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Lamin A/C mutations linked to muscular dystrophies, but not lipodystrophies, cause impaired nuclear structure and mechanics in cells and tissue.
M. Zwerger1, D. E. Jaalouk1, M. Lombardi1, P. Isermann1, J. Lammerding1,2;
1Medicine, Brigham and Women's Hospital, Boston, MA, 2Biomedical Engineering, Cornell
University, Ithaca, NY
Objective: Mutations in the LMNA gene that encodes the nuclear envelope proteins lamin A and C cause a plethora of human diseases (laminopathies), including muscular dystrophies, cardiomyopathies, and familial partial lipodystrophy. It remains unclear how mutations in a single gene can result in such a variety of diseases, many of them specifically affecting muscle tissues, while other mutations cause little or no muscle involvement. Since lamins A and C are the main contributors to nuclear stiffness, we hypothesized that lamin mutations associated with muscular phenotypes could impair the structural properties of the nuclear lamin network, weakening the nuclear integrity and resulting in cells more susceptible to mechanical stress. Methods: We measured nuclear stiffness in fibroblasts derived from patients with diverse laminopathies and in lamin A/C–deficient mouse embryonic fibroblasts (MEFs) engineered to stably express physiological levels of specific lamin A mutants. In a subset of cells, we also probed nucleo-cytoskeletal coupling with a custom-developed microneedle assay. To assess the in situ effect of lamin mutations in muscle, we measured nuclear stiffness in body wall muscle of Drosophila melanogaster expressing lamin C mutations associated with human laminopathies or wildtype lamin.
Results: Patient fibroblasts carrying LMNA mutations associated with muscular dystrophies had significantly softer nuclei than cells from healthy controls (P<0.001), while fibroblasts from lipodystrophy patients had normal nuclear mechanics. Extending our studies to MEFs expressing a broad panel of lamin A mutations, we found that 4 of the 15 tested lamin A mutations caused decreased nuclear stiffness. Importantly, all four mutations were associated with laminopathies affecting muscle tissue, whereas mutations linked to lipodystrophy had no effect on the structural function of lamin A. Of note, most mutations linked to muscular disease also showed disturbed nucleo-cytoskeletal coupling. Experiments with freshly isolated Drosophila melanogaster body wall muscle subjected to mechanical strain confirmed that specific lamin A mutations affect nuclear mechanics not only in isolated cells, but also in intact muscle tissue.
Conclusions: LMNA mutations associated with muscular laminopathies can cause impaired nuclear mechanics, which may lead to increased cellular sensitivity to mechanical stress and contribute to the muscle specific phenotype in these diseases. In conclusion, our results demonstrate the importance of lamins A and C on nuclear mechanics in laminopathies, but also indicate that additional factors such as altered nucleo-cytoskeletal coupling influence the disease outcome.
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Vinculin is important for p130Cas-mediated mechanotransduction.
T. Dey1, R. Janostiak2, D. Rosel2, J. Brabek2, B. Fabry1, W. H. Goldmann1; 1Biophysics, University of Erlangen-Nuremberg, Erlangen, Germany, 2Department of Cell Biology, Charles University Prague, Czech Republic
Fibroblasts, when subjected to mechanical stress, reinforce their focal adhesion contacts by activating the focal adhesion protein p130Cas, followed by binding of p130Cas with focal adhesion kinase (FAK) and talin, and subsequent activation of downstream pathways such as extracellular-signal-regulated kinase, ERK1/2 phosphorylation. A newly discovered phosphorylation site (Y12) on p130Cas has been suggested to play an important role in modulating the binding with FAK, and potentially with vinculin. In particular, phosphorylation of position Y12 or mutation with phospho-mimicking glutamate (Y12→E) suppresses binding to vinculin. Our question was how Y12 phosphorylation and vinculin binding affects the distribution of p130Cas in cells and its activation upon mechanical stress. The presence of p130Cas in the focal adhesion complexes was not altered in vinculin-deficient fibroblasts. Baseline and stretch- induced phosphorylation of p130Cas, however, was reduced compared to vinculin wildtype cells. Moreover, phospho-mimicking mutation (Y12→E) on p130Cas, which prevents vinculin binding, increases downstream phosphorylation of ERK1/2 in stretched cells. Taken together, these data demonstrate that vinculin is an important modulator of the p130Cas-mediated mechanotransduction pathway in cells.
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A mechanosensory system governs myosin II cleavage furrow accumulation.
Y. Kee1, Y. Ren1, D. Dorfman1, M. Iijima1, R. Firtel2, P. Iglesias3, D. Robinson1; 1Department of Cell Biology, Johns Hopkins University, Baltimore, MD, 2Section of Cell & Developmental Biology, University of California San Diego, CA, 3Department of Electrical and Computer Engineering, Johns Hopkins University, Baltimore, MD
The mitotic spindle is generally considered to be the initiator of furrow ingression. However, recent studies have shown that furrows can form in the absence of spindles, particularly during asymmetric cell division. In Dictyostelium, the mechanoenzyme myosin II and the actin crosslinker cortexillin I form a mechanosensor that responds to mechanical stress, which could account for spindle-independent contractile protein recruitment. Here, we show that the cleavage furrow regulatory and contractility network, composed of myosin II, cortexillin I, IQGAP1, IQGAP2, kinesin-6 (kif12) and INCENP, is a mechanical stress-responsive system. Myosin II and cortexillin I form the core mechanosensor, and mechanotransduction is mediated by IQGAP2 through kif12 and INCENP. Additionally, IQGAP2 is antagonized by IQGAP1 to modulate the mechanoresponsiveness of cortexillin I, suggesting a possible mechanism for discriminating between mechanical and biochemical inputs. Furthermore, IQGAP2 is important for maintaining normal spindle morphology and recruitment of kif12 and myosin II to the cleavage furrow. Finally, the mitotic spindle is dispensable for the system. Overall, we suggest that this mechanosensory system is structured like a control system characterized by mechanochemical feedback loops that regulate the myosin II levels at the cleavage furrow.
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Matrix compliance regulates epithelial-mesenchymal transition.
K. Lee1, Q. Chen1, C. Lui1, M. Cichon2, D. Radisky2, C. Nelson1; 1Chemical and Biological Engineering, Princeton University, Princeton, NJ 2Mayo Clinic Cancer Center
Epithelial-mesenchymal transition (EMT) is a phenotypic switch wherein cells detach from a polarized epithelium and acquire a motile, fibroblastic phenotype. Matrix metalloproteinases (MMPs) have been shown to be key factors associated with pathological EMT. We found that microenvironmental context, specifically substratum stiffness, plays a pivotal role in the regulation of EMT by MMP3. Soft substrata, with compliances comparable to that of normal mammary tissue, are protective against MMP3-induced EMT, whereas stiffer substrata, with compliances characteristic of breast tumors, promote EMT. Matrix rigidity governs EMT by controlling the subcellular localization of Rac1b, a highly activated splice variant of Rac1 found in breast and colorectal tumors. Culture on soft substrata inhibits focal adhesion formation and the membrane localization of Rac1b, and thereby interrupts its interaction with NADPH oxidase. This subsequently inhibits MMP3- and Rac1b-induced production of reactive oxygen species (ROS), expression of Snail, and activation of the EMT program. Altering Rac1b prenylation or integrin clustering using mutated forms of these proteins can bypass the signals induced by substratum compliance. These results suggest the role of a novel mechanotransduction pathway in the regulation of EMT.
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Bleaching/blinking assisted localization microscopy (BALM) for super-resolution imaging using standard fluorescent molecules.
D. Burnette1, P. Sengupta1, Y. Dai2, J. Lippincott-Schwartz1, B. Kachar2; 1CBMB, National Institute of Child Health and Human Development, NIH, Bethesda, MD, 2National Institute on Deafness and Other Communication Disorders, NIH, Bethesda, MD
Super-resolution imaging techniques circumvent the diffraction-limit of light microscopes and are useful in cell biological research because they bridge the gap between the image-resolution possible from diffraction-limited light microscopy (hundreds of nanometers) and the resolution possible from electron microscopy (less than 1 nanometer). Point localization techniques, such as Photoactivated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM), achieve a particularly high resolution by fitting single fluorescent molecules with a theoretical Gaussian to localize them with a precision on the order of tens of nanometers. PALM/STORM rely on photoactivatable proteins and photoswitching dyes, respectively, making them technically challenging and preventing them from being easily added to a cell biologist’s “tool box”. Here, we present a simple and practical way of producing point localization-based-super-resolution images, bleaching/blinking localization microscopy (BALM), that relies on the intrinsic bleaching (irreversibly turn off) and blinking (reversibly turn off and on) behaviors characteristic of all commonly used fluorescent molecules. To do BALM, the researcher first acquires a streaming recording of a fixed sample labeled with a fluorescent probe, which will bleach over time. Single molecule-turn off (bleach or blink off) events can be detected by subtracting from each image of the acquisition the next subsequent image. Similarly, blink on events are detectable by subtracting from each frame the previous one. To validate that BALM works using a standard EMCCD camera we first imaged microtubules in COS7 cells labeled with Alexa-488. After image subtractions, the localization precision of single Alexa-488 molecules was subsequently calculated (σ=26.6 +/- 14 nm) which is similar to that achieved by PALM. We then verified the increase in structural resolution by comparing microtubules reconstructed with BALM with those reconstructed with PALM. We then performed BALM on Myosin IIC-GFP to show that it works with exogenously expressed fluorescent
proteins. Finally, we performed BALM on microtubules labeled with Alexa-488, Alexa-561, and Alexa-647 to show that BALM can be used to localize several different fluorescent probes in the same cell. Thus, BALM is a practical and flexible super-resolution technique, which is only limited by the users ability to separate the emission of different fluorophores.
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Adipose-Derived Stem Cells Form Functional Myotubes Via Mechanical Induction whereas Marrow-derived cells Do Not.
Y. Choi1, L. Vincent1, A. Lee1, M. Dobke2, A. Engler1; 1Bioengineering, Univ California-San Diego, La Jolla, CA, 2Plastic Surgery, Univ California-San Diego, La Jolla, CA
Undifferentiated stem cells integrate cues around themselves to differentiate, but often for fibrotic muscle diseases that stiffen the microenvironment, e.g. muscular dystrophy and myocardial infarction, differentiation is misdirected. When co-delivered with exogenous growth factors, bone marrow-derived stem cells (BMSC) commit to their appropriate mesodermal- derived lineages, but efficiency is not sufficiently high enough to restore function. Biochemical methods overlook biophysical interactions between cells and extracellular matrix (ECM), which is stiffened by fibrosis and clearly impacts cell function in vivo. Though they express a similar molecular signature and can commit to mesodermal lineages via growth factors, differences between adipose-derived stem cell (ASC) and BMSCs may likely impact their differentiation efficiency in diseased muscle. Here we found that ASCs reflect the same qualitative stiffness sensitivity as BMSCs with morphological changes reflecting differentiated phenotypes (Engler et al, Cell 2006). However, quantitative analysis of lineage expression showed ASCs correctly express the appropriate temporal sequence of muscle transcriptional regulators, e.g. MyoD, Myogenin, MEF2C, etc., and do so at levels at least 10-fold higher than BMSCs. Moreover by 7 days in culture, 2.1% of ASCs form multi-nucleated myotubes with a continuous network that is not the result of misdirected cell division. This process mirrors that in primary muscle cells, and most importantly, BMSCs were never observed to undergo this process. Treatment with myoseverin, a microtubule depolymerizing drug that severs myotubes, could disrupt ASC- derived myotubes, but 7 days after drug washout, ASCs refused and formed myotubes at a rate similar to their pretreated value. To understand why ASC are more myogenic when stimulated by ECM, it was determined that they appear more contractile and form adhesions faster than BMSCs. Their fate is also alpha5 and alphaV integrin-dependent as they do not undergo myogenesis in their absence. Interestingly, ASC replated on non-permissive substrates maintain their state, which implies that they are less plastic and may likely be more successful in engrafting and restoring function in fibrotic muscle.