EFFECTS OF LAMINS A/C LOSS OF FUNCTION ON MICE AND PRIMARY MOUSE EMBRYO FIBROBLASTS

Abstract

The nuclear lamina is a peripheral filamentous layer underlying the inner nuclear membrane in vertebrates and invertebrates. Its principal components consist of three nuclear intermediate filament proteins called lamin A, B and C. Lamin A and C are derived from the same gene by alternative splicing. The lamins interact with the nuclear membrane, chromatin, nuclear matrix and associated proteins such as the lamin associated proteins (LAPS), lamin B receptor, nuclear actin, cytoplasmic intermediate filaments, emerin, and retinoblastoma protein (RB). The lamins are thought to provide stability to the nuclear membrane and to organize chromatin by involvement in the disassembly and reassembly of the nuclear envelope during mitosis and in DNA replication, either directly or indirectly through interactions with the nuclear matrix. Interestingly, lamins A and C are not expressed during mammalian embryogenesis until terminal differentiation of cells, whereas lamin B is constitutively expressed in all cells. In adults, lamins A and C are widely expressed, except for hemopoietic stem cells, stem cells of the gut and parts of the cerebellum. To understand the role of lamins in mammalian development, mice were derived by gene targeting that were null for lamins A and C. The mice developed to term, but around 12 days of age, the homozygous mice showed retarded growth and later died at 5-8 weeks. The homozygous mice displayed a hunched appearance, lack of visible fat and had a generalized weakness. Histological analysis revealed that the homozygous mice suffered from a type of muscular dystrophy in the skeletal and heart muscles. Ultrastructural analysis of homozygous lung, liver cells and primary mouse embryo fibroblasts (PMEFs) revealed perturbations of the nuclear membrane with undefined areas, loss of heterochromatin and extrusion of the chromatin contents indicating that the lamina had collapsed. Homozygous PMEF's in vitro cell growth and proliferation assays indicated that they had reduced proliferation with a high death rate with premature senescence and early death. Immunofluorescence staining of homozygous PMEFs showed lack of lamin B, nuclear pore protein (Nup 153) and LAP-2 protein localization to one or both nuclear poles, indicating that the lamina had collapsed in these areas, possibly because of locomotion related stress. Immunofluorescent staining of emerin, an inner nuclear membrane associated protein, revealed an increased localization to the cytoplasm in homozygotes and heterozygotes. Emerin localization to the nucleus in homozygous lamins A/C mouse skeletal muscle, tongue and heart tissues was decreased or absent. The loss of emerin expression in humans causes an X-linked disease called Emery-Dreifuss muscular dystrophy (EDMD). The syndrome is characterized by progressive muscle wasting and cardiac muscle abnormalities, with the lower leg and upper arm being the skeletal muscle most affected with conduction defects in the heart. The lamins A/C K/0 homozygous mice have remarkable similarities with this syndrome, therefore, it is suggested that the lamin A/C homozygous mice may have a type of EDMD. (The cause of the lamins A/C mice muscular dystrophy may be due to the increased fragility of the nuclear membranes and lamina because of the lack of lamin A and C and/or the increased cytoplasmic localization of emerin.) Recently Bonne, et al. (1999) reported that humans with the autosomal dominant form of EDMD (AD-EDMD) had mutations in the Lamin A/C gene and, therefore, implicated that Lamins A/C loss is the cause of AD-EDMD. Therefore, the lamins A/C null mice generated here, represent a mouse model for studying the autosomal dominant form of Emery- Dreifuss muscular dystrophy.