Cecilia Östlund

Diseases of the Nuclear Envelope

In the past decade, a wide range of fascinating monogenic diseases have been linked to mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins of the nuclear envelope. These diseases include dilated cardiomyopathy with variable muscular dystrophy, Dunnigan-type familial partial lipodystrophy, a Charcot-Marie-Tooth type 2 disease, mandibuloacral dysplasia, and Hutchinson-Gilford progeria syndrome. Several diseases are also caused by mutations in genes encoding B-type lamins and proteins that associate with the nuclear lamina. Studies of these so-called laminopathies or nuclear envelopathies, some of which phenocopy common human disorders, are providing clues about functions of the nuclear envelope and insights into disease pathogenesis and human aging.

Dynamics and molecular interactions of linker of nucleoskeleton and cytoskeleton (LINC) complex proteins

The linker of nucleoskeleton and cytoskeleton (LINC) complex is situated in the nuclear envelope and forms a connection between the lamina and cytoskeletal elements. Sun1, Sun2 and nesprin-2 are important components of the LINC complex. We expressed these proteins fused to green fluorescent protein in embryonic fibroblasts and studied their diffusional mobilities using fluorescence recovery after photobleaching. We show that they all are more mobile in embryonic fibroblasts from mice lacking A-type lamins than in cells from wild-type mice. Knockdown of Sun2 also increased the mobility of a short, chimeric form of nesprin-2 giant (mini-nesprin-2G), whereas the lack of emerin did not affect the mobility of Sun1, Sun2 or mini-nesprin-2G. Fluorescence resonance energy transfer experiments showed Sun1 to be more closely associated with lamin A than is Sun2. Sun1 and Sun2 had similar affinity for the nesprin-2 KASH domain in plasmon surface resonance (Biacore) experiments. This affinity was ten times higher than that previously reported between nesprin-2 and actin. Deletion of the actin-binding domain had no effect on mini-nesprin-2G mobility. Our data support a model in which A-type lamins and Sun2 anchor nesprin-2 in the outer nuclear membrane, whereas emerin, Sun1 and actin are dispensable for this anchoring.

Lamin A variants that cause striated muscle disease are defective in anchoring transmembrane actin-associated nuclear lines for nuclear movement

Mutations in LMNA, which encodes A-type lamins, result in disparate diseases, known collectively as laminopathies, that affect distinct tissues, including striated muscle and adipose tissue. Lamins provide structural support for the nucleus and sites of attachment for chromatin, and defects in these functions may contribute to disease pathogenesis. Recent studies suggest that A-type lamins may facilitate connections between the nucleus and the cytoskeleton mediated by nuclear envelope nesprin and SUN proteins. In mammalian cells, however, interfering with A-type lamins does not affect the localization of these proteins. Here, we used centrosome orientation in fibroblasts, which requires separate nuclear and centrosome positioning pathways, as a model system to understand how LMNA mutations affect nucleus-cytoskeletal connections. We find that LMNA mutations causing striated muscle diseases block actin-dependent nuclear movement, whereas most that affect adipose tissue inhibit microtubule-dependent centrosome positioning. Genetic deletion or transient depletion of A-type lamins also blocked nuclear movement, showing that mutations affecting muscle exhibit the null phenotype. Lack of A-type lamins, or expression of variants that cause striated muscle disease, did not affect assembly of nesprin-2G and SUN2 into transmembrane actin-associated nuclear (TAN) lines that attach the nucleus to retrogradely moving actin cables. Nesprin-2G TAN lines were less stable, however, and slipped over the nucleus rather than moving with it, indicating that they were not anchored. Nesprin-2G TAN lines also slipped in SUN2-depleted cells. Our results establish A-type lamins as anchors for nesprin-2G–SUN2 TAN lines to allow productive movement and proper positioning of the nucleus by actin.Nuclear position is actively controlled and can be adjusted according to the needs of a cell by nuclear movement. Microtubules mediate the majority of nuclear movements studied to date, although examples of nuclear movements mediated by the actin cytoskeleton have been described. One such actin-dependent nuclear movement occurs during centrosome orientation in fibroblasts polarizing for migration. Here, the centrosome is maintained at the cell center while the nucleus is moved to the cell rear by actin retrograde flow thus positioning the centrosome between the nucleus and the leading edge of the cell. We have explored the molecular mechanism for actin dependent movement of the nucleus during centrosome centration. We found that a novel linear array of nuclear envelope membrane proteins composed of nesprin-2G and SUN2, called transmembrane actin-associated nuclear (TAN) lines, couple the nucleus to moving actin cables resulting in the nucleus being positioned toward the cell rear. TAN lines are anchored by A-type lamins and this allows the forces generated by the actin cytoskeleton to be transmitted across the nuclear envelope to move the nucleus. Here we review the data supporting this mechanism for nuclear movement, discuss questions remaining to be addressed and consider how this new mechanism of nuclear movement may shed light on human disease.

Nuclear envelope proteins and neuromuscular diseases

Several neuromuscular diseases are caused by mutations in emerin and A‐type lamins, proteins of the nuclear envelope. Emery–Dreifuss muscular dystrophy is caused by mutations in emerin (X‐linked) or A‐type lamins (autosomal dominant). Mutations in A‐type lamins also cause limb‐girdle muscular dystrophy type 1B, dilated cardiomyopathy with conduction defect, and Charcot–Marie–Tooth disorder type 2B1. They also cause partial lipodystrophy syndromes. The functions of emerin and A‐type lamins and the mechanisms of how mutations in these proteins cause tissue‐specific diseases are not well understood. The mutated proteins may cause structural damage to cells but may also affect processes such as gene regulation. This review gives an overview of this topic and describes recent advances in identification of disease‐causing mutations, studies of cells and tissues from subjects with these diseases, and animal and cell culture models. Muscle Nerve 27: 393–406, 2003

Effect of pathogenic mis-sense mutations in lamin A on its interaction with emerin in vivo

Mutations in lamin A/C can cause Emery-Dreifuss muscular dystrophy (EDMD) or a related cardiomyopathy (CMD1A). Using transfection of lamin-A/C-deficient fibroblasts, we have studied the effects of nine pathogenic mutations on the ability of lamin A to assemble normally and to localize emerin normally at the nuclear rim. Five mutations in the rod domain (L85R, N195K, E358K, M371K and R386K) affected the assembly of the lamina. With the exception of mutant L85R, all rod domain mutants induced the formation of large nucleoplasmic foci in about 10% of all nuclei. The presence of emerin in these foci suggests that the interaction of lamin A with emerin is not directly affected by the rod domain mutations. Three mutations in the tail region, R453W, W520S and R527P, might directly affect emerin binding by disrupting the structure of the putative emerin-binding site, because mutant lamin A localized normally to the nuclear rim but its ability to trap emerin was impaired. Nucleoplasmic foci rarely formed in these three cases (<2%) but, when they did so, emerin was absent, consistent with a direct effect of the mutations on emerin binding. The lipodystrophy mutation R482Q, which causes a different phenotype and is believed to act through an emerin-independent mechanism, was indistinguishable from wild-type in its localization and its ability to trap emerin at the nuclear rim. The novel hypothesis suggested by the data is that EDMD/CMD1A mutations in the tail domain of lamin A/C work by direct impairment of emerin interaction, whereas mutations in the rod region cause defective lamina assembly that might or might not impair emerin capture at the nuclear rim. Subtle effects on the function of the lamina-emerin complex in EDMD/CMD1A patients might be responsible for the skeletal and/or cardiac muscle phenotype.

Altered protein dynamics of disease-associated lamin A mutants.

BackgroundRecent interest in the function of the nuclear lamina has been provoked by the discovery of lamin A/C mutations in the laminopathy diseases. However, it is not understood why mutations in lamin A give such a range of tissue-specific phenotypes. Part of the problem in rationalising genotype-phenotype correlations in the laminopathies is our lack of understanding of the function of normal and mutant lamin A. To investigate this we have used photobleaching in human cells to analyse the dynamics of wild-type and mutant lamin A protein at the nuclear periphery.ResultsWe have found that a large proportion of wild-type lamin A at the nuclear periphery is immobile, but that there is some slow movement of lamin A within the nuclear lamina. The mobility of an R482W mutant lamin A was indistinguishable from wild-type, but increased mobility of L85R and L530P mutant proteins within the nuclear lamina was found. However, the N195K mutant shows the most enhanced protein mobility, both within the nucleoplasm and within the lamina.ConclusionThe slow kinetics of lamin A movement is compatible with its incorporation into a stable polymer that only exchanges subunits very slowly. All of the myopathy-associated lamin A mutants that we have studied show increased protein movement compared with wild-type. In contrast, the dynamic behaviour of the lipodystrophy-associated lamin A mutant was indistinguishable from wild-type. This supports the hypothesis that the underlying defect in lamin A function is quite distinct in the laminopathies that affect striated muscle, compared to the diseases that affect adipose tissue. Our data are consistent with an alteration in the stability of the lamin A molecules within the higher-order polymer at the nuclear lamina in myopathies.

Subcellular localization of SREBP1 depends on its interaction with the C-terminal region of wild-type and disease related A-type lamins

Lamins A and C are nuclear intermediate filament proteins expressed in most differentiated somatic cells. Previous data suggested that prelamin A, the lamin A precursor, accumulates in some lipodystrophy syndromes caused by mutations in the lamin A/C gene, and binds and inactivates the sterol regulatory element binding protein 1 (SREBP1). Here we show that, in vitro, the tail regions of prelamin A, lamin A and lamin C bind a polypeptide of SREBP1. Such interactions also occur in HeLa cells, since expression of lamin tail regions impedes nucleolar accumulation of the SREBP1 polypeptide fused to a nucleolar localization signal sequence. In addition, the tail regions of A-type lamin variants that occur in Dunnigan-type familial partial lipodystrophy of (R482W) and Hutchison Gilford progeria syndrome (∆607-656) bind to the SREBP1 polypeptide in vitro, and the corresponding FLAG-tagged full-length lamin variants co-immunoprecipitate the SREBP1 polypeptide in cells. Overexpression of wild-type A-type lamins and variants favors SREBP1 polypeptide localization at the intranuclear periphery, suggesting its sequestration. Our data support the hypothesis that variation of A-type lamin protein level and spatial organization, in particular due to disease-linked mutations, influences the sequestration of SREBP1 at the nuclear envelope and thus contributes to the regulation of SREBP1 function.

Linker of nucleoskeleton and cytoskeleton (LINC) complex-mediated actin-dependent nuclear positioning orients centrosomes in migrating myoblasts

Myoblast migration is essential for muscle development and repair; however, the factors that contribute to the polarity of migrating myoblasts are relatively unknown. We find that randomly migrating C2C12 myoblasts orient their centrosomes in the direction of migration. Using wounded monolayers, we further show that centrosome orientation is stimulated by the serum factor lysophosphatidic acid (LPA) and involves the rearward movement of the nucleus while the centrosome is maintained at the cell centroid. The rate of nuclear movement correlated with that of actin retrograde flow and both cytochalasin D and blebbistatin prevented nuclear movement and centrosome orientation. Actin-dependent rearward nuclear movement in fibroblasts is mediated by assembly of nuclear membrane nesprin-2G and SUN2 LINC complexes into transmembrane actin-associated nuclear (TAN) lines anchored by A-type lamins and emerin. In C2C12 myoblasts, depletion of nesprin-2G, SUN2 or lamin A/C prevented nuclear movement and endogenous nesprin-2G and a chimeric GFP-mini-nesprin-2G formed TAN lines during nuclear movement. Depleting nesprin-2G strongly interfered with directed cell migration and reduced the efficiency of myoblast fusion into multinucleated myotubes. Our results show that nuclear movement contributes to centrosome orientation and polarity for efficient migration and fusion of myoblasts. Given that mutations in the genes encoding A-type lamins, nesprin-2 and SUN2 cause Emery-Dreifuss muscular dystrophy and related myopathies, our results have implications for understanding the mechanism of disease pathogenesis.

The R439C mutation in LMNA causes lamin oligomerization and susceptibility to oxidative stress

Dunnigan‐type familial partial lipodystrophy (FPLD) is a laminopathy characterized by an aberrant fat distribution and a metabolic syndrome for which oxidative stress has recently been suggested as one of the disease‐causing mechanisms. In a family affected with FPLD, we identified a heterozygous missense mutation c.1315C>T in the LMNA gene leading to the p.R439C substitution. Cultured patient fibroblasts do not show any prelamin A accumulation and reveal honeycomb‐like lamin A/C formations in a significant percentage of nuclei. The mutation affects a region in the C‐terminal globular domain of lamins A and C, different from the FPLD‐related hot spot. Here, the introduction of an extra cysteine allows for the formation of disulphide‐mediated lamin A/C oligomers. This oligomerization affects the interaction properties of the C‐terminal domain with DNA as shown by gel retardation assays and causes a DNA‐interaction pattern that is distinct from the classical R482W FPLD mutant. Particularly, whereas the R482W mutation decreases the binding efficiency of the C‐terminal domain to DNA, the R439C mutation increases it. Electron spin resonance spectroscopy studies show significantly higher levels of reactive oxygen species (ROS) upon induction of oxidative stress in R439C patient fibroblasts compared to healthy controls. This increased sensitivity to oxidative stress seems independent of the oligomerization and enhanced DNA binding typical for R439C, as both the R439C and R482W mutants show a similar and significant increase in ROS upon induction of oxidative stress by H2O2.

Loss of a DNA binding site within the tail of prelamin A contributes to altered heterochromatin anchorage by progerin

MINT‐7893957, MINT‐7893974, MINT‐7894055: H3 (uniprotkb:Q71DI3) binds (MI:0407) to progerin (uniprotkb:Q6UYC3) by surface plasmon resonance (MI:0107)