Differential Talin cleavage in transformed and non-transformed cells and its consequences.

This study investigates differences in focal adhesion (FA) morphology and Talin cleavage levels between transformed and non-transformed cell lines. Utilizing fluorescently tagged wild-type Talin and Talin mutants with calpain cleavage site mutations, FA structures were visualized. Mutations in different Talin cleavage sites showed varying impacts on FA morphology and distribution across melanoma cell lines (Meljuso, A375P, A2058) and a non-transformed cell line (HFF). Western blot analysis, ratiometric fluorescence intensity-based measurements, and FRAP experiments revealed higher Talin cleavage levels within FAs of transformed cell lines compared to non-transformed cells. Additionally, growth assays indicated that reducing calpain cleavage levels attenuated transformed cell growth. These findings suggest that Talin cleavage level is crucial for FA morphology and assembly, with higher levels observed in transformed cells, influencing their growth dynamics.

Actin polymerisation and crosslinking drive left-right asymmetry in single cell and cell collectives.

Deviations from mirror symmetry in the development of bilateral organisms are common but the mechanisms of initial symmetry breaking are insufficiently understood. The actin cytoskeleton of individual cells self-organises in a chiral manner, but the molecular players involved remain essentially unidentified and the relationship between chirality of an individual cell and cell collectives is unclear. Here, we analysed self-organisation of the chiral actin cytoskeleton in individual cells on circular or elliptical patterns, and collective cell alignment in confined microcultures. Screening based on deep-learning analysis of actin patterns identified actin polymerisation regulators, depletion of which suppresses chirality (mDia1) or reverses chirality direction (profilin1 and CapZß). The reversed chirality  is mDia1-independent but requires the function of actin-crosslinker α-actinin1. A robust correlation between the effects of a variety of actin assembly regulators on chirality of individual cells and cell collectives is revealed. Thus, actin-driven cell chirality may underlie tissue and organ asymmetry.

Assessing and enhancing migration of human myogenic progenitors using directed iPS cell differentiation and advanced tissue modelling.

Muscle satellite stem cells (MuSCs) are responsible for skeletal muscle growth and regeneration. Despite their differentiation potential, human MuSCs have limited in vitro expansion and in vivo migration capacity, limiting their use in cell therapies for diseases affecting multiple skeletal muscles. Several protocols have been developed to derive MuSC-like progenitors from human induced pluripotent stem (iPS) cells (hiPSCs) to establish a source of myogenic cells with controllable proliferation and differentiation. However, current hiPSC myogenic derivatives also suffer from limitations of cell migration, ultimately delaying their clinical translation. Here we use a multi-disciplinary approach including bioinformatics and tissue engineering to show that DLL4 and PDGF-BB improve migration of hiPSC-derived myogenic progenitors. Transcriptomic analyses demonstrate that this property is conserved across species and multiple hiPSC lines, consistent with results from single cell motility profiling. Treated cells showed enhanced trans-endothelial migration in transwell assays. Finally, increased motility was detected in a novel humanised assay to study cell migration using 3D artificial muscles, harnessing advanced tissue modelling to move hiPSCs closer to future muscle gene and cell therapies.

Advanced models of human skeletal muscle differentiation, development and disease: Three-dimensional cultures, organoids and beyond.

Advanced in vitro models of human skeletal muscle tissue are increasingly needed to model complex developmental dynamics and disease mechanisms not recapitulated in animal models or in conventional monolayer cell cultures. There has been impressive progress towards creating such models by using tissue engineering approaches to recapitulate a range of physical and biochemical components of native human skeletal muscle tissue. In this review, we discuss recent studies focussed on developing complex in vitro models of human skeletal muscle beyond monolayer cell cultures, involving skeletal myogenic differentiation from human primary myoblasts or pluripotent stem cells, often in the presence of structural scaffolding support. We conclude with our outlook on the future of advanced skeletal muscle three-dimensional cultures (e.g. organoids and biofabrication) to produce physiologically and clinically relevant platforms for disease modelling and therapy development in musculoskeletal and neuromuscular disorders.

Micro-stepping extended focus reduces photobleaching and preserves structured illumination super-resolution features.

Despite progress made in confocal microscopy, even fast systems still have insufficient temporal resolution for detailed live-cell volume imaging, such as tracking rapid movement of membrane vesicles in three-dimensional space. Depending on the shortfall, this may result in undersampling and/or motion artifacts that ultimately limit the quality of the imaging data. By sacrificing detailed information in the Z-direction, we propose a new imaging modality that involves capturing fast 'projections' from the field of depth and shortens imaging time by approximately an order of magnitude as compared to standard volumetric confocal imaging. With faster imaging, radiation exposure to the sample is reduced, resulting in less fluorophore photobleaching and potential photodamage. The implementation minimally requires two synchronized control signals that drive a piezo stage and trigger the camera exposure. The device generating the signals has been tested on spinning disk confocal and instant structured-illumination-microscopy (iSIM) microscopes. Our calibration images show that the approach provides highly repeatable and stable imaging conditions that enable photometric measurements of the acquired data, in both standard live imaging and super-resolution modes.This article has an associated First Person interview with the first author of the paper.

Actin cytoskeleton self-organization in single epithelial cells and fibroblasts under isotropic confinement.

Actin cytoskeleton self-organization in two cell types, fibroblasts and epitheliocytes, was studied in cells confined to isotropic adhesive islands. In fibroblasts plated onto islands of optimal size, an initially circular actin pattern evolves into a radial pattern of actin bundles that undergo asymmetric chiral swirling before finally producing parallel linear stress fibers. Epitheliocytes, however, did not exhibit succession through all the actin patterns described above. Upon confinement, the actin cytoskeleton in non-keratinocyte epitheliocytes was arrested at the circular stage, while in keratinocytes it progressed as far as the radial pattern but still could not break symmetry. Epithelial-mesenchymal transition pushed actin cytoskeleton development from circular towards radial patterns but remained insufficient to cause chirality. Knockout of cytokeratins also did not promote actin chirality development in keratinocytes. Left-right asymmetric cytoskeleton swirling could, however, be induced in keratinocytes by treatment with small doses of the G-actin sequestering drug, latrunculin A in a transcription-independent manner. Both the nucleus and the cytokeratin network followed the induced chiral swirling. Development of chirality in keratinocytes was controlled by DIAPH1 (mDia1) and VASP, proteins involved in regulation of actin polymerization.This article has an associated First Person interview with the first author of the paper.