To fully understand genome function, the linear genome map must be integrated with a spatial map of chromosomes in the nucleus. Distinct nuclear addresses for a few human chromosomes have been described. Previously we have demonstrated that the gene-rich human chromosome 19 is located in a more central position in the nucleus than the similarly sized, but gene-poor, chromosome 18. To determine whether these two chromosomes are a paradigm for the organization of chromatin in the human nucleus, we have now analysed the nuclear organization of every human chromosome in diploid lymphoblasts and primary fibroblasts. We find that the most gene-rich chromosomes concentrate at the centre of the nucleus, whereas the more gene-poor chromosomes are located towards the nuclear periphery. In contrast, we find no significant relationship between chromosome size and position within the nucleus. Proteins of the nuclear membrane or lamina are candidates for molecules that might anchor regions of the genome at the nuclear periphery and it has been suggested that disruption of this organization may play a role in some disease pathologies. We show that the intranuclear organization of chromosomes is not altered in cells that lack the integral nuclear membrane protein emerin, from an individual with X-linked Emery--Dreifuss muscular dystrophy. This suggests that emerin is not necessary for localizing chromosomes at the nuclear periphery and that the muscular dystrophy phenotype in such individuals is not due to grossly altered nuclear organization of chromatin.

Emery-Dreifuss muscular dystrophy (EDMD) is a heterogeneous late-onset disease involving skeletal muscle wasting and heart defects caused, in a minority of cases, by mutations in either of two genes encoding the inner nuclear membrane (INM) proteins, emerin and lamins A/C. Nesprin-1 and -2 are multi-isomeric, spectrin-repeat proteins that bind both emerin and lamins A/C and form a network in muscle linking the nucleoskeleton to the INM, the outer nuclear membrane, membraneous organelles, the sarcomere and the actin cytoskeleton. Thus, disruptions in nesprin/lamin/emerin interactions might play a role in the muscle-specific pathogenesis of EDMD. Screening for DNA variations in the genes encoding nesprin-1 (SYNE1) and nesprin-2 (SYNE2) in 190 probands with EDMD or EDMD-like phenotypes identified four heterozygous missense mutations. Fibroblasts from these patients exhibited nuclear morphology defects and specific patterns of emerin and SUN2 mislocalization. In addition, diminished nuclear envelope localization of nesprins and impaired nesprin/emerin/lamin binding interactions were common features of all EDMD patient fibroblasts. siRNA knockdown of nesprin-1 or -2 in normal fibroblasts reproduced the nuclear morphological changes and mislocalization of emerin and SUN2 observed in patient fibroblasts. Taken together, these data suggest that EDMD may be caused, in part, by uncoupling of the nucleoskeleton and cytoskeleton because of perturbed nesprin/emerin/lamin interactions.

Nesprin-2 is a multi-isomeric, modular protein composed of variable numbers of spectrin-repeats linked to a C-terminal transmembrane domain and/or to N-terminal paired calponin homology (CH) domains. The smaller isoforms of nesprin-2 co-localize with and bind lamin A and emerin at the inner nuclear envelope (NE). In SW-13 cells, which lack lamin A/C, nesprin-2 epitopes and emerin were both mislocalized and formed aggregates in the endoplasmic reticulum (ER). The larger isoforms and other CH-domain-containing isoforms co-localize with heterochromatin within the nucleus and are also present at the outer NE and in multiple cytoplasmic compartments. Nesprin-2 isoforms relocalize during in vitro muscle differentiation of C2C12 myoblasts to the sarcomere of myotubes. Immunogold electron microscopy using antibodies specific for three different epitopes detected nesprin-2 isoforms at multiple locations including intranuclear foci, both membranes of the NE, mitochondria, sarcomeric structures and plasma membrane foci. In adult skeletal muscle, confocal immunolocalization studies demonstrated that nesprin-2 epitopes were present at the Z-line and were also associated with the sarcoplasmic reticulum (SR) in close apposition to SERCA2. These data suggest that nesprin-2 isoforms form a linking network between organelles and the actin cytoskeleton and thus may be important for maintaining sub-cellular spatial organisation. Moreover, its association at the NE with lamin and emerin, the genes mutated in Emery-Dreifuss muscular dystrophy, suggests a mechanism to explain how disruption of the NE leads to muscle dysfunction.

Emerin is a type II inner nuclear membrane (INM) protein of unknown function. Emerin function is likely to be important because, when it is mutated, emerin promotes both skeletal muscle and heart defects. Here we show that one function of Emerin is to regulate the flux of beta-catenin, an important transcription coactivator, into the nucleus. Emerin interacts with beta-catenin through a conserved adenomatous polyposis coli (APC)-like domain. When GFP-emerin is expressed in HEK293 cells, beta-catenin is restricted to the cytoplasm and beta-catenin activity is inhibited. In contrast, expression of an emerin mutant, lacking its APC-like domain (GFP-emerinDelta), dominantly stimulates beta-catenin activity and increases nuclear accumulation of beta-catenin. Human fibroblasts that are null for emerin have an autostimulatory growth phenotype. This unusual growth phenotype arises through enhanced nuclear accumulation and activity of beta-catenin and can be replicated in wild-type fibroblasts by transfection with constitutively active beta-catenin. Our results support recent findings that suggest that INM proteins can influence signalling pathways by restricting access of transcription coactivators to the nucleus.

The nuclear envelope (NE) LINC complex, in mammals comprised of SUN domain and nesprin proteins, provides a direct connection between the nuclear lamina and the cytoskeleton, which contributes to nuclear positioning and cellular rigidity. SUN1 and SUN2 interact with lamin A, but lamin A is only required for NE localization of SUN2, and it remains unclear how SUN1 is anchored. Here, we identify emerin and short nesprin-2 isoforms as novel nucleoplasmic binding partners of SUN1/2. These have overlapping binding sites distinct from the lamin A binding site. However, we demonstrate that tight association of SUN1 with the nuclear lamina depends upon a short motif within residues 209–228, a region that does not interact significantly with known SUN1 binding partners. Moreover, SUN1 localizes correctly in cells lacking emerin. Importantly then, the major determinant of SUN1 NE localization has yet to be identified. We further find that a subset of lamin A mutations, associated with laminopathies Emery-Dreifuss muscular dystrophy (EDMD) and Hutchinson-Gilford progeria syndrome (HGPS), disrupt lamin A interaction with SUN1 and SUN2. Despite this, NE localization of SUN1 and SUN2 is not impaired in cell lines from either class of patients. Intriguingly, SUN1 expression at the NE is instead enhanced in a significant proportion of HGPS but not EDMD cells and strongly correlates with pre-lamin A accumulation due to preferential interaction of SUN1 with pre-lamin A. We propose that these different perturbations in lamin A-SUN protein interactions may underlie the opposing effects of EDMD and HGPS mutations on nuclear and cellular mechanics.

Satellite cells are the resident stem cells of adult skeletal muscle. To date though, there is a paucity of native markers that can be used to easily identify quiescent satellite cells, with Pax7 probably being the best that is currently available. Here we have further characterized a number of recently described satellite cell markers, and also describe novel ones. Caveolin-1, integrin α7 and the calcitonin receptor proved reliable markers for quiescent satellite cells, being expressed by all satellite cells identified with Pax7. These three markers remained expressed as satellite cells were activated and underwent proliferation. The nuclear envelope proteins lamin A/C and emerin, mutations in which underlie Emery-Dreifuss muscular dystrophy, were also expressed in both quiescent and proliferating satellite cells. Conversely, Jagged-1, a Notch ligand, was not expressed in quiescent satellite cells but was induced upon activation. These findings further contribute to defining the molecular signature of muscle satellite cells.

NADPH-dependent superoxide production by intact human neutrophils is inhibited by DPI (diphenyleneiodonium), when stimulated by either FMLP (N-formylmethionyl-leucyl-phenylalanine) or PMA (phorbol 12-myristate 13-acetate). Addition of 10 microM-DPI abolished the reduction of both the FAD and the cytochrome b components of the NADPH oxidase. DPI inhibition of the oxidase was associated with defective aerobic killing of staphylococci by human neutrophils. Anaerobic killing, phagocytosis, chemotaxis and motility were relatively unaffected by 10 microM-DPI. Degranulation of the azurophil and specific granules, induced by the soluble stimuli FMLP or PMA, and by particulate stimuli was decreased by the presence of DPI. The above effects of DPI on human neutrophils are similar to those found in chronic granulomatous disease.

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