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Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy.

Sullivan T, Escalante-Alcalde D, Bhatt H, Anver M, Bhat N, Nagashima K, Stewart CL, Burke B - J. Cell Biol. (1999)

Bottom Line: Whereas B-type lamins are found in all mammalian cells, A-type lamin expression is developmentally regulated.This phenotype is associated with ultrastructural perturbations to the nuclear envelope.In skeletal and cardiac muscles, this is manifest as a dystrophic condition related to EDMD.

View Article: PubMed Central - PubMed

Affiliation: Advanced BioScience Laboratories-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland 21702, USA.

ABSTRACT
The nuclear lamina is a protein meshwork lining the nucleoplasmic face of the inner nuclear membrane and represents an important determinant of interphase nuclear architecture. Its major components are the A- and B-type lamins. Whereas B-type lamins are found in all mammalian cells, A-type lamin expression is developmentally regulated. In the mouse, A-type lamins do not appear until midway through embryonic development, suggesting that these proteins may be involved in the regulation of terminal differentiation. Here we show that mice lacking A-type lamins develop to term with no overt abnormalities. However, their postnatal growth is severely retarded and is characterized by the appearance of muscular dystrophy. This phenotype is associated with ultrastructural perturbations to the nuclear envelope. These include the mislocalization of emerin, an inner nuclear membrane protein, defects in which are implicated in Emery-Dreifuss muscular dystrophy (EDMD), one of the three major X-linked dystrophies. Mice lacking the A-type lamins exhibit tissue-specific alterations to their nuclear envelope integrity and emerin distribution. In skeletal and cardiac muscles, this is manifest as a dystrophic condition related to EDMD.

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Targeting of the Lmna gene. (a) Structure of the mouse Lmna gene, together with the targeting vector and homologous recombinant containing the PgkNeo cassette. (b) Southern analysis of the representative genotypes from Lmna heterozygote crosses. (c) Northern analysis from wild-type and Lmna  fibroblasts showing loss of full length forms of lamin A and C mRNAs. These are replaced by truncated transcripts at levels 10 fold below those of wild type. (d) Western analysis, with the XB10 antibody (to the central domain of lamin A/C) of nuclear extracts from MEFs of all three genotypes showing that lamin A and C proteins were undetectable in the −/− fibroblasts, whereas lamin B levels were unaffected. P19 EC cells were used as a negative control. (e) Western analysis, using the 1E4 antibody (against the lamin A/C amino-terminal region) of liver nuclei and nuclear envelopes showing, as in the MEFs' absence of the lamin A proteins.
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Figure 1: Targeting of the Lmna gene. (a) Structure of the mouse Lmna gene, together with the targeting vector and homologous recombinant containing the PgkNeo cassette. (b) Southern analysis of the representative genotypes from Lmna heterozygote crosses. (c) Northern analysis from wild-type and Lmna fibroblasts showing loss of full length forms of lamin A and C mRNAs. These are replaced by truncated transcripts at levels 10 fold below those of wild type. (d) Western analysis, with the XB10 antibody (to the central domain of lamin A/C) of nuclear extracts from MEFs of all three genotypes showing that lamin A and C proteins were undetectable in the −/− fibroblasts, whereas lamin B levels were unaffected. P19 EC cells were used as a negative control. (e) Western analysis, using the 1E4 antibody (against the lamin A/C amino-terminal region) of liver nuclei and nuclear envelopes showing, as in the MEFs' absence of the lamin A proteins.

Mentions: To mutate the mouse lamin A/C (Lmna) gene, we deleted a region extending from exon 8 to the middle of exon 11. This removed 114 codons as well as the 3′ untranslated sequence, including the polyadenylation signal of lamin C, whereas 152 codons were eliminated from the lamin A coding region. The deletion was introduced by homologous recombination into ES cells and six homologous recombinant clones were identified (Fig. 1, a and b). After blastocyst injection of two clones, chimeric offspring were derived with subsequent germline transmission of the mutated allele. Heterozygotes were intercrossed to derive viable homozygous offspring. At birth, these were indistinguishable from their heterozygous or wild-type siblings.


Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy.

Sullivan T, Escalante-Alcalde D, Bhatt H, Anver M, Bhat N, Nagashima K, Stewart CL, Burke B - J. Cell Biol. (1999)

Targeting of the Lmna gene. (a) Structure of the mouse Lmna gene, together with the targeting vector and homologous recombinant containing the PgkNeo cassette. (b) Southern analysis of the representative genotypes from Lmna heterozygote crosses. (c) Northern analysis from wild-type and Lmna  fibroblasts showing loss of full length forms of lamin A and C mRNAs. These are replaced by truncated transcripts at levels 10 fold below those of wild type. (d) Western analysis, with the XB10 antibody (to the central domain of lamin A/C) of nuclear extracts from MEFs of all three genotypes showing that lamin A and C proteins were undetectable in the −/− fibroblasts, whereas lamin B levels were unaffected. P19 EC cells were used as a negative control. (e) Western analysis, using the 1E4 antibody (against the lamin A/C amino-terminal region) of liver nuclei and nuclear envelopes showing, as in the MEFs' absence of the lamin A proteins.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169344&req=5

Figure 1: Targeting of the Lmna gene. (a) Structure of the mouse Lmna gene, together with the targeting vector and homologous recombinant containing the PgkNeo cassette. (b) Southern analysis of the representative genotypes from Lmna heterozygote crosses. (c) Northern analysis from wild-type and Lmna fibroblasts showing loss of full length forms of lamin A and C mRNAs. These are replaced by truncated transcripts at levels 10 fold below those of wild type. (d) Western analysis, with the XB10 antibody (to the central domain of lamin A/C) of nuclear extracts from MEFs of all three genotypes showing that lamin A and C proteins were undetectable in the −/− fibroblasts, whereas lamin B levels were unaffected. P19 EC cells were used as a negative control. (e) Western analysis, using the 1E4 antibody (against the lamin A/C amino-terminal region) of liver nuclei and nuclear envelopes showing, as in the MEFs' absence of the lamin A proteins.
Mentions: To mutate the mouse lamin A/C (Lmna) gene, we deleted a region extending from exon 8 to the middle of exon 11. This removed 114 codons as well as the 3′ untranslated sequence, including the polyadenylation signal of lamin C, whereas 152 codons were eliminated from the lamin A coding region. The deletion was introduced by homologous recombination into ES cells and six homologous recombinant clones were identified (Fig. 1, a and b). After blastocyst injection of two clones, chimeric offspring were derived with subsequent germline transmission of the mutated allele. Heterozygotes were intercrossed to derive viable homozygous offspring. At birth, these were indistinguishable from their heterozygous or wild-type siblings.

Bottom Line: Whereas B-type lamins are found in all mammalian cells, A-type lamin expression is developmentally regulated.This phenotype is associated with ultrastructural perturbations to the nuclear envelope.In skeletal and cardiac muscles, this is manifest as a dystrophic condition related to EDMD.

View Article: PubMed Central - PubMed

Affiliation: Advanced BioScience Laboratories-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland 21702, USA.

ABSTRACT
The nuclear lamina is a protein meshwork lining the nucleoplasmic face of the inner nuclear membrane and represents an important determinant of interphase nuclear architecture. Its major components are the A- and B-type lamins. Whereas B-type lamins are found in all mammalian cells, A-type lamin expression is developmentally regulated. In the mouse, A-type lamins do not appear until midway through embryonic development, suggesting that these proteins may be involved in the regulation of terminal differentiation. Here we show that mice lacking A-type lamins develop to term with no overt abnormalities. However, their postnatal growth is severely retarded and is characterized by the appearance of muscular dystrophy. This phenotype is associated with ultrastructural perturbations to the nuclear envelope. These include the mislocalization of emerin, an inner nuclear membrane protein, defects in which are implicated in Emery-Dreifuss muscular dystrophy (EDMD), one of the three major X-linked dystrophies. Mice lacking the A-type lamins exhibit tissue-specific alterations to their nuclear envelope integrity and emerin distribution. In skeletal and cardiac muscles, this is manifest as a dystrophic condition related to EDMD.

Show MeSH
Related in: MedlinePlus