Optogenetic control of YAP can enhance the rate of wound healing.

BACKGROUND: Tissues need to regenerate to restore function after injury. Yet, this regenerative capacity varies significantly between organs and between species. For example, in the heart, some species retain full regenerative capacity throughout their lifespan but human cardiac cells display a limited ability to repair the injury. After a myocardial infarction, the function of cardiomyocytes is impaired and reduces the ability of the heart to pump, causing heart failure. Therefore, there is a need to restore the function of an injured heart post myocardial infarction. We investigate in cell culture the role of the Yes-associated protein (YAP), a transcriptional co-regulator with a pivotal role in growth, in driving repair after injury. METHODS: We express optogenetic YAP (optoYAP) in three different cell lines. We characterised the behaviour and function of optoYAP using fluorescence imaging and quantitative real-time PCR of downstream YAP target genes. Mutant constructs were generated using site-directed mutagenesis. Nuclear localised optoYAP was functionally tested using wound healing assay. RESULTS: Utilising optoYAP, which enables precise control of pathway activation, we show that YAP induces the expression of downstream genes involved in proliferation and migration. optoYAP can increase the speed of wound healing in H9c2 cardiomyoblasts. Interestingly, this is not driven by an increase in proliferation, but by collective cell migration. We subsequently dissect specific phosphorylation sites in YAP to identify the molecular driver of accelerated healing. CONCLUSIONS: This study shows that optogenetic YAP is functional in H9c2 cardiomyoblasts and its controlled activation can potentially enhance wound healing in a range of conditions.

Slow muscles guide fast myocyte fusion to ensure robust myotome formation despite the high spatiotemporal stochasticity of fusion events.

Skeletal myogenesis is dynamic, and it involves cell-shape changes together with cell fusion and rearrangements. However, the final muscle arrangement is highly organized with striated fibers. By combining live imaging with quantitative analyses, we dissected fast-twitch myocyte fusion within the zebrafish myotome in toto. We found a strong mediolateral bias in fusion timing; however, at a cellular scale, there was heterogeneity in cell shape and the relationship between initial position of fast myocytes and resulting fusion partners. We show that the expression of the fusogen myomaker is permissive, but not instructive, in determining the spatiotemporal fusion pattern. Rather, we observed a close coordination between slow muscle rearrangements and fast myocyte fusion. In mutants that lack slow fibers, the spatiotemporal fusion pattern is substantially noisier. We propose a model in which slow muscles guide fast myocytes by funneling them close together, enhancing fusion probability. Thus, despite fusion being highly stochastic, a robust myotome structure emerges at the tissue scale.

Optogenetic control of YAP cellular localisation and function.

YAP, an effector of the Hippo signalling pathway, promotes organ growth and regeneration. Prolonged YAP activation results in uncontrolled proliferation and cancer. Therefore, exogenous regulation of YAP activity has potential translational applications. We present a versatile optogenetic construct (optoYAP) for manipulating YAP localisation, and consequently its activity and function. We attach a LOV2 domain that photocages a nuclear localisation signal (NLS) to the N-terminus of YAP. In 488 nm light, the LOV2 domain unfolds, exposing the NLS, which shuttles optoYAP into the nucleus. Nuclear import of optoYAP is reversible and tuneable by light intensity. In cell culture, activated optoYAP promotes YAP target gene expression and cell proliferation. Similarly, optofYap can be used in zebrafish embryos to modulate target genes. We demonstrate that optoYAP can override a cell's response to substrate stiffness to generate anchorage-independent growth. OptoYAP is functional in both cell culture and in vivo, providing a powerful tool to address basic research questions and therapeutic applications in regeneration and disease.

DNA-damage induced cell death in yap1;wwtr1 mutant epidermal basal cells.

In a previous study, it was reported that Yap1 and Wwtr1 in zebrafish regulates the morphogenesis of the posterior body and epidermal fin fold (Kimelman et al., 2017). We report here that DNA damage induces apoptosis of epidermal basal cells (EBCs) in zebrafish yap1-/-;wwtr1-/- embryos. Specifically, these mutant EBCs exhibit active Caspase-3, Caspase-8, and γH2AX, consistent with DNA damage serving as a stimulus of the extrinsic apoptotic pathway in epidermal cells. Live imaging of zebrafish epidermal cells reveals a steady growth of basal cell size in the developing embryo, but this growth is inhibited in mutant basal cells followed by apoptosis, leading to the hypothesis that factors underscoring cell size play a role in this DNA damage-induced apoptosis phenotype. We tested two of these factors using cell stretching and substrate stiffness assays, and found that HaCaT cells cultured on stiff substrates exhibit more numerous γH2AX foci compared to ones cultured on soft substrates. Thus, our experiments suggest that substrate rigidity may modulate genomic stress in epidermal cells, and that Yap1 and Wwtr1 promotes their survival.

Enhancement of Endothelialization by Topographical Features Is Mediated by PTP1B-Dependent Endothelial Adherens Junctions Remodeling.

Endothelial Cells (ECs) form cohesive cellular lining of the vasculature and play essential roles in both developmental processes and pathological conditions. Collective migration and proliferation of endothelial cells (ECs) are key processes underlying endothelialization of vessels as well as vascular graft, but the complex interplay of mechanical and biochemical signals regulating these processes are still not fully elucidated. While surface topography and biochemical modifications have been used to enhance endothelialization in vitro, thus far such single-modality modifications have met with limited success. As combination therapy that utilizes multiple modalities has shown improvement in addressing various intractable and complex biomedical conditions, here, we explore a combined strategy that utilizes topographical features in conjunction with pharmacological perturbations. We characterized EC behaviors in response to micrometer-scale grating topography in concert with pharmacological perturbations of endothelial adherens junctions (EAJ) regulators. We found that the protein tyrosine phosphatase, PTP1B, serves as a potent regulator of EAJ stability, with PTP1B inhibition synergizing with grating topographies to modulate EAJ rearrangement, thereby augmenting global EC monolayer sheet orientation, proliferation, connectivity, and collective cell migration. Our data delineates the crosstalk between cell-ECM topography sensing and cell-cell junction integrity maintenance and suggests that the combined use of grating topography and PTP1B inhibitor could be a promising strategy for promoting collective EC migration and proliferation.

Biomimetic niches reveal the minimal cues to trigger apical lumen formation in single hepatocytes.

The symmetry breaking of protein distribution and cytoskeleton organization is an essential aspect for the development of apicobasal polarity. In embryonic cells this process is largely cell autonomous, while differentiated epithelial cells collectively polarize during epithelium formation. Here, we demonstrate that the de novo polarization of mature hepatocytes does not require the synchronized development of apical poles on neighbouring cells. De novo polarization at the single-cell level by mere contact with the extracellular matrix and immobilized cadherin defining a polarizing axis. The creation of these single-cell liver hemi-canaliculi allows unprecedented imaging resolution and control and over the lumenogenesis process. We show that the density and localization of cadherins along the initial cell-cell contact act as key triggers of the reorganization from lateral to apical actin cortex. The minimal cues necessary to trigger the polarization of hepatocytes enable them to develop asymmetric lumens with ectopic epithelial cells originating from the kidney, breast or colon.