Mathematical modeling of the YAP/TAZ shuttling as a response to cell density, actin dynamics, and liver damaging drugs

The Hippo pathway facilitates its biological function through the regulation of the transcriptional coactivators yes-associated protein (YAP) and WW domain containing transcription regulator 1 (WWTR1 or TAZ). However, both YAP and TAZ may respond differently to activating cues with respect to gene expression, their subcellular localization, and biological properties. To address the question, how YAP and TAZ deferentially respond to external stimuli, we utilized live-cell imaging, quantitative image analysis and mathematical modeling techniques.

Using lentiviruses, we stably transfected Hep3B cells with cerulean-tagged histon 2B (H2B), venus-tagged YAP and mCherry-tagged TAZ reporter constructs that allow to track nuclear-cytoplasmic shuttling of both factors via time-lapse microscopy. The time-resolved data was analyzed in a quantitative manner using Fiji. Image segmentation was performed with the Trainable Weka segmentation plugin, which performs object segmentation based on training of the machine learning algorithms. The output of the classification algorithm is probability maps that are thresholded, thus obtaining binary maps, which are used for the calculation of the nuclear to cytoplasmic ratio of YAP localisation. First, confirmatory experiments revealed that upon increasing cell density YAP clearly shifts from nucleus to cytoplasm. In addition, perturbation of the actin stress fibers by Latrunculin B (LatB) or Cytochalasin D (CytoD) under low cell density conditions led to the clear cytoplasmic enrichment in a dose-dependent manner. The liver-damaging drugs (acetaminophen) lead to slight nuclear enrichment of YAP. Using the measured data, we created an ordinary differential equation-based computational model, which describes Hippo pathway activation upon increasing cell density and perturbations of actin dynamics. In addition, we modeled the interactions between phosphorylated and unphosphorylated YAP and TAZ, actin, 14 – 3-3 and intracellular shuttling and degradation of YAP and TAZ.

In summary, we developed a tool that quantitatively describes the cellular YAP/TAZ response of hepatocellular cells on drugs and liver-damaging substances. Furthermore, this work for the first time shows, how the dynamic regulation of actin affects the subcellular localization of YAP and TAZ and thus activating Hippo pathway in a live-cell setting. In the future, we aim to investigate how Hippo pathway-directed cancer therapies predominantly affect YAP or TAZ.