Quantitative Action Spectroscopy Reveals ARPE-19 Sensitivity to Long-Wave 1 Ultraviolet Radiation at 350 nm and 380 nm

The role of ultraviolet radiation (UVR) exposure in the aetiology of retinal degeneration has been debated for decades with epidemiological evidence failing to find a clear consensus for or against it playing a role. A key reason for this is a lack of foundational research into the response of living retinal tissue to UVR in regard to modern ageing-specific parameters of tissue function. We therefore explored the response of cultured retinal pigmented epithelium (RPE), the loss of which heralds advanced visual decline, to specific wavelengths of UVR across the UV-B and UV-A bands found in natural sunlight. Using a bespoke in vitro UVR exposure apparatus coupled with bandpass filters we exposed the immortalised RPE cell line, ARPE-19, to 10 nm bands of UVR between 290 and 405 nm. Physical cell dynamics were assessed during exposure in cells cultured upon specialist electrode culture plates which allow for continuous, non-invasive electrostatic interrogation of key cell parameters during exposure such as monolayer coverage and tight-junction integrity. UVR exposures were also utilised to quantify wavelength-specific effects using a rapid cell viability assay and a phenotypic profiling assay which was leveraged to simultaneously quantify intracellular reactive oxygen species (ROS), nuclear morphology, mitochondrial stress, epithelial integrity and cell viability as part of a phenotypic profiling approach to quantifying the effects of UVR. Electrical impedance assessment revealed unforeseen detrimental effects of UV-A, beginning at 350 nm, alongside previously demonstrated UV-B impacts. Cell viability analysis also highlighted increased effects at 350 nm as well as 380 nm. Effects at 350 nm were further substantiated by high content image analysis which highlighted increased mitochondrial dysfunction and oxidative stress. We conclude that ARPE-19 cells exhibit a previously uncharacterised sensitivity to UV-A radiation, specifically at 350 nm and somewhat less at 380 nm. If upheld in vivo, such sensitivity will have impacts upon geoepidemiological risk scoring of macular sensitivity

The Hippo pathway drives the cellular response to hydrostatic pressure

Cells need to rapidly and precisely react to multiple mechanical and chemical stimuli in order to ensure precise context‐dependent responses. This requires dynamic cellular signalling events that ensure homeostasis and plasticity when needed. A less well‐understood process is cellular response to elevated interstitial fluid pressure, where the cell senses and responds to changes in extracellular hydrostatic pressure. Here, using quantitative label‐free digital holographic imaging, combined with genome editing, biochemical assays and confocal imaging, we analyse the temporal cellular response to hydrostatic pressure. Upon elevated cyclic hydrostatic pressure, the cell responds by rapid, dramatic and reversible changes in cellular volume. We show that YAP and TAZ, the co‐transcriptional regulators of the Hippo signalling pathway, control cell volume and that cells without YAP and TAZ have lower plasma membrane tension. We present direct evidence that YAP/TAZ drive the cellular response to hydrostatic pressure, a process that is at least partly mediated via clathrin‐dependent endocytosis. Additionally, upon elevated oscillating hydrostatic pressure, YAP/TAZ are activated and induce TEAD‐mediated transcription and expression of cellular components involved in dynamic regulation of cell volume and extracellular matrix. This cellular response confers a feedback loop that allows the cell to robustly respond to changes in interstitial fluid pressure

Optical Cellular Micromotion: A New Paradigm to Measure Tumor Cells Invasion within Gels Mimicking the 3D Tumor Environments

Published online: June 28, 2022Measuring tumor cell invasiveness through 3D tissues, particularly at the single-cell level, can provide important mechanistic understanding and assist in identifying therapeutic targets of tumor invasion. However, current experimental approaches, including standard in vitro invasion assays, have limited physiolog-ical relevance and offer insufficient insight into the vast heterogeneity in tumor cell migration through tissues. To address these issues, here the concept of optical cellular micromotion is reported on, where digital holographic microscopy is used to map the optical nano- to submicrometer thickness fluctuations within single-cells. These fluctuations are driven by the dynamic movement of subcel-lular structures including the cytoskeleton and inherently associated with the biological processes involved in cell invasion within tissues. It is experimentally demonstrated that the optical cellular micromotion correlates with tumor cells motility and invasiveness both at the population and single-cell levels. In addi-tion, the optical cellular micromotion significantly reduced upon treatment with migrastatic drugs that inhibit tumor cell invasion. These results demonstrate that micromotion measurements can rapidly and non-invasively determine the inva-sive behavior of single tumor cells within tissues, yielding a new and powerful tool to assess the efficacy of approaches targeting tumor cell invasiveness.Zhaobin Guo, Chih-Tsung Yang, Chia-Chi Chien, Luke A. Selth, Pierre O. Bagnaninchi, and Benjamin Thierr