Nuclear deformation regulates YAP dynamics in cancer associated fibroblasts.

Cells cultured on stiff 2D substrates exert high intracellular force, resulting in mechanical deformation of their nuclei. This nuclear deformation (ND) plays a crucial role in the transport of Yes Associated Protein (YAP) from the cytoplasm to the nucleus. However, cells in vivo are in soft 3D environment with potentially much lower intracellular forces. Whether and how cells may deform their nuclei in 3D for YAP localization remains unclear. Here, by culturing human colon cancer associated fibroblasts (CAFs) on 2D, 2.5D, and 3D substrates, we differentiated the effects of stiffness, force, and ND on YAP localization. We found that nuclear translocation of YAP depends on the degree of ND irrespective of dimensionality, stiffness and total force. ND induced by the perinuclear force, not the total force, and nuclear membrane curvature correlate strongly with YAP activation. Immunostained slices of human tumors further supported the association between ND and YAP nuclear localization, suggesting ND as a potential biomarker for YAP activation in tumors. Additionally, we conducted quantitative analysis of the force dynamics of CAFs on 2D substrates to construct a stochastic model of YAP kinetics. This model revealed that the probability of YAP nuclear translocation, as well as the residence time in the nucleus follow a power law. This study provides valuable insights into the regulatory mechanisms governing YAP dynamics and highlights the significance of threshold activation in YAP localization. STATEMENT OF SIGNIFICANCE: Yes Associated Protein (YAP), a transcription cofactor, has been identified as one of the drivers of cancer progression. High tumor stiffness is attributed to driving YAP to the nucleus, wherein it activates pro-metastatic genes. Here we show, using cancer associated fibroblasts, that YAP translocation to the nucleus depends on the degree of nuclear deformation, irrespective of stiffness. We also identified that perinuclear force induced membrane curvature correlates strongly with YAP nuclear transport. A novel stochastic model of YAP kinetics unveiled a power law relationship between the activation threshold and persistence time of YAP in the nucleus. Overall, this study provides novel insights into the regulatory mechanisms governing YAP dynamics and the probability of activation that is of immense clinical significance.

Dose-independent threshold illumination for non-invasive time-lapse fluorescence imaging of live cells.

Fluorescent microscopy employs monochromatic light for excitation, which can adversely affect the cells being observed. We reported earlier that fibroblasts relax their contractile force in response to green light of typical intensity. Here we show that such effects are independent of extracellular matrix and cell lines. In addition, we establish a threshold intensity that elicits minimal or no adverse effect on cell contractility even for long-time exposure. This threshold intensity is wavelength dependent. We cultured fibroblasts on soft 2D elastic hydrogels embedded with fluorescent beads to trace substrate deformation and cell forces. The beads move towards cell center when cells contract, but they move away when cells relax. We use relaxation/contraction ratio (λ r), in addition to traction force, as measures of cell response to red (wavelength, λ=635-650 nm), green (λ=545-580 nm) and blue (λ=455-490 nm) lights with varying intensities. Our results suggest that intensities below 57, 31 and 3.5 W/m2 for red, green and blue lights, respectively, do not perturb force homeostasis. To our knowledge, these intensities are the lowest reported safe thresholds, implying that cell traction is a highly sensitive readout of the effect of light on cells. Most importantly, we find these threshold intensities to be dose-independent; i.e., safe regardless of the energy dosage or time of exposure. Conversely, higher intensities result in widespread force-relaxation in cells with λ r > 1. Furthermore, we present a photo-reaction based model that simulates photo-toxicity and predicts threshold intensity for different wavelengths within the visible spectra. In conclusion, we recommend employing illumination intensities below aforementioned wavelength-specific thresholds for time-lapse imaging of cells and tissues in order to avoid light-induced artifacts in experimental observations.