Biology subjects

Toh, P. J.

Publications and source records attributed to Toh, P. J..

3 recordsLinked to original sources

bioRxiv2022

Optogenetic control of YAP can enhance the rate of wound healing

BackgroundTissues 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 limited ability to repair 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 YAP, a transcriptional co-regulator with a pivotal role in growth, in driving repair after injury. MethodsWe 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 and anchorage-independent colony formation assay. ResultsUtilising 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. ConclusionsThis study shows that optogenetic YAP is functional in H9c2 cardiomyoblasts and its controlled activation can potentially enhance wound healing in a range of conditions.

cell biology↗

bioRxiv2021

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, D., 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 {gamma}H2AX, 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 {gamma}H2AX foci compared to ones cultured on soft substrates. Thus, we propose that substrate rigidity modulates genomic stress in the developing epidermal cell, and that Yap1 and Wwtr1 are required for its survival.

developmental biology↗

bioRxiv2021

Optogenetic manipulation 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 attached 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, cell proliferation, and anchorage-independent growth. Similarly, we can utilise optoYAP in zebrafish embryos to modulate target genes. 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.

cell biology↗