Myopathic lamin mutations impair nuclear stability in cells and tissue and disrupt nucleo-cytoskeletal coupling.

Lamins are intermediate filament proteins that assemble into a meshwork underneath the inner nuclear membrane, the nuclear lamina. Mutations in the LMNA gene, encoding lamins A and C, cause a variety of diseases collectively called laminopathies. The disease mechanism for these diverse conditions is not well understood. Since lamins A and C are fundamental determinants of nuclear structure and stability, we tested whether defects in nuclear mechanics could contribute to the disease development, especially in laminopathies affecting mechanically stressed tissue such as muscle. Using skin fibroblasts from laminopathy patients and lamin A/C-deficient mouse embryonic fibroblasts stably expressing a broad panel of laminopathic lamin A mutations, we found that several mutations associated with muscular dystrophy and dilated cardiomyopathy resulted in more deformable nuclei; in contrast, lamin mutants responsible for diseases without muscular phenotypes did not alter nuclear deformability. We confirmed our results in intact muscle tissue, demonstrating that nuclei of transgenic Drosophila melanogaster muscle expressing myopathic lamin mutations deformed more under applied strain than controls. In vivo and in vitro studies indicated that the loss of nuclear stiffness resulted from impaired assembly of mutant lamins into the nuclear lamina. Although only a subset of lamin mutations associated with muscular diseases caused increased nuclear deformability, almost all mutations tested had defects in force transmission between the nucleus and cytoskeleton. In conclusion, our results indicate that although defective nuclear stability may play a role in the development of muscle diseases, other factors, such as impaired nucleo-cytoskeletal coupling, likely contribute to the muscle phenotype.

Keeping the LINC: the importance of nucleocytoskeletal coupling in intracellular force transmission and cellular function.

Providing a stable physical connection between the nucleus and the cytoskeleton is essential for a wide range of cellular functions and it could also participate in mechanosensing by transmitting intra- and extra-cellular mechanical stimuli via the cytoskeleton to the nucleus. Nesprins and SUN proteins, located at the nuclear envelope, form the LINC (linker of nucleoskeleton and cytoskeleton) complex that connects the nucleus to the cytoskeleton; underlying nuclear lamins contribute to anchoring LINC complex components at the nuclear envelope. Disruption of the LINC complex or loss of lamins can result in disturbed perinuclear actin and intermediate filament networks and causes severe functional defects, including impaired nuclear positioning, cell polarization and cell motility. Recent studies have identified the LINC complex as the major force-transmitting element at the nuclear envelope and suggest that many of the aforementioned defects can be attributed to disturbed force transmission between the nucleus and the cytoskeleton. Thus mutations in nesprins, SUN proteins or lamins, which have been linked to muscular dystrophies and cardiomyopathies, may weaken or completely eliminate LINC complex function at the nuclear envelope and result in impaired intracellular force transmission, thereby disrupting critical cellular functions.

Biophysical assays to probe the mechanical properties of the interphase cell nucleus: substrate strain application and microneedle manipulation.

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.(1) On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.(2) Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.(3) To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

Cell-surface sensors for real-time probing of cellular environments.

The ability to explore cell signalling and cell-to-cell communication is essential for understanding cell biology and developing effective therapeutics. However, it is not yet possible to monitor the interaction of cells with their environments in real time. Here, we show that a fluorescent sensor attached to a cell membrane can detect signalling molecules in the cellular environment. The sensor is an aptamer (a short length of single-stranded DNA) that binds to platelet-derived growth factor (PDGF) and contains a pair of fluorescent dyes. When bound to PDGF, the aptamer changes conformation and the dyes come closer to each other, producing a signal. The sensor, which is covalently attached to the membranes of mesenchymal stem cells, can quantitatively detect with high spatial and temporal resolution PDGF that is added in cell culture medium or secreted by neighbouring cells. The engineered stem cells retain their ability to find their way to the bone marrow and can be monitored in vivo at the single-cell level using intravital microscopy.

The interaction between nesprins and sun proteins at the nuclear envelope is critical for force transmission between the nucleus and cytoskeleton.

Maintaining physical connections between the nucleus and the cytoskeleton is important for many cellular processes that require coordinated movement and positioning of the nucleus. Nucleo-cytoskeletal coupling is also necessary to transmit extracellular mechanical stimuli across the cytoskeleton to the nucleus, where they may initiate mechanotransduction events. The LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, formed by the interaction of nesprins and SUN proteins at the nuclear envelope, can bind to nuclear and cytoskeletal elements; however, its functional importance in transmitting intracellular forces has never been directly tested. This question is particularly relevant since recent findings have linked nesprin mutations to muscular dystrophy and dilated cardiomyopathy. Using biophysical assays to assess intracellular force transmission and associated cellular functions, we identified the LINC complex as a critical component for nucleo-cytoskeletal force transmission. Disruption of the LINC complex caused impaired propagation of intracellular forces and disturbed organization of the perinuclear actin and intermediate filament networks. Although mechanically induced activation of mechanosensitive genes was normal (suggesting that nuclear deformation is not required for mechanotransduction signaling) cells exhibited other severe functional defects after LINC complex disruption; nuclear positioning and cell polarization were impaired in migrating cells and in cells plated on micropatterned substrates, and cell migration speed and persistence time were significantly reduced. Taken together, our findings suggest that the LINC complex is critical for nucleo-cytoskeletal force transmission and that LINC complex disruption can result in defects in cellular structure and function that may contribute to the development of muscular dystrophies and cardiomyopathies.

HLA-Cw allele frequencies in northern and southern Italy.

The evidence for HLA-Cw antigens' involvement in the modulation of the immune response in bone marrow transplantation, NK alloreactivity and the susceptibility and follow-up for different diseases has been growing in the recent years, but very few data on HLA-Cw distribution in healthy Italian subjects are available to date. This report presents an updated description of HLA-Cw frequencies in Italy, comparing data from the northern (Lombardia) and southern (Campania and Puglia) parts of the country. A total of 1101 healthy subjects of Italian origin were genotyped, and the results showed that HLA-Cw*04, Cw*07, Cw*12, and, in particular, Cw*0401, Cw*0701, Cw*1203, were the most frequent alleles found in all three regions analysed. Nevertheless, statistically significant differences were observed in Cw*07 distribution, which was more frequent in the southern than in the northern part of Italy (28.8% vs 22.4%; p=0.001; OR: 1.4; 95%CI: 1.14-1.73), and in Cw*12 distribution, which was more frequent in the north than the south (17.0% vs 12.4%; p=0.007, OR: 1.4; 95%CI: 1.10-1.91). These results, which give an improved pattern of distribution of HLA-Cw alleles in the Italian population, would be useful in bone marrow transplantation and anthropological studies. Moreover, due to the important role of HLA-Cw antigens in modulation of the immune response and NK alloreactivity, these data would be of interest in studies on susceptibility, follow-up and/or protection against different diseases.

Traction force microscopy in Dictyostelium reveals distinct roles for myosin II motor and actin-crosslinking activity in polarized cell movement.

Continuous cell movement requires the coordination of protrusive forces at the leading edge with contractile forces at the rear of the cell. Myosin II is required to generate the necessary contractile force to facilitate retraction; however, Dictyostelium cells that lack myosin II (mhcA-) are still motile. To directly investigate the role of myosin II in contractility we used a gelatin traction force assay to measure the magnitude and dynamic redistribution of traction stresses generated by randomly moving wild-type, myosin II essential light chain null (mlcE-) and mhcA- cells. Our data show that for each cell type, periods of rapid, directed cell movement occur when an asymmetrical distribution of traction stress is present, in which traction stresses at the rear are significantly higher than those at the front. We found that the major determinants of cell speed are the rate and frequency at which traction stress asymmetry develops, not the absolute magnitude of traction stress. We conclude that traction stress asymmetry is important for rapid, polarized cell movement because high traction stresses at the rear promote retraction, whereas low traction at the front allows protrusion. We propose that myosin II motor activity increases the rate and frequency at which traction stress asymmetry develops, whereas actin crosslinking activity is important for stabilizing it.

Human melanoma metastases express functional CXCR4.

PURPOSE: The chemokine receptor CXCR4 was identified as an independent predictor of poor prognosis in primary melanoma. The aim of the study was to investigate the role of CXCR4 in human melanoma metastases. EXPERIMENTAL DESIGN: CXCR4 expression was evaluated in melanoma metastases and in metastatic cell lines through immunohistochemistry, immunoblotting, immunofluorescence, and reverse transcription-PCR. The function of CXCR4 was tested in the presence of the ligand, CXCL12, through induction of extracellular signal-regulated kinase-1 and -2 (Erk-1 and -2) phosphorylation, proliferation, apoptosis, and migration capabilities. RESULTS: CXCR4 expression was detected in 33 out of 63 (52.4%) metastases from cutaneous melanomas. Metastatic melanoma cell lines expressed cell surface CXCR4; PES 43, Alo 40, and COPA cell lines showed the highest levels of CXCR4 (>90% of positive cells); PES 41, Alo 39, PES 47, POAG, and CIMA cell lines showed low to moderate degrees of expression (5-65% of positive cells). Other chemokine receptors, CCR7 and CCR10, were detected on the melanoma cell lines; CXCL12 activated Erk-1 and Erk-2, the whose induction was specifically inhibited by AMD3100 treatment. CXCL12 increased the growth in PES 41, PES 43, and PES 47 cells under suboptimal (1% serum) and serum-free culture conditions; AMD3100 (1 mumol/L) inhibited the spontaneous and CXCL12-induced proliferation. No rescue from apoptosis was shown but PES 41, PES 43, and PES 47 cells migrate toward CXCL12. CONCLUSIONS: These findings indicate that CXCR4 is expressed and active in human melanoma metastases, suggesting that active inhibitors such as AMD3100 may be experienced in human melanoma.