Decreased mechanical stiffness in LMNA-/- cells is caused by defective nucleo-cytoskeletal integrity: implications for the development of laminopathies
Decreased mechanical stiffness in LMNA-/- cells is caused by defective nucleo-cytoskeletal integrity: implications for the development of laminopathies
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DOI：
10.1093/hmg/ddh295
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发表时间：
2004-11-01
影响因子：
3.5
通讯作者：
Ramaekers, FCS
中科院分区：
文献类型：
--
作者：
Broers, JLV;Peeters, EAG;Ramaekers, FCS
核纤层蛋白病包括一组具有可变临床表型的遗传性疾病，由核纤层蛋白A/C基因（LMNA）中的突变引起。这些疾病中的几种的突出特征是肌肉萎缩，如在Emery-Dreifuss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。虽然这种表型的机制在很大程度上仍然模糊，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性。在这项研究中，使用新开发的细胞压缩装置，我们已经测试了后者的假设。该装置允许将机械载荷受控地施加到单个活细胞上，同时可视化细胞变形和定量阻力。使用该装置，我们比较了野生型（MEF+/+）和LMNA敲除（MEF-/-）小鼠胚胎成纤维细胞（MEF），发现MEF-/-细胞显示出显著降低的机械刚度和显著降低的破裂力。通过用核纤层蛋白A或核纤层蛋白C转染的表型的部分拯救防止了如在MEF-/-细胞中所见的总体核破坏，但不能完全恢复这些细胞中的机械刚度。我们的研究表明，在活细胞中，LMNA蛋白的缺失与核脆性之间存在直接相关性。通过共聚焦显微镜同时记录显示，MEF-/-细胞中的细胞核，与MEF+/+细胞相反，在压痕时表现出各向同性变形，尽管作为一个整体的细胞各向异性变形。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，肌动蛋白，波形蛋白和微管蛋白为基础的细丝的三维组织的仔细调查显示，这些结构在MEF-/-细胞的干扰相互作用。因此，我们认为，除了核硬度的损失，核结构（即核纤层蛋白）和细胞骨架之间的物理相互作用的损失，造成更普遍的细胞虚弱，并强调核纤层蛋白在维持细胞张力整体性的潜在关键功能。
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.