Effects of expressing lamin A mutant protein causing Emery-Dreifuss muscular dystrophy and familial partial lipodystrophy in HeLa cells.
Patients with the autosomal dominant form of Emery-Dreifuss muscular dystrophy (EDMD) or familial partial lipodystrophy (FPLD) have specific mutations in the lamin A gene. Three such point mutations, G465D (FPLD), R482L, (FPLD), or R527P (EDMD), were introduced by site-specific mutagenesis in the C-terminal tail domain of a FLAG-tagged full-length lamin A construct. HeLa cells were transfected with mutant and wild-type constructs. Lamin A accumulated in nuclear aggregates and the number of cells with aggregates increased with time after transfection. At 72 h post transfection 60-80% of cells transfected with the mutant lamin A constructs had aggregates, while only 35% of the cells transfected with wild-type lamin A revealed aggregates. Mutant transfected cells expressed 10-24x, and wild-type transfected cells 20x, the normal levels of lamin A. Lamins C, B1 and B2, Nup153, LAP2, and emerin were recruited into aggregates, resulting in a decrease of these proteins at the nuclear rim. Aggregates were also characterized by electron microscopy and found to be preferentially associated with the inner nuclear membrane. Aggregates from mutant constructs were larger than those formed by the wild-type constructs, both in immunofluorescence and electron microscopy. The combined results suggest that aggregate formation is in part due to overexpression, but that there are also mutant-specific effects.
Bechert
Kim
K
Max Planck Institute for Biophysical Chemistry, Department of Biochemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Lagos-Quintana
Mariana
M
Harborth
Jens
J
Weber
Klaus
K
Osborn
Mary
M
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D034904:Lamin Type A / Q000235:genetics* / Q000378:metabolism*; D034921:Lamin Type B / Q000378:metabolism; D008060:Lipodystrophy / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D020389:Muscular Dystrophy, Emery-Dreifuss / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism; D009687:Nuclear Proteins; D017354:Point Mutation; D013946:Thymopoietins / Q000378:metabolism; D014162:Transfection
D004268:DNA-Binding Proteins; D034904:Lamin Type A; D034921:Lamin Type B; D008565:Membrane Proteins; C079377:NUP153 protein, human; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D013946:Thymopoietins; C092710:emerin; C093413:lamina-associated polypeptide 2