Human Molecular Genetics Advance Access originally published online on September 14, 2004
Human Molecular Genetics 2004 13(21):2567-2580; doi:10.1093/hmg/ddh295
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Human Molecular Genetics, Vol. 13, No. 21 © Oxford University Press 2004; all rights reserved
Decreased mechanical stiffness in LMNA–/– cells is caused by defective nucleo-cytoskeletal integrity: implications for the development of laminopathies
1Department of Molecular Cell Biology, Cardiovascular Research Institute Maastricht, University Maastricht, PO Box 616, 6200 MD Maastricht, The Netherlands and 2Department of Biomedical Engineering, Biomechanics and Tissue Engineering, Eindhoven University of Technology, The Netherlands
Received June 22, 2004; Accepted September 6, 2004
Laminopathies comprise a group of inherited diseases with variable clinical phenotypes, caused by mutations in the lamin A/C gene (LMNA). A prominent feature in several of these diseases is muscle wasting, as seen in Emery–Dreifuss muscle dystrophy, dilated cardiomyopathy and limb-girdle muscular dystrophy. Although the mechanisms underlying this phenotype remain largely obscure, two major working hypotheses are currently being investigated, namely, defects in gene regulation and/or abnormalities in nuclear architecture causing cellular fragility. In this study, using a newly developed cell compression device we have tested the latter hypothesis. The device allows controlled application of mechanical load onto single living cells, with simultaneous visualization of cellular deformation and quantitation of resistance. With the device, we have compared wild-type (MEF+/+) and LMNA knockout (MEF–/–) mouse embryonic fibroblasts (MEFs), and found that MEF–/– cells show a significantly decreased mechanical stiffness and a significantly lower bursting force. Partial rescue of the phenotype by transfection with either lamin A or lamin C prevented gross nuclear disruption, as seen in MEF–/– cells, but was unable to fully restore mechanical stiffness in these cells. Our studies show a direct correlation between absence of LMNA proteins and nuclear fragility in living cells. Simultaneous recordings by confocal microscopy revealed that the nuclei in MEF–/– cells, in contrast to MEF+/+ cells, exhibited an isotropic deformation upon indentation, despite an anisotropic deformation of the cell as a whole. This nuclear behaviour is indicative for a loss of interaction of the disturbed nucleus with the surrounding cytoskeleton. In addition, careful investigation of the three-dimensional organization of actin-, vimentin- and tubulin-based filaments showed a disturbed interaction of these structures in MEF–/– cells. Therefore, we suggest that in addition to the loss of nuclear stiffness, the loss of a physical interaction between nuclear structures (i.e. lamins) and the cytoskeleton is causing more general cellular weakness and emphasizes a potential key function for lamins in maintaining cellular tensegrity.
* To whom correspondence should be addressed at: Department of Molecular Cell Biology, Cardiovascular Research Institute, University Maastricht, PO Box 616, NL-6200 MD Maastricht, The Netherlands. Tel: +31 433881366; Fax: +31 433884151; Email: jos.broers{at}molcelb.unimaas.nl
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