Nuclear envelopathies: a complex LINC between nuclear envelope and pathology

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

Nuclear compartmentalization seems to have an important role in regulating metazoan genes. Although studies on immunoglobulin and other loci have shown a correlation between positioning at the nuclear lamina and gene repression, the functional consequences of this compartmentalization remain untested. We devised an approach for inducible tethering of genes to the inner nuclear membrane (INM), and tested the consequences of such repositioning on gene activity in mouse fibroblasts. Here, using three-dimensional DNA-immunoFISH, we demonstrate repositioning of chromosomal regions to the nuclear lamina that is dependent on breakdown and reformation of the nuclear envelope during mitosis. Moreover, tethering leads to the accumulation of lamin and INM proteins, but not to association with pericentromeric heterochromatin or nuclear pore complexes. Recruitment of genes to the INM can result in their transcriptional repression. Finally, we use targeted adenine methylation (DamID) to show that, as is the case for our model system, inactive immunoglobulin loci at the nuclear periphery are contacted by INM and lamina proteins. We propose that these molecular interactions may be used to compartmentalize and to limit the accessibility of immunoglobulin loci to transcription and recombination factors.

The nuclear envelope (NE) is connected to the different types of cytoskeletal elements by linker of nucleoskeleton and cytoskeleton (LINC) complexes. LINC complexes exist from yeast to humans, and have preserved their general architecture throughout evolution. They are composed of SUN and KASH domain proteins of the inner and the outer nuclear membrane, respectively. These SUN–KASH bridges are used for the transmission of forces across the NE and support diverse biological processes. Here, we review the function of SUN and KASH domain proteins in various unicellular and multicellular species. Specifically, we discuss their influence on nuclear morphology and cytoskeletal organization. Further, emphasis is given on the role of LINC complexes in nuclear anchorage and migration as well as in genome organization.

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 migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Changes in nuclear morphology are observed in diverse developmental processes as well as in pathological conditions. Modification of nuclear membrane and nuclear lamina protein levels results in altered nuclear shapes, as it has been demonstrated in experimental systems ranging from yeast to human cells. The important role of nuclear membrane components in regulating nuclear morphology is additionally highlighted by the abnormally shaped nuclei observed in diseases where nuclear lamina proteins are mutated. Even though the effect of nuclear envelope components on nuclear shape has been thoroughly described, not much is known about the molecular mechanisms that govern these events. In addition to the known role of intermediate filament formation by lamins, here we discuss several mechanisms that might alone or in combination participate in the regulation of nuclear shape observed upon modification of the levels of nuclear membrane and lamina proteins. Based on recent work with the two farnesylated nuclear membrane Drosophila proteins, kugelkern and lamin Dm0, we propose that the direct interaction of farnesylated nuclear membrane proteins with the phospholipid bilayer leads to nuclear envelope deformation. In addition to this mechanism, we suggest that the interaction of nuclear membrane and lamina proteins with cytoskeletal elements and chromatin, and modifications in lipid biosynthesis might also be involved in the formation of abnormally shaped nuclei.

Employing avian erythrocytes, we have previously isolated a multimeric complex consisting of the lamin B receptor (LBR, or p58), the nuclear lamins, an LBR-specific kinase, a 34-kDa protein, and an 18-kDa polypeptide termed p18. As the LBR kinase and the 34-kDa component have been recently characterized, we now proceed in the characterization of p18. We show here that p18 is an integral membrane protein specific to the erythrocyte nuclear envelope which binds to LBR and B-type lamins. NH2-terminal sequencing indicates that p18 is distinct from other nuclear envelope components, but has similarity to the mitochondrial isoquinoline-binding protein. In situ analysis by immunoelectron microscopy and examination of digitonin-permeabilized cells by indirect immunofluorescence show that p18, unlike LBR and other lamin-binding proteins, is equally distributed between the inner and outer nuclear membrane. Furthermore, cycloheximide inhibition experiments reveal that the fraction of p18 that resides in the outer nuclear membrane does not represent nascent chains en route to the inner nuclear membrane, but rather material in equilibrium with the p18 that partitions with the inner nuclear membrane. The paradigm of p18 suggests that transmembrane complexes formed by the nuclear lamins and LBR provide potential docking sites for integral membrane proteins of the nuclear envelope that equilibrate between the rough endoplasmic reticulum and the inner nuclear membrane.

Invaginations of the nuclear membrane occur in different shapes, sizes, and compositions. Part of these pleiomorphic invaginations make up the nucleoplasmic reticulum (NR), while others are merely nuclear folds. We define the NR as tubular invaginations consisting of either both the inner and outer nuclear membrane, or only the inner nuclear membrane. Specifically, invaginations of both the inner and outer nuclear membrane are also called type II NR, while those of only the inner nuclear membrane are defined as type I NR. The formation and structure of the NR is determined by proteins associated to the nuclear membrane, which induce a high membrane curvature leading to tubular invaginations. Here we review and discuss the current knowledge of nuclear invaginations and the NR in particular. An increase in tubular invaginations of the nuclear envelope is associated with several pathologies, such as laminopathies, cancer, (reversible) heart failure, and Alzheimer’s disease. Furthermore, viruses can induce both type I and II NR. In laminopathies, the amount of A-type lamins throughout the nucleus is generally decreased or the organization of lamins or lamin-associated proteins is disturbed. Also, lamin overexpression or modulation of lamin farnesylation status impacts NR formation, confirming the importance of lamin processing in NR formation. Virus infections reorganize the nuclear lamina via (de)phosphorylation of lamins, leading to an uneven thickness of the nuclear lamina and in turn lobulation of the nuclear membrane and the formation of invaginations of the inner nuclear membrane. Since most studies on the NR have been performed with cell cultures, we present additional proof for the existence of these structures in vivo, focusing on a variety of differentiated cardiovascular and hematopoietic cells. Furthermore, we substantiate the knowledge of the lamin composition of the NR by super-resolution images of the lamin A/C and B1 organization. Finally, we further highlight the essential role of lamins in NR formation by demonstrating that (over)expression of lamins can induce aberrant NR structures.

The nuclear envelope defines the barrier between the nucleus and cytoplasm and features inner and outer membranes separated by a perinuclear space (PNS). The inner nuclear membrane contains specific integral proteins that include Sun1 and Sun2. Although the outer nuclear membrane (ONM) is continuous with the endoplasmic reticulum, it is nevertheless enriched in several integral membrane proteins, including nesprin 2 Giant (nesp2G), an 800-kD protein featuring an NH2-terminal actin-binding domain. A recent study (Padmakumar, V.C., T. Libotte, W. Lu, H. Zaim, S. Abraham, A.A. Noegel, J. Gotzmann, R. Foisner, and I. Karakesisoglou. 2005. J. Cell Sci. 118:3419–3430) has shown that localization of nesp2G to the ONM is dependent upon an interaction with Sun1. In this study, we confirm and extend these results by demonstrating that both Sun1 and Sun2 contribute to nesp2G localization. Codepletion of both of these proteins in HeLa cells leads to the loss of ONM-associated nesp2G, as does overexpression of the Sun1 lumenal domain. Both treatments result in the expansion of the PNS. These data, together with those of Padmakumar et al. (2005), support a model in which Sun proteins tether nesprins in the ONM via interactions spanning the PNS. In this way, Sun proteins and nesprins form a complex that links the nucleoskeleton and cytoskeleton (the LINC complex).

Pancreatic cancer is an aggressive and intractable malignancy with high mortality. This is due in part to a high resistance to chemotherapeutics and radiation treatment conferred by diverse regulatory mechanisms. Among these, constituents of the nuclear envelope play a significant role in regulating oncogenesis and pancreatic tumor biology, and this review focuses on three specific components and their roles in cancer. The LINC complex is a nuclear envelope component formed by proteins with SUN and KASH domains that interact in the periplasmic space of the nuclear envelope. These interactions functionally and structurally couple the cytoskeleton to chromatin and facilitates gene regulation informed by cytoplasmic activity. Furthermore, cancer cell invasiveness is impacted by LINC complex biology. The nuclear lamina is adjacent to the inner nuclear membrane of the nuclear envelope and can actively regulate chromatin in addition to providing structural integrity to the nucleus. A disrupted lamina can impart biophysical compromise to nuclear structure and function, as well as form dysfunctional micronuclei that may lead to genomic instability and chromothripsis. In close relationship to the nuclear lamina is the nuclear pore complex, a large megadalton structure that spans both outer and inner membranes of the nuclear envelope. The nuclear pore complex mediates bidirectional nucleocytoplasmic transport and is comprised of specialized proteins called nucleoporins that are overexpressed in many cancers and are diagnostic markers for oncogenesis. Furthermore, recent demonstration of gene regulatory functions for discrete nucleoporins independent of their nuclear trafficking function suggests that these proteins may contribute more to malignant phenotypes beyond serving as biomarkers. The nuclear envelope is thus a complex, intricate regulator of cell signaling, with roles in pancreatic tumorigenesis and general oncogenic transformation.

Overexpression of the lamina proteins Lamin and Kugelkern induces specific ultrastructural alterations in the morphology of the nuclear envelope of intestinal stem cells and enterocytes

In most metazoan cell types, the nucleus occupies a clearly defined region of the cytoplasm. For instance in epithelial cells, it is commonly positioned close to the basal membrane, whereas in skeletal muscle fibres, the majority of nuclei are uniformly distributed adjacent to the plasma membrane. A few muscle cell nuclei, however, cluster beneath the postsynaptic membrane. In certain cell types, the nucleus may be repositioned according to cell cycle phase or cell activity. Each of these situations requires that nuclei be physically coupled to the cytoskeleton. This is accomplished in large part by LINC (linker of the nucleoskeleton and cytoskeleton) complexes. These structures consist of inner nuclear membrane SUN (Sad1p‐UNC‐84)‐domain proteins that function as transluminal tethers for outer nuclear membrane KASH ‐domain proteins. Together, SUN–KASH pairs function as links in a molecular chain that couples cytoskeletal elements, including motor proteins, to nuclear components. In addition to having obligate roles in nuclear positioning and chromatin organisation, LINC complexes may also function in mechanotransduction. The fact that a number of human diseases have been associated with LINC complex defects indicates that in many cell types these represent essential structures at the interface between the nucleus and cytoplasm. Key Concepts The nuclear envelope forms the interface between the nucleus and cytoplasm. Its most prominent features are the inner and outer nuclear membranes (INM and ONM) separated by a narrow (∼50 nm) gap, the perinuclear space (PNS). SUN domain proteins of the INM function as transluminal tethers for KASH domain proteins in the ONM. The nucleoplasmic domain of SUN proteins provides binding sites for both chromatin proteins and components of the nuclear lamina. KASH domain proteins represent attachment sites at the nuclear envelope for a variety of cytoskeletal elements, including motor proteins. SUN–KASH pairs, which span both nuclear membranes, are known as LINC (linker of the nucleoskeleton and cytoskeleton) complexes and mechanically couple nuclear structures to the cytoskeleton. LINC complexes have essential roles in nuclear migration and positioning in multiple cell types. During meiosis, LINC complexes have an essential role in telomere‐led chromosome movement, a process that facilitates homologous chromosome pairing and synapsis. LINC complexes are thought to mediate the transmission of mechanical signals to the nucleus: mechanotransduction.

The normal distribution of nuclear envelope transmembrane proteins (NETs) is disrupted in several human diseases. NETs are synthesized on the endoplasmic reticulum and then transported from the outer nuclear membrane (ONM) to the inner nuclear membrane (INM). Quantitative determination of the distribution of NETs on the ONM and INM is limited in available approaches, which moreover provide no information about translocation rates in the two membranes. Here we demonstrate a single-point single-molecule FRAP microscopy technique that enables determination of distribution and translocation rates for NETs in vivo.

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.

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.