Lamins are nuclear intermediate filaments that, together with lamin-associated proteins, maintain nuclear shape and provide a structural support for chromosomes and replicating DNA. We have determined the solution structure of the human lamin A/C C-terminal globular domain which contains specific mutations causing four different heritable diseases. This domain encompasses residues 430-545 and adopts an Ig-like fold of type s. We have also characterized by NMR and circular dichroism the structure and thermostability of three mutants, R453W and R482W/Q, corresponding to "hot spots" causing Emery-Dreifuss muscular dystrophy and Dunnigan-type lipodystrophy, respectively. Our structure determination and mutant analyses clearly show that the consequences of the mutations causing muscle-specific diseases or lipodystrophy are different at the molecular level.
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 actinassociated 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.nesprin SUN | linker of nucleoskeleton and cytoskeleton complex | muscular dystrophy L amin A and lamin C, the predominant A-type lamins, are expressed in most differentiated somatic cells. Yet, different mutations in the LMNA gene encoding these proteins result in a variety of diseases that affect specific tissues. LMNA mutations cause autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) and related diseases with dilated cardiomyopathy (DCM) that affect cardiac muscle and skeletal muscle to variable degrees (1). Other LMNA mutations cause Dunnigan-type familial partial lipodystrophy (FPLD) that affects adipose tissue (2), Charcot-Marie-Tooth type 2B1 disease that affects peripheral neurons (3), and progeroid syndromes with features of accelerated aging (4, 5). The mechanism whereby mutations in a single gene that is widely expressed cause such diverse diseases remains a puzzle.There are two prevailing hypotheses to explain the pathogenesis of laminopathies. The mechanical stress hypothesis proposes that alterations in A-type lamins compromise nuclear integrity in tissues susceptible to stress, such as striated muscle. This model is supported by findings that A-type lamin deficiency disrupts nuclear integrity in model systems (6, 7). The secon...
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 CharcotMarie-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.
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.
Lamins A and C are intermediate filament proteins which polymerize into the nucleus to form the nuclear lamina network. The lamina is apposed to the inner nuclear membrane and functions in tethering chromatin to the nuclear envelope and in maintaining nuclear shape. We have recently characterized a globular domain that adopts an immunoglobulin fold in the carboxyl-terminal tail common to lamins A and C. Using an electrophoretic mobility shift assay (EMSA), we show that a peptide containing this domain interacts in vitro with DNA after dimerization through a disulfide bond, but does not interact with the core particle or the dinucleosome. The covalent dimer binds a 30-40 bp DNA fragment with a micromolar affinity and no sequence specificity. Using nuclear magnetic resonance (NMR) and an EMSA, we observed that two peptide regions participate in the DNA binding: the unstructured amino-terminal part containing the nuclear localization signal and a large positively charged region centered around amino acid R482 at the surface of the immunoglobulin-like domain. Mutations R482Q and -W, which are responsible for Dunnigan-type partial lipodystrophy, lower the affinity of the peptide for DNA. We conclude that the carboxyl-terminal end of lamins A and C binds DNA and suggest that alterations in lamin-DNA interactions may play a role in the pathophysiology of some lamin-linked diseases.
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