Broers Jos L V, Peeters Emiel A G, Kuijpers Helma J H, Endert Jorike, Bouten Carlijn V C, Oomens Cees W J, Baaijens Frank P T, Ramaekers Frans C S
Department of Moecular Cell Biology, Cardiovascular Research Institute Maastricht, University Maastricht, PO Box 616, NL-6200 MD Maastricht, The Netherlands.
Hum Mol Genet. 2004 Nov 1;13(21):2567-80. doi: 10.1093/hmg/ddh295. Epub 2004 Sep 14.
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.
核纤层蛋白病是一组由核纤层蛋白A/C基因（LMNA）突变引起的、具有不同临床表型的遗传性疾病。这些疾病中的几种的一个突出特征是肌肉萎缩，如在埃默里-德赖富斯肌营养不良症、扩张型心肌病和肢带型肌营养不良症中所见。尽管这种表型背后的机制在很大程度上仍不清楚，但目前正在研究两个主要的工作假设，即基因调控缺陷和/或核结构异常导致细胞脆弱性。在这项研究中，我们使用新开发的细胞压缩装置对后一个假设进行了测试。该装置允许对单个活细胞施加可控的机械负荷，同时可视化细胞变形并定量阻力。使用该装置，我们比较了野生型（MEF+/+）和LMNA基因敲除（MEF-/-）小鼠胚胎成纤维细胞（MEF），发现MEF-/-细胞表现出明显降低的松质骨硬度和明显较低的破裂力。用核纤层蛋白A或核纤层蛋白C转染对表型的部分挽救可防止出现如MEF-/-细胞中所见的严重核破坏，但无法完全恢复这些细胞的松质骨硬度。我们的研究表明，活细胞中LMNA蛋白的缺失与核脆弱性之间存在直接相关性。共聚焦显微镜的同步记录显示，与MEF+/+细胞相比，MEF-/-细胞的细胞核在压痕时表现出各向同性变形，尽管细胞整体表现出各向异性变形。这种核行为表明受干扰的细胞核与周围细胞骨架的相互作用丧失。此外，对基于肌动蛋白、波形蛋白和微管蛋白的细丝的三维组织的仔细研究表明，这些结构在MEF-/-细胞中的相互作用受到干扰。因此，我们认为除了核硬度的丧失外，核结构（即核纤层蛋白）与细胞骨架之间物理相互作用的丧失导致了更普遍的细胞脆弱性，并强调了核纤层蛋白在维持细胞张力完整性方面的潜在关键作用。
