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Expression and localization of nuclear proteins in autosomal-dominant Emery-Dreifuss muscular dystrophy with LMNA R377H mutation.

Reichart B, Klafke R, Dreger C, Krüger E, Motsch I, Ewald A, Schäfer J, Reichmann H, Müller CR, Dabauvalle MC - BMC Cell Biol. (2004)

Bottom Line: Cellular levels of A-type lamins were reduced compared to control cells.Cell synchronization experiments showed that the reduction of the cellular level of A-type lamin was due to instability of lamin A.From our results we conclude: Firstly, that structural alterations of the nuclei which are found only in a minor fraction of lymphoblastoid cells and mature muscle fibres are not sufficient to explain the clinical pathology of EDMD; Secondly, that wild type lamin A is required not only for the retention of LBR in the inner nuclear membrane but also for a correct localization of the transcriptionally active RNA pol II in muscle cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Developmental Biology, University of Würzburg, Germany. beatereichart@web.de

ABSTRACT

Background: The autosomal dominant form of Emery-Dreifuss muscular dystrophy (AD-EDMD) is caused by mutations in the gene encoding for the lamins A and C (LMNA). Lamins are intermediate filament proteins which form the nuclear lamina underlying the inner nuclear membrane. We have studied the expression and the localization of nuclear envelope proteins in three different cell types and muscle tissue of an AD-EDMD patient carrying a point mutation R377H in the lamin A/C gene.

Results: Lymphoblastoid cells, skin fibroblasts, primary myoblasts and muscle thin sections were studied by immunocytochemistry and electron microscopy. Cellular levels of A-type lamins were reduced compared to control cells. In contrast, the amount of emerin and lamin B appeared unaltered. Cell synchronization experiments showed that the reduction of the cellular level of A-type lamin was due to instability of lamin A. By electron microscopy, we identified a proportion of nuclei with morphological alterations in lymphoblastoid cells, fibroblasts and mature muscle fibres. Immunofluorescence microscopy showed that a major population of the lamin B receptor (LBR), an inner nuclear membrane protein, was recovered in the cytoplasm in association with the ER. In addition, the intranuclear organization of the active form of RNA polymerase II was markedly different in cells of this AD-EDMD patient. This aberrant intranuclear distribution was specifically observed in muscle cells where the pathology of EDMD predominates.

Conclusions: From our results we conclude: Firstly, that structural alterations of the nuclei which are found only in a minor fraction of lymphoblastoid cells and mature muscle fibres are not sufficient to explain the clinical pathology of EDMD; Secondly, that wild type lamin A is required not only for the retention of LBR in the inner nuclear membrane but also for a correct localization of the transcriptionally active RNA pol II in muscle cells. We speculate that a rearrangement of the internal chromatin could lead to muscle-specific disease symptoms by interference with proper mRNA transcription.

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Some myoblasts cell nuclei from the AD-EDMD patient 99-3 are negative for antibodies to lamin A/C. Immunolabelling of myoblasts cells with mutation LMNA R377H (a-c) and control cells (d-f) was done by using antibodies against emerin (a,d), lamin A/C (b,e) and lamin B (c,f). Not all nuclei stain for lamin A/C in patient cells (compare b with e). The corresponding phase-contrast images (a"-c"; d"-f") and Hoechst fluorescences (a'-c'; d'-f') are shown. Bars (c",f") 10 µm.
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Figure 4: Some myoblasts cell nuclei from the AD-EDMD patient 99-3 are negative for antibodies to lamin A/C. Immunolabelling of myoblasts cells with mutation LMNA R377H (a-c) and control cells (d-f) was done by using antibodies against emerin (a,d), lamin A/C (b,e) and lamin B (c,f). Not all nuclei stain for lamin A/C in patient cells (compare b with e). The corresponding phase-contrast images (a"-c"; d"-f") and Hoechst fluorescences (a'-c'; d'-f') are shown. Bars (c",f") 10 µm.

Mentions: In the immunoblot experiments we had detected a reduced amount of lamin A/C in lymphoblastoid and fibroblast cells but surprisingly not in myoblasts and mature muscle (see Fig. 1). However, by immunofluorescence microscopy of myoblasts (Fig. 4) or frozen sections of a muscle biopsy of patient 99-3 (Fig. 5), about 5% (169 out of 3290) of myoblast nuclei (Fig. 4b) and 5% (79 out of 1580) of mature muscle nuclei showed only a faint staining with lamin A/C antibodies (Fig. 5b, arrow). In contrast, we never saw a reduction of lamin A/C staining in control myoblasts (3430 nuclei) or mature muscle (1650 nuclei). Nuclear periphery staining with emerin or lamin B2 antibodies was normal (Figs. 4, 5a,5c) as was the staining of control myoblasts and muscle sections by all antibodies (Figs. 4, 5d,5e,5f). It is interesting to note that the reduced amount of lamin A/C has been observed not only in cultured cells (Figs. 2, 3, 4), but also on frozen muscle sections (Fig. 5). This indicates that the observed reduction of lamin A/C staining is not caused by our cell culture conditions. Given that only 5% of nuclei in myoblasts and mature muscle fibres showed a reduction of lamin A/C staining it is not surprising that immunoblotting failed to demonstrate a reduction of the level of lamin A/C by (Fig. 1, lane 5).


Expression and localization of nuclear proteins in autosomal-dominant Emery-Dreifuss muscular dystrophy with LMNA R377H mutation.

Reichart B, Klafke R, Dreger C, Krüger E, Motsch I, Ewald A, Schäfer J, Reichmann H, Müller CR, Dabauvalle MC - BMC Cell Biol. (2004)

Some myoblasts cell nuclei from the AD-EDMD patient 99-3 are negative for antibodies to lamin A/C. Immunolabelling of myoblasts cells with mutation LMNA R377H (a-c) and control cells (d-f) was done by using antibodies against emerin (a,d), lamin A/C (b,e) and lamin B (c,f). Not all nuclei stain for lamin A/C in patient cells (compare b with e). The corresponding phase-contrast images (a"-c"; d"-f") and Hoechst fluorescences (a'-c'; d'-f') are shown. Bars (c",f") 10 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC407848&req=5

Figure 4: Some myoblasts cell nuclei from the AD-EDMD patient 99-3 are negative for antibodies to lamin A/C. Immunolabelling of myoblasts cells with mutation LMNA R377H (a-c) and control cells (d-f) was done by using antibodies against emerin (a,d), lamin A/C (b,e) and lamin B (c,f). Not all nuclei stain for lamin A/C in patient cells (compare b with e). The corresponding phase-contrast images (a"-c"; d"-f") and Hoechst fluorescences (a'-c'; d'-f') are shown. Bars (c",f") 10 µm.
Mentions: In the immunoblot experiments we had detected a reduced amount of lamin A/C in lymphoblastoid and fibroblast cells but surprisingly not in myoblasts and mature muscle (see Fig. 1). However, by immunofluorescence microscopy of myoblasts (Fig. 4) or frozen sections of a muscle biopsy of patient 99-3 (Fig. 5), about 5% (169 out of 3290) of myoblast nuclei (Fig. 4b) and 5% (79 out of 1580) of mature muscle nuclei showed only a faint staining with lamin A/C antibodies (Fig. 5b, arrow). In contrast, we never saw a reduction of lamin A/C staining in control myoblasts (3430 nuclei) or mature muscle (1650 nuclei). Nuclear periphery staining with emerin or lamin B2 antibodies was normal (Figs. 4, 5a,5c) as was the staining of control myoblasts and muscle sections by all antibodies (Figs. 4, 5d,5e,5f). It is interesting to note that the reduced amount of lamin A/C has been observed not only in cultured cells (Figs. 2, 3, 4), but also on frozen muscle sections (Fig. 5). This indicates that the observed reduction of lamin A/C staining is not caused by our cell culture conditions. Given that only 5% of nuclei in myoblasts and mature muscle fibres showed a reduction of lamin A/C staining it is not surprising that immunoblotting failed to demonstrate a reduction of the level of lamin A/C by (Fig. 1, lane 5).

Bottom Line: Cellular levels of A-type lamins were reduced compared to control cells.Cell synchronization experiments showed that the reduction of the cellular level of A-type lamin was due to instability of lamin A.From our results we conclude: Firstly, that structural alterations of the nuclei which are found only in a minor fraction of lymphoblastoid cells and mature muscle fibres are not sufficient to explain the clinical pathology of EDMD; Secondly, that wild type lamin A is required not only for the retention of LBR in the inner nuclear membrane but also for a correct localization of the transcriptionally active RNA pol II in muscle cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Developmental Biology, University of Würzburg, Germany. beatereichart@web.de

ABSTRACT

Background: The autosomal dominant form of Emery-Dreifuss muscular dystrophy (AD-EDMD) is caused by mutations in the gene encoding for the lamins A and C (LMNA). Lamins are intermediate filament proteins which form the nuclear lamina underlying the inner nuclear membrane. We have studied the expression and the localization of nuclear envelope proteins in three different cell types and muscle tissue of an AD-EDMD patient carrying a point mutation R377H in the lamin A/C gene.

Results: Lymphoblastoid cells, skin fibroblasts, primary myoblasts and muscle thin sections were studied by immunocytochemistry and electron microscopy. Cellular levels of A-type lamins were reduced compared to control cells. In contrast, the amount of emerin and lamin B appeared unaltered. Cell synchronization experiments showed that the reduction of the cellular level of A-type lamin was due to instability of lamin A. By electron microscopy, we identified a proportion of nuclei with morphological alterations in lymphoblastoid cells, fibroblasts and mature muscle fibres. Immunofluorescence microscopy showed that a major population of the lamin B receptor (LBR), an inner nuclear membrane protein, was recovered in the cytoplasm in association with the ER. In addition, the intranuclear organization of the active form of RNA polymerase II was markedly different in cells of this AD-EDMD patient. This aberrant intranuclear distribution was specifically observed in muscle cells where the pathology of EDMD predominates.

Conclusions: From our results we conclude: Firstly, that structural alterations of the nuclei which are found only in a minor fraction of lymphoblastoid cells and mature muscle fibres are not sufficient to explain the clinical pathology of EDMD; Secondly, that wild type lamin A is required not only for the retention of LBR in the inner nuclear membrane but also for a correct localization of the transcriptionally active RNA pol II in muscle cells. We speculate that a rearrangement of the internal chromatin could lead to muscle-specific disease symptoms by interference with proper mRNA transcription.

Show MeSH
Related in: MedlinePlus