Publications

Gene editing methods are an attractive therapeutic option for Duchenne muscular dystrophy, and they have an immediate application in the generation of research models. To generate myoblast cultures that could be useful in in vitro drug screening, we have optimised a CRISPR/Cas9 gene edition protocol. We have successfully used it in wild type immortalised myoblasts to delete exon 52 of the dystrophin gene, modelling a common Duchenne muscular dystrophy mutation; and in patient’s immortalised cultures we have deleted an inhibitory microRNA target region of the utrophin UTR, leading to utrophin upregulation. We have characterised these cultures by demonstrating, respectively, inhibition of dystrophin expression and overexpression of utrophin, and evaluating the expression of myogenic factors (Myf5 and MyH3) and components of the dystrophin associated glycoprotein complex (α-sarcoglycan and β-dystroglycan). To demonstrate their use in the assessment of DMD treatments, we have performed exon skipping on the DMDΔ52-Model and have used the unedited DMD cultures/ DMD-UTRN-Model combo to assess utrophin overexpression after drug treatment. While the practical use of DMDΔ52-Model is limited to the validation to our gene editing protocol, DMD-UTRN-Model presents a possible therapeutic gene edition target as well as a useful positive control in the screening of utrophin overexpression drugs.

JTD Keywords: expression, in-vitro, mouse model, muscle, mutations, phenotype, quantification, sarcolemma, therapy, 3' untranslated regions, Cells, cultured, Crispr-cas systems, Cytoskeletal proteins, Drug discovery, Dystroglycans, Dystrophin, Gene editing, Hek293 cells, Humans, Muscular dystrophy, duchenne, Myoblasts, Myogenic regulatory factor 5, Primary cell culture, Sarcoglycans, Utrophin, Utrophin up-regulation

Cell response to force regulates essential processes in health and disease. However, the fundamental mechanical variables that cells sense and respond to remain unclear. Here we show that the rate of force application (loading rate) drives mechanosensing, as predicted by a molecular clutch model. By applying dynamic force regimes to cells through substrate stretching, optical tweezers, and atomic force microscopy, we find that increasing loading rates trigger talin-dependent mechanosensing, leading to adhesion growth and reinforcement, and YAP nuclear localization. However, above a given threshold the actin cytoskeleton softens, decreasing loading rates and preventing reinforcement. By stretching rat lungs in vivo, we show that a similar phenomenon may occur. Our results show that cell sensing of external forces and of passive mechanical parameters (like tissue stiffness) can be understood through the same mechanisms, driven by the properties under force of the mechanosensing molecules involved. Cells sense mechanical forces from their environment, but the precise mechanical variable sensed by cells is unclear. Here, the authors show that cells can sense the rate of force application, known as the loading rate, with effects on YAP nuclear localization and cytoskeletal stiffness remodelling.

JTD Keywords: Actin cytoskeleton, Actin filament, Actin-filament, Adhesion, Animal, Animals, Atomic force microscopy, Breathing, Cell, Cell adhesion, Cell culture, Cell nucleus, Cells, cultured, Cytoplasm, Extracellular-matrix, Fibroblast, Fibroblasts, Fibronectin, Frequency, Gene knockdown, Gene knockdown techniques, Genetics, Germfree animal, Integrin, Intracellular signaling peptides and proteins, Knockout mouse, Lung, Male, Mechanotransduction, Mechanotransduction, cellular, Metabolism, Mice, Mice, knockout, Microscopy, atomic force, Mouse, Optical tweezers, Paxillin, Physiology, Primary cell culture, Pxn protein, mouse, Rat, Rats, Rats, sprague-dawley, Respiration, Signal peptide, Softening, Specific pathogen-free organisms, Sprague dawley rat, Stress, Substrate, Substrate rigidity, Talin, Talin protein, mouse, Tln2 protein, mouse, Traction, Transmission, Ultrastructure, Yap1 protein, rat