphological changes. Recent studies demonstrated that LEM-domain protein family mediates interactions among inner nuclear membrane, nuclear lamina protein and chromatin by interacting with barrier-to-autointegration-factor (BAF). The structure and function of the ternary complex formed by LEM-domain protein, nuclear lamina protein and BAF are dependent on each other. Moreover, the network formation based on this structure and function is critical for the development of basic biological processes of nuclear, and it plays important roles in chromatin separation in late metaphase and anaphase, NE reassembly after mitosis, morphological maintenance of nuclear and NE in interphase, regulation of DNA replication and DNA damage repair, regulation of gene expression and signaling pathway, and infection of retrovirus. Mutations in genes encoding LEM family proteins have important impacts on development and progression of laminopathic diseases and tumorigenesis. This review provides a detailed summary of structural and functional studies of the LEM family proteins.

ELCS in Ice: Cryo-electron Microscopy of Nuclear Envelope-Limited Chromatin Sheets

Nuclear envelope-limited chromatin sheets (ELCS) form during excessive interphase nuclear envelope growth in a variety of cells. ELCS appear as extended sheets within the cytoplasm connecting distant nuclear lobes. Cross-section stained images of ELCS, viewed by transmission electron microscopy, resemble a sandwich of apposed nuclear envelopes separated by ∼30 nm, containing a layer of parallel chromatin fibers. In this study, the ultrastructure of ELCS was compared by three different methods: (1) aldehyde fixation/dehydration/plastic embedding/sectioning and staining, (2) high-pressure freezing/freeze substitution into plastic/sectioning and staining, and (3) high-pressure freezing/cryo-sectioning/cryo-electron microscopy. ELCS could be clearly visualized by all three methods and, consequently, must exist in vivo and are not fixation artifacts. The ∼30-nm chromatin fibers could only be observed following aldehyde fixation; none were seen in cryo-sections. Electron microscopic tomography tangential views of aldehyde-fixed ELCS suggested an ordering of the separate chromatin fibers adjacent to the nuclear envelope. Possible mechanisms of this chromatin ordering are discussed.

ELCS in ice: cryo-electron microscopy of nuclear envelope-limited chromatin sheets

Nuclear envelope-limited chromatin sheets (ELCS) form during excessive interphase nuclear envelope growth in a variety of cells. ELCS appear as extended sheets within the cytoplasm connecting distant nuclear lobes. Cross-section stained images of ELCS, viewed by transmission electron microscopy, resemble a sandwich of apposed nuclear envelopes separated by ∼30nm, containing a layer of parallel chromatin fibers. In this study, the ultrastructure of ELCS was compared by three different methods: (1) aldehyde fixation/dehydration/plastic embedding/sectioning and staining, (2) high-pressure freezing/freeze substitution into plastic/sectioning and staining, and (3) high-pressure freezing/cryo-sectioning/cryo-electron microscopy. ELCS could be clearly visualized by all three methods and, consequently, must exist in vivo and are not fixation artifacts. The ∼30-nm chromatin fibers could only be observed following aldehyde fixation; none were seen in cryo-sections. Electron microscopic tomography tangential views of aldehyde-fixed ELCS suggested an ordering of the separate chromatin fibers adjacent to the nuclear envelope. Possible mechanisms of this chromatin ordering are discussed.

Spindle pole bodies

Spindle pole bodies

Brr6 drives the Schizosaccharomyces pombe spindle pole body nuclear envelope insertion/extrusion cycle

The fission yeast interphase spindle pole body (SPB) is a bipartite structure in which a bulky cytoplasmic domain is separated from a nuclear component by the nuclear envelope. During mitosis, the SPB is incorporated into a fenestra that forms within the envelope during mitotic commitment. Closure of this fenestra during anaphase B/mitotic exit returns the cytoplasmic component to the cytoplasmic face of an intact interphase nuclear envelope. Here we show that Brr6 is transiently recruited to SPBs at both SPB insertion and extrusion. Brr6 is required for both SPB insertion and nuclear envelope integrity during anaphase B/mitotic exit. Genetic interactions with apq12 and defective sterol assimilation suggest that Brr6 may alter envelope composition at SPBs to promote SPB insertion and extrusion. The restriction of the Brr6 domain to eukaryotes that use a polar fenestra in an otherwise closed mitosis suggests a conserved role in fenestration to enable a single microtubule organizing center to nucleate both cytoplasmic and nuclear microtubules on opposing sides of the nuclear envelope.

Nuclear envelope dynamics during plant cell division suggest common mechanisms between kingdoms

Behaviour of the NE (nuclear envelope) during open mitosis has been explored extensively in metazoans, but lack of native markers has limited similar investigations in plants. In the present study, carried out using living synchronized tobacco BY-2 suspension cultures, the non-functional NE marker LBR (lamin B receptor)-GFP (green fluorescent protein) and two native, functional NE proteins, AtSUN1 [Arapidopsis thaliana SUN (Sad1/UNC84) 1] and AtSUN2, we provide evidence that the ER (endoplasmic reticulum)-retention theory for NE membranes is applicable in plants. We also observe two apparently unique plant features: location of the NE-membrane components in close proximity to chromatin throughout division, and spatially distinct reformation of the NE commencing at the chromatin surface facing the spindle poles and concluding at the surface facing the cell plate. Mobility of the proteins was investigated in the interphase NE, during NE breakdown and reformation, in the spindle membranes and the cell plate. A role for AtSUN2 in nuclear envelope breakdown is suggested.

AtFH8 Is Involved in Root Development under Effect of Low-Dose Latrunculin B in Dividing Cells

Formins have been paid much attention for their potent nucleating activity. However, the connection between the in vivo functions of AtFHs (Arabidopsis thaliana formin homologs) and their effects on actin organization is poorly understood. In this study, we characterized the bundling activity of AtFH8 in vitro and in vivo. Biochemical analysis showed that AtFH8(FH1FH2) could form dimers and bundle preformed actin filaments or induce stellar structures during actin polymerization. Expression of truncated forms of AtFH8 and immunolocalization analysis showed that AtFH8 localized primarily to nuclear envelope in interphase and to the new cell wall after cytokinesis, depending primarily on its N-terminal transmembrane domain. GUS histochemical staining showed AtFH8 was predominantly expressed in Arabidopsis root meristem, vasculature, and outgrowth points of lateral roots. The primary root growth and lateral root initiation of atfh8 could be decreased by latrunculin B (LatB). Analysis of the number of dividing cells in Arabidopsis root tips showed that much fewer dividing cells in Lat B-treated atfh8 plants than wild-type plants, which indicates that AtFH8 was involved in cell division. Actin cytoskeleton in root meristem of atfh8-1 was more sensitive to LatB treatment than that of wild-type. Altogether, our results indicate that AtFH8 is an actin filament nucleator and bundler that functions in cell division and root development.

Going green: plants' alternative way to position the Ran gradient

Ran is a multi-functional small GTPase of the Ras super-family involved in nucleocytoplasmic transport, mitotic spindle assembly, cell cycle control and nuclear envelope (NE) formation. Its roles are accomplished by the asymmetric distribution of its GTP- and GDP-bound forms, enabled by the specific localization of Ran accessory proteins, the Ran GTPase-activating protein RanGAP and the nucleotide exchange factor RCC1. Mammalian RanGAP1 is targeted to the NE during interphase and to the spindle and kinetochores during mitosis via a SUMOylated C-terminal domain and interaction with the nucleoporin Nup358/RanBP2. Arabidopsis RanGAP1 (AtRanGAP1) lacks the SUMOylated C-terminal domain of vertebrate RanGAP, but contains a plant-specific N-terminal domain (WPP domain), which is necessary and sufficient for its targeting to the NE in interphase. AtRanGAP1 has a mitotic trafficking pattern uniquely different from that of vertebrate RanGAP, which includes targeting to the outward-growing rim of the cell plate. The WPP domain is necessary and sufficient for this targeting. Now, a novel family of plant-specific, nuclear pore-associated proteins has been identified in Arabidopsis, which is essential for anchoring RanGAP to the Arabidopsis nuclear envelope at the root meristem. This suggests that RanGAP anchoring to the nuclear pore has been solved in two fundamentally different ways in animals and plants. These findings support a separate evolution of RanGAP targeting mechanisms in different kingdoms, possibly related to different functional geometries of the Ran gradient in animal and higher plant cell division.

Localized Regulation of Axonal RanGTPase Controls Retrograde Injury Signaling in Peripheral Nerve

Peripheral sensory neurons respond to axon injury by activating an importin-dependent retrograde signaling mechanism. How is this mechanism regulated? Here, we show that Ran GTPase and its associated effectors RanBP1 and RanGAP regulate the formation of importin signaling complexes in injured axons. A gradient of nuclear RanGTP versus cytoplasmic RanGDP is thought to be fundamental for the organization of eukaryotic cells. Surprisingly, we find RanGTP in sciatic nerve axoplasm, distant from neuronal cell bodies and nuclei, and in association with dynein and importin-alpha. Following injury, localized translation of RanBP1 stimulates RanGTP dissociation from importins and subsequent hydrolysis, thereby allowing binding of newly synthesized importin-beta to importin-alpha and dynein. Perturbation of RanGTP hydrolysis or RanBP1 blockade at axonal injury sites reduces the neuronal conditioning lesion response. Thus, neurons employ localized mechanisms of Ran regulation to control retrograde injury signaling in peripheral nerve.

NUCLEAR PORE ANCHOR, theArabidopsisHomolog of Tpr/Mlp1/Mlp2/Megator, Is Involved in mRNA Export and SUMO Homeostasis and Affects Diverse Aspects of Plant Development

Vertebrate Tpr and its yeast homologs Mlp1/Mlp2, long coiled-coil proteins of nuclear pore inner basket filaments, are involved in mRNA export, telomere organization, spindle pole assembly, and unspliced RNA retention. We identified Arabidopsis thaliana NUCLEAR PORE ANCHOR (NUA) encoding a 237-kD protein with similarity to Tpr. NUA is located at the inner surface of the nuclear envelope in interphase and in the vicinity of the spindle in prometaphase. Four T-DNA insertion lines were characterized, which comprise an allelic series of increasing severity for several correlating phenotypes, such as early flowering under short days and long days, increased abundance of SUMO conjugates, altered expression of several flowering regulators, and nuclear accumulation of poly(A)+ RNA. nua mutants phenocopy mutants of EARLY IN SHORT DAYS4 (ESD4), an Arabidopsis SUMO protease concentrated at the nuclear periphery. nua esd4 double mutants resemble nua and esd4 single mutants, suggesting that the two proteins act in the same pathway or complex, supported by yeast two-hybrid interaction. Our data indicate that NUA is a component of nuclear pore-associated steps of sumoylation and mRNA export in plants and that defects in these processes affect the signaling events of flowering time regulation and additional developmental processes.

Plant‐specific mitotic targeting of RanGAP requires a functional WPP domain

The small GTPase Ran is involved in nucleocytoplasmic transport, spindle formation, nuclear envelope (NE) formation, and cell-cycle control. In vertebrates, these functions are controlled by a three-dimensional gradient of Ran-GTP to Ran-GDP, established by the spatial separation of Ran GTPase-activating protein (RanGAP) and the Ran guanine nucleotide exchange factor RCC1. While this spatial separation is established by the NE during interphase, it is orchestrated during mitosis by association of RCC1 with the chromosomes and RanGAP with the spindle and kinetochores. SUMOylation of vertebrate RanGAP1 is required for NE, spindle, and centromere association. Arabidopsis RanGAP1 (AtRanGAP1) lacks the SUMOylated C-terminal domain of vertebrate RanGAP, but contains a plant-specific N-terminal domain (WPP domain), which is necessary and sufficient for its targeting to the NE in interphase. Here we show that the human and plant RanGAP-targeting domains are kingdom specific. AtRanGAP1 has a mitotic trafficking pattern uniquely different from that of vertebrate RanGAP, which includes targeting to the outward-growing rim of the cell plate. The WPP domain is necessary and sufficient for this targeting. Point mutations in conserved residues of the WPP domain also abolish targeting to the nuclear rim and the cell plate, suggesting that the same mechanism is involved in both targeting events. These results indicate that plant and animal RanGAPs undergo different migration patterns during cell division, which require their kingdom-specific targeting domains.

Importin β is transported to spindle poles during mitosis and regulates Ran-dependent spindle assembly factors in mammalian cells

Spatial control is a key issue in cell division. The Ran GTPase regulates several fundamental processes for cell life, largely acting through importin molecules. The best understood of these is protein import through the nuclear envelope in interphase, but roles in mitotic spindle assembly are also established. In mammalian cells, in which centrosomes are major spindle organizers, a link is emerging between the Ran network, centrosomes and spindle poles. Here, we show that, after nuclear envelope breakdown, importin beta is transported to the spindle poles in mammalian cells. This localization is temporally regulated from prometaphase until anaphase, when importin beta dissociates from poles and is recruited back around reforming nuclei. Importin beta sediments with mitotic microtubules in vitro and its accumulation at poles requires microtubule integrity and dynamics in vivo. Furthermore, RNA interference-dependent inactivation of TPX2, the major Ran-dependent spindle organizer, abolishes importin beta accumulation at poles. Importin beta has a functional role in spindle pole organization, because overexpression yields mitotic spindles with abnormal, fragmented poles. Coexpression of TPX2 with importin beta mitigates these abnormalities. Together, these results indicate that the balance between importins and spindle regulators of the TPX2 type is crucial for spindle formation. Targeting of TPX2/importin-beta complexes to poles is a key aspect in Ran-dependent control of the mitotic apparatus in mammalian cells.