Broers, J. L. et al. Decreased mechanical stiffness in LMNA-/- cells is caused by defective nucleo-cytoskeletal integrity: implications for the development of laminopathies. Hum. Mol. Genet. 13, 2567-2580

Department of Moecular Cell Biology, Cardiovascular Research Institute Maastricht, University Maastricht, PO Box 616, NL-6200 MD Maastricht, The Netherlands.
Human Molecular Genetics (Impact Factor: 6.39). 12/2004; 13(21):2567-80. DOI: 10.1093/hmg/ddh295
Source: PubMed


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.

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Available from: Carlijn Bouten, Oct 09, 2015
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    • "The mechanical properties of the nucleus are highly dependent on the molecular and structural composition of the nuclear lamina. For example, nuclear mechanics is impaired in lamin A/C-deficient cells and cells devoid of emerin, an integral protein of the INM that interacts with lamins (Broers et al. 2004; Lammerding et al. 2004, 2005; Rowat et al. 2006; Lee et al. 2007). Furthermore, it appears that the mechanical stiffness of the nucleus and its viscosity are solely dependent on A-type but not B-type lamins, while B-type lamins provide elasticity to the nucleus and the ability to deform. "
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    ABSTRACT: The intermediate filament proteins, A- and B-type lamins, form the nuclear lamina scaffold adjacent to the inner nuclear membrane. B-type lamins confer elasticity, while A-type lamins lend viscosity and stiffness to nuclei. Lamins also contribute to chromatin regulation and various signaling pathways affecting gene expression. The mechanical roles of lamins and their functions in gene regulation are often viewed as independent activities, but recent findings suggest a highly cross-linked and interdependent regulation of these different functions, particularly in mechanosignaling. In this newly emerging concept, lamins act as a "mechanostat" that senses forces from outside and responds to tension by reinforcing the cytoskeleton and the extracellular matrix. A-type lamins, emerin, and the linker of the nucleoskeleton and cytoskeleton (LINC) complex directly transmit forces from the extracellular matrix into the nucleus. These mechanical forces lead to changes in the molecular structure, modification, and assembly state of A-type lamins. This in turn activates a tension-induced "inside-out signaling" through which the nucleus feeds back to the cytoskeleton and the extracellular matrix to balance outside and inside forces. These functions regulate differentiation and may be impaired in lamin-linked diseases, leading to cellular phenotypes, particularly in mechanical load-bearing tissues. © 2015 Osmanagic-Myers et al.; Published by Cold Spring Harbor Laboratory Press.
    Genes & Development 02/2015; 29(3):225-237. DOI:10.1101/gad.255968.114 · 10.80 Impact Factor
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    • "Since most NE genes are expressed ubiquitously, a main challenge is to understand how laminopathy mutations result in tissue-specific phenotypes [5,6]. One proposed hypothesis argues that an alteration of the integrity of the NE - for example, by affecting the nuclear lamina or the connection of the NE with the cytoskeleton - could lead to a structural weakness and a decrease in the ability of the nucleus to resist mechanical stress [10-12]. This could be of special relevance in muscle and skeletal disorders. "
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    ABSTRACT: Laminopathies are diseases characterized by defects in nuclear envelope structure. A well-known example is Emery-Dreifuss muscular dystrophy, which is caused by mutations in the human lamin A/C and emerin genes. While most nuclear envelope proteins are ubiquitously expressed, laminopathies often affect only a subset of tissues. The molecular mechanisms underlying these tissue-specific manifestations remain elusive. We hypothesize that different functional subclasses of genes might be differentially affected by defects in specific nuclear envelope components. Here we determine genome-wide DNA association profiles of two nuclear envelope components, lamin/LMN-1 and emerin/EMR-1 in adult Caenorhabditis elegans. Although both proteins bind to transcriptionally inactive regions of the genome, EMR-1 is enriched at genes involved in muscle and neuronal function. Deletion of either EMR-1 or LEM-2, another integral envelope protein, causes local changes in nuclear architecture as evidenced by altered association between DNA and LMN-1. Transcriptome analyses reveal that EMR-1 and LEM-2 are associated with gene repression, particularly of genes implicated in muscle and nervous system function. We demonstrate that emr-1, but not lem-2, mutants are sensitive to the cholinesterase inhibitor aldicarb, indicating altered activity at neuromuscular junctions. We identify a class of elements that bind EMR-1 but do not associate with LMN-1, and these are enriched for muscle and neuronal genes. Our data support a redundant function of EMR-1 and LEM-2 in chromatin anchoring to the nuclear envelope and gene repression. We demonstrate a specific role of EMR-1 in neuromuscular junction activity that may contribute to Emery-Dreifuss muscular dystrophy in humans.
    Genome biology 02/2014; 15(2):R21. DOI:10.1186/gb-2014-15-2-r21 · 10.81 Impact Factor
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    • "Work in cell culture has demonstrated that in the absence of Lamin A/C, nuclear movement is inhibited (Lee et al., 2007; Hale et al., 2008; Houben et al., 2009; Folker et al., 2011), the ability of the nucleus to withstand physical stress is limited (Broers et al., 2004; Lammerding et al., 2004), and the ability of the cell to organize its genome is compromised (Gnocchi et al., 2011; Mattout et al., 2011). Each of these biological functions has been, and continues to be, explored as possible pathogenic mechanisms of LMNA mutations and significant data support each of these hypotheses. "
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    ABSTRACT: Muscle disease as a group is characterized by muscle weakness, muscle loss, and impaired muscle function. Although the phenotype is the same, the underlying cellular pathologies, and the molecular causes of these pathologies, are diverse. One common feature of many muscle disorders is the mispositioning of myonuclei. In unaffected individuals, myonuclei are spaced throughout the periphery of the muscle fiber such that the distance between nuclei is maximized. However, in diseased muscles, the nuclei are often clustered within the center of the muscle cell. Although this phenotype has been acknowledged for several decades, it is often ignored as a contributor to muscle weakness. Rather, these nuclei are taken only as a sign of muscle repair. Here we review the evidence that mispositioned myonuclei are not merely a symptom of muscle disease but also a cause. Additionally, we review the working models for how myonuclei move from two different perspectives: from that of the nuclei and from that of the cytoskeleton. We further compare and contrast these mechanisms with the mechanisms of nuclear movement in other cell types both to draw general themes for nuclear movement and to identify muscle-specific considerations. Finally, we focus on factors that can be linked to muscle disease and find that genes that regulate myonuclear movement and positioning have been linked to muscular dystrophy. Although the cause-effect relationship is largely speculative, recent data indicate that the position of nuclei should no longer be considered only a means to diagnose muscle disease.
    Frontiers in Physiology 12/2013; 4:363. DOI:10.3389/fphys.2013.00363 · 3.53 Impact Factor
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