Effects of expressing lamin A mutant protein causing Emery-Dreifuss muscular dystrophy and familial partial lipodystrophy in Hela cells

• Published in: Experimental cell research 2003-05, Vol.286 (1), p.75-86
• Main Authors: Bechert, Kim, Lagos-Quintana, Mariana, Harborth, Jens, Weber, Klaus, Osborn, Mary
• Format: Article
• Language: English
• Subjects: Animals; Cell Nucleus - metabolism; DNA-Binding Proteins - metabolism; Emerin; Emery-Dreifuss muscular dystrophy; Familial partial lipodystrophy; HeLa Cells; Humans; Immunohistochemistry; Lamin A; Lamin Type A - genetics; Lamin Type A - metabolism; Lamin Type B - metabolism; Lamins; Lipodystrophy - genetics; Lipodystrophy - metabolism; Membrane Proteins - metabolism; Microscopy, Electron; Muscular Dystrophy, Emery-Dreifuss - genetics; Muscular Dystrophy, Emery-Dreifuss - metabolism; Mutagenesis, Site-Directed; Nuclear aggregates; Nuclear Pore Complex Proteins - metabolism; Nuclear Proteins; Point Mutation; Thymopoietins - metabolism; Transfection
• ISSN: 0014-4827; 1090-2422
• DOI: 10.1016/S0014-4827(03)00104-6

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 10–24×, and wild-type transfected cells 20×, 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.