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Cecilia Ostlund

Publications and source records attributed to Cecilia Ostlund.

10 recordsLinked to original sources
PubMed2006

Dependence of diffusional mobility of integral inner nuclear membrane proteins on A-type lamins.

Integral proteins of the nuclear envelope inner membrane have been proposed to reach their sites by diffusion after their co-translational insertion in the rough endoplasmic reticulum. They are then retained in the inner nuclear membrane by binding to nuclear structures. One such structure is the nuclear lamina, an intermediate filament meshwork composed of A-type and B-type lamin proteins. Emerin, MAN1, and LBR are three integral inner nuclear membrane proteins. We expressed these proteins fused to green fluorescent protein in embryonic fibroblasts from wild-type mice and Lmna -/- mice, which lack A-type lamins. We then studied the diffusional mobilities of emerin, MAN1, and LBR using fluorescence recovery after photobleaching. We show that emerin and MAN1, but not LBR, are more mobile in the inner nuclear membrane of cells from Lmna -/- mice than in cells from wild-type mice. In cells from Lmna -/- mice expressing exogenous lamin A, the protein mobilities were similar to those in cells from wild-type mice. This supports a model where emerin and MAN1 are at least partly retained in the inner nuclear membrane by binding to A-type lamins, while LBR depends on other binding partners for its retention.

Animals↗
PubMed2005

Intracellular trafficking and dynamics of double homeodomain proteins.

Double homeodomain (DUX) proteins are encoded by a family of 3.3-kilobase repeated elements dispersed in the human genome. One of these elements named D4Z4 is found in a tandem repeat array on chromosome 4 that is partially deleted in facioscapulohumeral muscular dystrophy. We have evaluated the trafficking and mobility of two DUX proteins, DUX1 and DUX4. We transfected C2C12 myoblasts with cDNA encoding these proteins fused to the green fluorescent protein and studied their intracellular localization and diffusional mobilities using fluorescence recovery after photobleaching and fluorescence loss in photobleaching. We also studied truncated forms of the proteins, containing one or both homeodomains or a region outside the homeodomains. We show that both full-length proteins are actively transported into the nucleus, and that the homeodomains contain the signals required for this localization. DUX1 is more mobile than DUX4 within the nucleus (t(1/2) = 4.8 s for DUX1 and 13.4 s for DUX4), suggesting differences in the way the two proteins interact with nuclear components.

Active Transport, Cell Nucleus↗
PubMed2004

Altered protein dynamics of disease-associated lamin A mutants.

BACKGROUND: Recent 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. RESULTS: We 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. CONCLUSION: The 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.

Cell Line, Tumor↗
PubMed2003

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.

Animals↗
PubMed2003

The carboxyl-terminal region common to lamins A and C contains a DNA binding domain.

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.

Amino Acid Substitution↗
PubMed2003

Expression of lamin A mutated in the carboxyl-terminal tail generates an aberrant nuclear phenotype similar to that observed in cells from patients with Dunnigan-type partial lipodystrophy and Emery-Dreifuss muscular dystrophy.

Autosomal dominantly inherited missense mutations in lamins A and C cause familial partial lipodystrophy of the Dunnigan-type (FPLD), and myopathies including Emery-Dreifuss muscular dystrophy (EDMD). While mutations responsible for FPLD are restricted to the carboxyl-terminal tails, those responsible for EDMD are spread throughout the molecules. We observed here the same structural abnormalities in the nuclear envelope and chromatin of fibroblasts from patients with FPLD and EDMD, harboring missense mutations at codons 482 and 453, respectively. Similar nuclear alterations were generated in fibroblasts, myoblasts, and preadipocytes mouse cell lines overexpressing lamin A harboring either of these two mutations. A large variation in sensitivity to lamin A overexpression was observed among the three cell lines, which was correlated with their variable endogenous content in A-type lamins and emerin. The occurrence of nuclear abnormalities was reduced when lamin B1 was coexpressed with mutant lamin A, emphasizing the functional interaction of the two types of lamins. Transfected cells therefore develop similar phenotypes when expressing lamins mutated in the carboxyl-terminal tail at sites responsible for FPLD or EDMD.

Adipocytes↗
PubMed2003

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.

Animals↗
PubMed2002

The Ig-like structure of the C-terminal domain of lamin A/C, mutated in muscular dystrophies, cardiomyopathy, and partial lipodystrophy.

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.

Amino Acid Sequence↗