Molecular Pathology of Laminopathies.

141st ENMC International Workshop Inaugural Meeting of the EURO-Laminopathies Project Nuclear Envelope-linked Rare Human Diseases: From Molecular Pathophysiology towards Clinical Applications 10–12 March 2006, Naarden, The Netherlands

The double membrane nuclear envelope (NE), which is contiguous with the ER, contains nuclear pore complexes (NPCs) – the channels for nucleocytoplasmic transport, and the nuclear lamina (NL) – a scaffold for NE and chromatin organization. Since numerous human diseases linked to NE proteins occur in mesenchyme-derived cells, we used proteomics to characterize NE and other subcellular fractions isolated from mesenchymal stem cells and from adipocytes and myocytes. Based on spectral abundance, we calculated enrichment scores for proteins in the NE fractions. We demonstrated by quantitative immunofluorescence microscopy that five little-characterized proteins with high enrichment scores are substantially concentrated at the NE, with Itprip exposed at the outer nuclear membrane, Smpd4 enriched at the NPC, and Mfsd10, Tmx4, and Arl6ip6 likely residing in the inner nuclear membrane. These proteins provide new focal points for studying the functions of the NE. Moreover, our datasets provide a resource for evaluating additional potential NE proteins.

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 G(1) 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.

We investigated the role of LMNA in adipose tissue by developing a novel mouse model of lipodystrophy. Transgenic mice were generated that express the LMNA mutation that causes familial partial lipodystrophy of the Dunnigan type (FPLD2). The phenotype observed in FPLD-transgenic mice resembles many of the features of human FPLD2, including lack of fat accumulation, insulin resistance, and enlarged, fatty liver. Similar to the human disease, FPLD-transgenic mice appear to develop normally, but after several weeks they are unable to accumulate fat to the same extent as their wild-type littermates. One poorly understood aspect of lipodystrophies is the mechanism of fat loss. To this end, we have examined the effects of the FPLD2 mutation on fat cell function. Contrary to the current literature, which suggests FPLD2 results in a loss of fat, we found that the key mechanism contributing to the lack of fat accumulation involves not a loss, but an apparent inability of the adipose tissue to renew itself. Specifically, preadipocytes are unable to differentiate into mature and fully functional adipocytes. These findings provide insights not only for the treatment of lipodystrophies, but also for the study of adipogenesis, obesity, and insulin resistance.

Nuclear Envelope, Nuclear Lamina, and Inherited Disease

The Nuclear Envelope as a Signaling Node in Development and Disease

Lamins are nuclear intermediate filament proteins. They provide mechanical stability, organize chromatin and regulate transcription, replication, nuclear assembly and nuclear positioning. Recent studies provide new insights into the role of lamins in development, differentiation and tissue response to mechanical, reactive oxygen species and thermal stresses. These studies also propose the existence of separate filament networks for A- and B-type lamins and identify new roles for the different networks. Furthermore, they show changes in lamin composition in different cell types, propose explanations for the more than 14 distinct human diseases caused by lamin A and lamin C mutations and propose a role for lamin B1 in these diseases.

Nuclear pore complexes (NPCs) are supramolecular nanomachines that mediate the exchange of macromolecules and inorganic ions between the nucleus and the cytosol. Although there is no doubt that large cargo is transported through the centrally located channel, the route of ions and small molecules remains debatable. We thus tested the hypothesis that there are two separate pathways by imaging NPCs using atomic force microscopy, NPC electrical conductivity measurements, and macromolecule permeability assays. Our data indicate a spatial separation between the active transport of macromolecules through the central channel and the passive transport of ions and small macromolecules through the pore periphery.

The present study demonstrates a major remodeling of the nuclear envelope and its underlying lamina during bovine preimplantation development. Up to the onset of major embryonic genome activation (MGA) at the 8-cell stage nuclei showed a non-uniform distribution of nuclear pore complexes (NPCs). NPCs were exclusively present at sites where DNA contacted the nuclear lamina. Extended regions of the lamina, which were not contacted by DNA, lacked NPCs. In post-MGA nuclei the whole lamina was contacted rather uniformly by DNA. Accordingly, NPCs became uniformly distributed throughout the entire nuclear envelope. These findings shed new light on the conditions which control the integration of NPCs into the nuclear envelope. The switch from maternal to embryonic production of mRNAs was accompanied by multiple invaginations covered with NPCs, which may serve the increased demands of mRNA export and protein import. Other invaginations, as well as interior nuclear segments and vesicles without contact to the nuclear envelope, were exclusively positive for lamin B. Since the abundance of these invaginations and vesicles increased in concert with a massive nuclear volume reduction, we suggest that they reflect a mechanism for fitting the nuclear envelope and its lamina to a shrinking nuclear size during bovine preimplantation development. In addition, a deposit of extranuclear clusters of NUP153 (a marker for NPCs) without associated lamin B was frequently observed from the zygote stage up to MGA. Corresponding RNA-Seq data revealed deposits of spliced, maternally provided NUP153 mRNA and little unspliced, newly synthesized RNA prior to MGA, which increased strongly at the initiation of embryonic expression of NUP153 at MGA.

Nuclear transport pathways use soluble receptors to mediate the translocation of protein and cargo through the nuclear pore complex (NPC). In October 2005, investigators from around the globe braved the threat of hurricane Wilma and presented their latest findings at the Nucleocytoplasmic Transport Meeting at Jekyll Island, GA, USA. The meeting was organized by Drs Maureen Powers, Chuck Cole, and Douglass Forbes. Major themes within the meeting included architecture of the NPC, structural analysis of receptors, regulation of nuclear transport, models for translocation, and emerging roles for the NPC and lamina in gene expression. The transport of proteins and RNAs between the cytoplasm and nucleus is mediated by nuclear pore complexes (NPCs), large proteinaceous structures embedded in the double membrane of the nuclear envelope (NE) (1–4). The NPC structure is conserved in all eukaryotes and is composed of approximately 30 separate proteins termed nucleoporins (Nups), most of which are present in multiple copies per NPC. Nups provide docking sites for soluble transport receptors that mediate the translocation of protein and RNA cargoes through the NPC. The karyopherin family is composed of receptors for import (importins) and export (exportins). These receptors mediate most protein import and export pathways in the cell, as well as the export of some RNAs. Transport receptors recognize specific sequences termed nuclear localization signals (NLSs) or nuclear export signals (NESs) contained in the cargo protein. mRNAs are exported by a class of transport receptors that are distinct from the karyopherin family. An important regulator of nuclear transport is the small GTPase Ran, and its GTPase activating protein (RanGAP) and guanine nucleotide exchange factor (RanGEF or RCC1). Previous models of the NPC have been based primarily on low-resolution cryo-electron microscopy (EM) studies and the immuno-EM localization of a handful of NPC proteins (Figure 1A) (4). More recently, proteomic approaches have established the identities of virtually all the protein constituents of the NPC, setting the stage for a more complete picture of the NPC (5,6). Michael Rout (The Rockefeller University, New York, NY, USA) and colleagues used a computational approach to reconstruct the arrangement of 30 different Nups in the NPC. The model combined EM immunolocalization data with protein–protein interaction data obtained by immunoprecipitation of tagged Nups. This exciting model gives us the first glimpse of an integrated view of the locations of individual Nups and various subcomplexes of the NPC. Of course, solving the atomic structure of the NPC is one of the ‘holy grails’ of the field, and Thomas Schwartz (Massachusetts Institute of Technology, Boston, MA, USA) updated the meeting on his progress toward solving the structure of the p62 complex, a subcomplex located in the central channel of the NPC. Given its enormous size (>100 MDa), it might be necessary to piece together subcomplexes to solve the complete NPC structure. Antibody studies have allowed the localization of a small number of Nups that are asymmetrically positioned on either the nuclear or cytoplasmic sides of the NPC, and Nups with biased locations are good candidates for specialized transport functions. Two nucleoplasmic Nups in vertebrates are the large nucleoporins Nup153 and TPR. Volker Cordes (ZMBH, Heidelberg, Germany) used domain-specific antibodies to analyze the structural arrangement of TPR at the NPC by scanning and transmission EM. He found that different TPR domains correspond to distinct segments of the nuclear basket, with TPR homodimers forming a central loop to contact Nup153 at the NPC nuclear ring, while both N- and C-terminus of TPR project toward the nucleoplasm. Volker's antibody data provide strong evidence that TPR is the key component of the nuclear basket. Gallery of images illustrating some diverse aspects of nuclear structure and function.(A) NPCs on the surface of the NE membrane of Xenopus oocytes visualized by field emission scanning electron microscopy. Cytoplasmic filaments of the NPC appear as globular structures that line the perimeter of the pore. The image is courtesy of Dr Terry Allen. (B) Mitotic embryo fibroblast from a rae1+/–nup98+/– mouse imaged during anaphase B. While most of the chromosomes (green) have moved to the spindle poles, lagging chromosomes (red arrow) are visible and give rise to aneuploidy. Image is courtesy of Dr Jan van Duersen. (C) Model of transmembrane linkages between proteins in the cytoplasm and nucleus. Nesprins bind actin filaments in the cytoplasm and contact SUN domain proteins in the NE lumen. SUN domain proteins also contact lamins; these interactions are thought to provide a continuous linkage between the cytoskeleton and the nucleoskeleton. The image is courtesy of Dr Brian Burke and is reproduced from Crisp et al. (26) with permission from Rockefeller University Press. A subset of Nups contain repeats of the amino acids phenylalanine-glycine, and many studies have established that these FG repeats are binding sites for the karyopherins moving through the central channel of the NPC (1,2,7). It is estimated that the central channel of the NPC contains several hundred FG repeats. There is much debate within the field as to what this channel looks like and how it mediates a selective gating mechanism. Models include a central tube lined with FG repeats that acts as an affinity gate (also called virtual gating), or a sieve-like channel where disordered FG repeats mediate hydrophobic interactions between Nups creating a gel-like phase that generates a permeability barrier within the NPC (also called the selective phase model) (1,2). Michael Rexach (University of California, Santa Cruz, CA, USA) used a new bead-based, real-time equilibrium-binding assay to test for low-affinity interactions between FG regions of Nups. Binding required phenylalanine residues in FG motifs and was disrupted by hexanediol, an alcohol known to weaken the NPC permeability barrier in transport assays. He concluded that FG regions of Nups interact with each other, supporting the ‘selective phase’ model for NPC gating. Dirk Gorlich (ZMBH, Heidelberg, Germany) reported on efforts by his group to address the properties of the central channel envisioned to be important for the selective phase model. He surprised the meeting by showing that a single FG nucleoporin, Nsp1p, can assemble into a three-dimensional ‘hydrogel’ with molecular sieve-like properties. Dirk also showed that a mutant version of Nsp1p where the >50 FG repeats had been mutated fails to form the ‘hydrogel’. These results were consistent with the idea that interactions between hydrophobic repeats in Nups could play structural and functional roles in the permeability barrier. Future experiments will determine whether this ‘hydrogel’ displays other properties of the central channel and help address longstanding questions regarding the central channel, including how gating of the NPC is achieved. Earlier studies by several groups have indicated that different karyopherins display distinct binding specificities for FG Nups in vitro. An extensive collection of FG-domain deletion strains created by the Wente laboratory is being used to probe the interactions between different karyopherins and the central channel in Saccharomyces cerevisiae (8). It has been shown that Kap104p-mediated translocation is more sensitive than Kap95p/Kap60p-mediated translocation to certain FG-domain deletions (8). David Goldfarb (University of Rochester, Rochester, NY, USA) presented recent experiments that are most consistent with the notion that Kap95p/Kap60p does better than Kap104p in these FG-domain deletion strains, because it competes better for remaining FG-binding sites. This advantage may normally occur under stress, because elevated levels of Hsp70 favor the translocation of Kap95p/Kap60p over Kap104p. The karyopherin family comprises the major family of transport receptors. In yeast, there are 14 members of this family, and in human cells, there are more than 20 members (1,2). Although the amino acid sequence identities are low, each Kap has similar large size (approximately 100 kDa) and are all predicted to consist of multiple (approximately 20) HEAT motifs. The HEAT motif is a helix-loop-helix structure that owes its name to the proteins in which it was originally identified (9). Kaps contain an N-terminal domain that interacts with RanGTP and nucleoporins. Kaps also bind directly (or via adapters) to the NLSs or NESs of their cargoes. The crystal structures of several karyopherins, empty, with cargo, and with RanGTP, have all been solved. At the meeting, several groups focused on further analysis of karyopherins at the atomic level. The overall theme in these talks was that karyopherins have great conformational flexibility and that members of this family can have different conformations despite the fact that they all are composed of tandem HEAT repeats. Murray Stewart (Medical Research Council, Cambridge, UK) discussed the crystal structure of Kap95p. He proposed that this importin is flexible when binding to cargoes, but the interaction with RanGTP, which precludes cargo binding, introduces inflexibility into the protein. Murray continued this theme in discussing his structure of the Cse1p, the receptor that mediates nuclear export of importin-α (10). Elena Conti's laboratory (EMBL, Heidelberg, Germany) has solved the structure of unliganded Cse1p and found it to form a relatively rigid ring-like structure. In this closed conformation, the binding sites for either importin-α or RanGTP are distorted. Indeed, Cse1p binds either importin-α or Ran with micromolar affinity, while the simultaneous interaction of Cse1p with importin-α and RanGTP occurs with nanomolar affinity and is correlated with the open conformation of Cse1p observed in Murray's structure. This conformational regulation provides a rationale for why Cse1p fails to interact with importin-α in the cytosol, in the absence of RanGTP. Reporting on a low-resolution study of conformations of different transport factors, Elena dismissed the idea of a general importin-like or exportin-like structure. For example, the exportin Xpo-t is more similar in overall structure to importin-β than it is to the exportin Cse1 (11). It appears that the most common feature of karyopherins is their flexibility in acquiring distinct conformations depending on whether they are empty or interacting with RanGTP or cargo. Conformational differences were to be expected due to acidic loops and insertions found between HEAT repeats in assorted family members. Lucy Pemberton (University of Virginia, Charlottesville, VA, USA) reported that a single karyopherin, Kap114p, can bind multiple cargoes simultaneously in vitro, and hopefully future structures will address how these 7interactions take place at the atomic level. Collectively, these studies suggest that we still have a lot to learn about the complexity of karyopherin–cargo interactions. There was a very lively session describing efforts to analyze the mechanism of nuclear transport at the single-molecule level, as well as efforts to model nuclear transport. Although much is known about individual components of the transport machinery, and many important binding interactions have been characterized, tracking individual cargoes as they transit through the NPC should provide clues about the transport mechanism. Ulrich Kubitschek (University of Bonn, Bonn, Germany) used single-molecule far-field fluorescence microscopy to visualize nuclear transport through NPCs of both permeabilized and living cells. Intracellular single-molecule detection in the nanometer range is feasible using this technique. Binding of cargo reduced the dwell times of the transport receptors in the NPC, suggesting that cargo has an important influence on karyopherin interactions with Nups. Seigfried Musser (Texas A&M, College Station, TX, USA) presented his measurements of import efficiency, which is the number of molecules that are transported divided by the number of molecules that interact with an NPC. In the permeabilized cell system, he found that both import efficiency and translocation time were dependent on the concentration of the importin-β, as import efficiency was higher and the translocation time was faster with increasing importin-β concentrations. Seigfried speculated that the cargo-free importin-β concentration within the NPC may regulate nucleocytoplasmic transport rates. Ruti Kapon (Weizmann Institute of Science, Rehovot, Israel) examined receptor-mediated import through the NPC using a non-equilibrium statistical mechanics model where all reactions are modeled using kinetic rates. Her model suggested that the selectivity of the channel against receptor-free cargo, in itself, leads to accumulation of the cargo in the destination compartment even if the translocation process itself is unbiased. A major question in the field is how the NPC coordinates import and export through the same channel. Ruti suggested that synchronization of import and export is based on stochastic fluctuations in the NPC. She posited the NPC is unidirectional for a characteristic time, but it can spontaneously change its directionality. Thus, for given periods, an individual NPC can be dedicated to either import or export. Experimental analysis is required to establish the validity of these models. Ian Macara (University of Virginia, Charlottesville, VA, USA) presented his ongoing studies that combine computer simulation and microinjection assays to monitor fluorescent cargoes and recombinant receptors in real time. Ian discussed his previous observation that in living cells, high levels of importin-β or RanGEF inhibit import of an NLS-cargo (12). Excess RanGEF probably sequesters Ran in the nucleus, whereas excess importin-β may deplete Ran from the nucleus by co-opting it for futile nucleoplasmic shuttling cycles. In his new studies, Ian used a shuttling NES-NLS cargo that can reach steady-state localization in the cell. Excess importin-β and Crm1 decreased the N:C ratio of the reporter, whereas importin-α increased the N:C ratio. Components of the Ran system had little effect most likely because the reporter reflected both import and export function. There was an interesting discussion regarding how importin-α-dependent transport is less efficient and energetically more costly given the need to recycle importin-α to the cytoplasm, but importin-α proteins endow the cell with greater flexibility in terms of binding diverse cargoes (13). Nuclear export of mRNA is mediated principally by Mex67p/TAP/NXF1, a highly conserved receptor family that is unrelated to the karyopherin family of import and export receptors (14). Functional coupling between mRNA export and upstream reactions (transcription and mRNA processing) is mediated by several multiprotein complexes. The TREX complex consists of proteins involved in transcription elongation and mRNA export and includes the THO protein complex as well as the mRNA export factors Yra1p/Aly and Sub2p/Uap56 (14). There were several talks that addressed the links between mRNA transcription, processing and nuclear export. Andres Aguilera (University of Seville, Spain) showed that the THO protein complex functions in co-transcriptional mRNP formation, and he described a new component of the THO complex, Tho1p. Tho1p is a conserved RNA-binding protein that interacts with transcribed chromatin in a THO- and RNA-dependent manner. These results suggested that Tho1p, similar to Sub2p, assembles onto nascent mRNA during transcription and participates in mRNP biogenesis and nuclear export. Nuclear export of mRNA is also coordinated with mRNA splicing and 5′ capping. Robin Reed (Harvard Medical School, Boston, MA, USA) showed that the human TREX complex is recruited to the 5′-end of mRNA in a splicing- and cap-dependent manner. The TREX complex binds to the 5′-methyl cap-binding protein, CBP80, an interaction that provides a plausible mechanism for TREX recruitment to the 5′-end of mRNA. The findings presented by Robin suggest a model of mRNA export that differs from models from Melissa Moore's laboratory, which were based on coupling the export machinery to the exon junction complex (15). Francoise Stutz (University of Geneva, Geneva, Switzerland) continued the theme of coupled reactions by describing links between the RNA export factor Yra1p/Aly, and Mlp1p and Mlp2p, yeast homologs of TPR that face the nucleoplasm. Previously, Francoise identified genetic interactions between the MLP1/2 and YRA1 that suggested that the Mlp1/2p proteins provide a scaffold at the NPC for Yra1p-dependent docking of mRNPs (16). Yra1p appears to directly regulate transcription, as recruitment of TBP to an induced promoter is decreased in a yra1 mutant. Francoise found that this effect could be reversed in a yra1/mlp2 double mutant, suggesting that Mlp1/2p negatively regulates the transcriptional function of Yra1p. Collectively, these talks emphasized the close links between nuclear export, transcription and RNA processing and suggested that the NPC may regulate transcription by several mechanisms. John Aitchison (Institute for Systems Biology, Seattle, WA, USA) focused on the role of nucleoporin Nup2p, which displays dynamic associations with the NPC. John investigated the previous observation that Nup2p has boundary activity when tethered to chromatin (17). John and colleagues used a variety of assays, including chromatin immunoprecipitation and gene arrays, to show that Nup2p regulates gene expression and gene silencing. Another component of the nuclear transport machinery, the RanGEF Prp20p, was found to have similar chromatin-related functions. The human homolog of Prp20p is known to interact with core histones, and as Nup2p interacts with both the NPC and Prp20p, these data argue for linkage between the NPC and chromatin. As the association of Nup2p with the NPC is transient, the data suggest that the NPC might play an anchoring role and help direct the partitioning of chromatin into inactive and active regions. The mRNA export events downstream of TREX and TAP/Nxf1/Mex67p are poorly defined. Some exported hnRNPs are enormous, suggesting that this must be an energy-requiring process. RanGTP does not appear to regulate this process, suggesting that other important regulatory mechanisms might exist. Three talks discussed Dbp5p, an RNA helicase that plays a role in mRNA export and transiently associates with the cytoplasmic side of the NPC. Karsten Weis (University of California, Berkeley, CA, USA) showed that the DExD/H-box protein Dbp5p has intrinsically low ATPase activity and is unable to associate stably with RNA in the absence of ATP, suggesting that Dbp5p alone is an inactive enzyme. He also showed that the NPC-associated protein Gle1p the ATPase activity of Dbp5p and presented a model in which these proteins as a complex to mRNA export. Wente University, USA) reported a similar that Gle1p and Dbp5p but in was to show that the the activity of Dbp5p by in the of the This is interesting in of analysis of the role of in nuclear transport, where showed that cytoplasmic is required for mRNA export Collectively, these findings to how small molecules may regulate mRNA export. Chuck Medical School, USA) focused on the role of Dbp5p in mRNA processing at distinct from export through the NPC. mRNA is increased in a mutant, and a of Dbp5p with He described new between Dbp5p and which are cytoplasmic structures that are to be necessary for mRNA and these data the model that Dbp5p may play important roles in mRNA export and processing both at the NPC and in the factors that play important roles in mRNA export have been and (University of California, CA, USA) described analysis of the yeast mRNA export factor This protein is an membrane protein that to the Although is not thought to be a of the NPC, it interacts with several which is consistent with a role in transport. She showed that interacts with mRNPs in and is required for the NE localization of the mRNA export factor She proposed a model where this membrane protein provides binding sites to the NPC to help mRNPs and in the for export. these studies suggest that mRNA export is a process a of factors that appear to regulate this process from transcription to talks addressed nucleocytoplasmic transport of components required for protein Medical USA) discussed the import and export of which are one of the most of In previous showed that in the nucleus in the exportin mutant can be into the nucleus in a mechanism that is dependent on Ran and the importin that of might be of an mRNA or it could be of a mechanism for given that in the nucleus in to (University of TX, USA) focused on nuclear export of the which on as an for export. of the from the cytoplasmic GTPase and the protein In in the nucleus, and accumulation in the These suggest a mechanism of the export complex can occur in the focused on the nuclear export and cytoplasmic processing of RNA component of the He showed that processing of the of the in occurs in the cytoplasm nuclear export. This is a of models that show that processing is in the nucleus. This is necessary for the activity of the in and might be important for interaction with in the nucleus. consist of core proteins and an RNA and are for biogenesis and mRNA It is not known how these but University, New York, NY, USA) the role of the protein in the process. between nucleus and cytoplasm and one of the core to the nascent is by core protein, to in and transcription factors of import and export as a of cytoplasmic signals with nuclear import export can have a influence on the nuclear concentration of transcription factors The receptor and (University of Virginia, Charlottesville, VA, USA) presented showing that export is by of a to the export (University of Germany) described his molecular analysis of nuclear export of nuclear factor of of nucleoporins or export of RNA export or protein and are on the cytoplasmic side of the NPC these Nups may be the sites where export complexes are and into the nucleoporins a that has on a number of transcription factors and The of the that this has the stage for how regulates protein USA) reported that reduced levels of in the in the nuclear transport of a transcription factor that is a downstream of transcription factors to on nuclear import mediated by karyopherins, but the of the protein can an that direct interaction with John USA) presented findings from and yeast that a model nuclear localization of high group transcription factors as than the observation that nuclear transport can be correlated with the in cells, the of whether there are in the nuclear transport machinery during in is University, used in and to show that there are in the expression of importin-α and importin-β receptors during This that nuclear transport might be at the of receptor during links between cell the import receptor and the RNA-binding protein were reported by University, of the import was shown to accumulation in the cytoplasm of in As can bind and factors and the effect of cytoplasmic was to and protein cell The study of and their regulatory mechanisms has many aspects of cell and the field of nuclear transport has been of the first export signals to be is by the human protein and to be a cargo for cell nuclear export. an RNA export function that is for the it is a David (University of Virginia, Charlottesville, VA, USA) presented his new results from a to of the small molecules in the display micromolar of function in cells. David an that to one of the small of that function can be identified in this of of regulation in has to a based on signals that nuclear export of RNA to the The transport in which this is by the export receptor The is to be to higher but the for RNA export signals in mRNAs has been by the in these signals at the sequence level. have for the of RNA export (University of Virginia, Charlottesville, VA, USA) used a system to for and identified in the human The have sequence to the with one the for a which that mediates nuclear export of its mRNA. In previous USA) had identified a nucleotide RNA transport and showed that its activity is to the from used a to in in the mouse RNA export, like RNA export, on the appears to contact with the structural between and should provide into the important of of mRNAs by direct mechanisms. Two talks presented at the meeting addressed how to the nucleus. In the of proteins are and the is through the NPC into the cell nucleus. (The Research CA, USA) has to the import mechanism and has identified a key role for protein which contains NLSs and the interaction between and cell import

Since the identification of the first disease causing mutation in the gene coding for emerin, a transmembrane protein of the inner nuclear membrane, hundreds of mutations and variants have been found in genes encoding for nuclear envelope components. These proteins can be part of the inner nuclear membrane (INM), such as emerin or SUN proteins, outer nuclear membrane (ONM), such as Nesprins, or the nuclear lamina, such as lamins A and C. However, they physically interact with each other to insure the nuclear envelope integrity and mediate the interactions of the nuclear envelope with both the genome, on the inner side, and the cytoskeleton, on the outer side. The core of this complex, called LINC (LInker of Nucleoskeleton to Cytoskeleton) is composed of KASH and SUN homology domain proteins. SUN proteins are INM proteins which interact with lamins by their N-terminal domain and with the KASH domain of nesprins located in the ONM by their C-terminal domain.Although most of these proteins are ubiquitously expressed, their mutations have been associated with a large number of clinically unrelated pathologies affecting specific tissues. Moreover, variants in SUN proteins have been found to modulate the severity of diseases induced by mutations in other LINC components or interactors. For these reasons, the diagnosis and the identification of the molecular explanation of “nuclear envelopathies” is currently challenging.The aim of this review is to summarize the human diseases caused by mutations in genes coding for INM proteins, nuclear lamina, and ONM proteins, and to discuss their potential physiopathological mechanisms that could explain the large spectrum of observed symptoms.

In contrast to the well-studied process of nucleocytoplasmic transport of soluble macromolecules, little is known about the targeting of integral membrane proteins to the inner nuclear membrane (INM), therefore the targeting of INM-proteins was studied in this work. Two major models haven been proposed for INM-protein transport, the diffusion-retention model, which assumes passive diffusion of proteins from the ER membrane, across the nuclear pore complex (NPC) to the INM and the transport factor-mediated model, where membrane proteins are believed to translocate across the NPC into the nucleus upon binding to nuclear import receptors. In this work, the targeting of proteins to the INM was studied for several tail-anchored (TA)-proteins, which contain only a single C-terminal transmembrane domain (TMD). As a model protein, the well-established INM-protein emerin was used. With the help of membrane insertion assays, we showed that emerin, expressed in bacteria or a cell-free lysate, can be post-translationally integrated into ER membranes by the TRC40-pathway. The involvement of the TRC40-system was shown by use of inhibitory fragments of the ER-membrane receptors WRB and CAML in the membrane insertion reaction. An interaction between emerin and TRC40 could further be confirmed by proximity ligation assays. Additionally, TRC40 knockdown experiments demonstrated a reduction of emerin localized at the nuclear envelope. An important objective of this work was to verify correct targeting of emerin and other integral membrane proteins to the INM. To accomplish this, we performed differential permeabilization experiments, and established a rapamycin-based dimerization assay. These experiments allowed us to confirm the localization of emerin at the INM in a microscopy based approach. In addition to wild type emerin, we also analyzed mutants. Mutations in the emerin gene in the TMD or in the C-terminal part lead to the neuromuscular disease Emery-Dreifuss muscular dystrophy. As the TMD of emerin is crucial for anchoring of emerin in a membrane, several C-terminal emerin mutations were selected and studied for their post-translational membrane insertion and localization at the INM. Several of these mutants showed severely disturbed TRC40-binding, membrane integration and INM-targeting. The efficiency of targeting to the INM might therefore rely on properties within the TMD or at least the C-terminal region of emerin. The disease could therefore be a consequence of an impaired transport process or a reduced retention at the INM. Apart from emerin, other putative tail-anchored INM-proteins like LAP2β, LEMD1, PTP1B, VAPB and LRRC59 were confirmed to localize to the INM using the rapamycin assay. Furthermore, some of them were shown to use a post-translational membrane insertion mechanism at the ER.

Nuclear lamins and nuclear pore complexes (NPCs) are major components of the nuclear envelope in metazoan cells. The objectives of this thesis were first to study interactions between nuclear lamins and the nuclear pore protein Nup153 (Chapters 2 and 3) and second to determine lamin assembly conditions of the Xenopus oocytes LIII in vitro (Chapter 4). Nuclear lamins are major constituents of the nuclear lamina underlying the nuclear periphery along with inner nuclear membrane proteins. The nuclear lamina provides stability and determines the nuclear architecture and spacing of the NPCs. NPCs form supramolecular assemblies that regulate nucleocytoplasmic transport. An overview of the functional aspects associated with the nuclear lamina and NPCs in health and disease is provided in Chapter 1. In depth analysis of the interaction of nuclear lamins with the nucleoporin Nup153 is revealed in Chapters 2 and 3. Using in vitro solution binding assays as well as immunoprecipitation assays, in chapter 2 we show direct associations between Nup153 and nuclear lamins. This work is explored even further in chapter 3 using binding assays and immunofluorescence microscopy as well as immunoprecipitation assays; we examined the interaction in the presence of lamin related mutations. Finally, in chapter 4 we established buffer conditions for LIII assembly in vitro, analysed by electron microscopy (EM) using glycerol spraying/low-angle rotary metal shadowing and negative staining. Our results presented in this thesis contribute to expand our current knowledge of the interactions of the NPCs with the nuclear lamins, as well as to increase our understanding of the impact of mutations in lamins that can cause laminopathies. In addition, the studies on the assembly conditions of LIII provide a vehicle for further characterisation of the influence of binding partners and the importance of lamin sub-fragments on the formation of higher order assemblies.

Brl1 and Brr6 are integral membrane proteins of the yeast nuclear envelope (NE) that transiently associate with nuclear pore complexes (NPCs). The exact roles of Brl1 and Brr6 during NPC assembly are unclear. Here, we demonstrate that Brr6 operates at both early and late stages of NPC assembly. Its early function is supported by amphipathic α-helix mutants, which impact nucleoporin recruitment without nuclear envelope deformation, whereas mutations in conserved cysteine residues result in NE deformation accompanied by defective NE fusion. The N-terminus of Brl1 interacts with the nucleoporin Nic96, promoting Nic96 recruitment to early assembly sites. AlphaFold predictions, the essential role of the conserved PAL motif, and the inhibition of NE fusion upon overexpression of PAL mutants together suggest that the perinuclear domains of Brl1 and Brr6 interact across the perinuclear space. Extending the length of the perinuclear-space regions of Brl1 and Brr6 causes uncontrolled NE fusion, as indicated by nuclear envelope disintegration dependent on the conserved cysteine residues. Together, Brl1 and Brr6 promote NE fusion by bridging the perinuclear space through PAL motif interactions, followed by nuclear envelope fusion and NPC insertion.

The replicative cycle of poliovirus occurs entirely in the cytoplasm of the infected cell. Poliovirus-infected cells exhibit redistribution of several nuclear proteins to the cytosol. Gustin and Sarnow show that poliovirus disrupts multiple nuclear import pathways in virally infected cells: (i) the classical import pathway that recognizes proteins with a nuclear localization signal (NLS), (ii) the transportin import pathway that recognizes proteins with an M9 NLS consisting of a glycine-rich motif, (iii) and the KNS import pathway involved in transporting the heterogenous nuclear ribonucleoprotein K (hnRNP K). In cell-free import and export assays, poliovirus inhibited an early step in nuclear translocation that prevents docking at the nuclear pore complex (NPC), but did not inhibit nuclear export mediated by the nuclear export signal receptor Crm1. Examination of components of the NPC recognized by antibody 414 suggested that at least two NPC proteins were specifically degraded in poliovirus-infected cells; however, the nuclear envelope was not generally disrupted based on normal immunofluorescent staining by an antibody against nuclear lamins. Thus, selective degradation of components of the NPC may disrupt specific nuclear import pathways. This could disrupt cellular signaling pathways, subvert apoptotic responses to infection, as well as redistribute nuclear proteins necessary for viral replication. K. E. Gustin, P. Sarnow, Effects of poliovirus infection on nucleo-cytoplasmic trafficking and nuclear pore complex composition. EMBO J. 20 , 240-249 (2001). [Abstract] [Full Text]