Fig. 1
From: Dynamic single cell measurements of kinase activity by synthetic kinase activity relocation sensors
SKARS relocation depends on an intact docking site and phosphorylation of NLS by MAPK. a Schematic of the three domains of the SKARS sensor: the docking site (DS), the nuclear localization signal (NLS), and the fluorescent protein (RFP). The Ste7DS-NLS-RFP SKARS is composed of the Ste7 docking site (Ste7DS, amino acids 1-33), a double NLS, and the mCherry protein. b Microscopy images of cells before and after stimulation with α-factor. In the red channel, one can observe the nuclear exit of the sensor after stimulus while the CFP histone tag signal remains stable. c–f After quantification of the time-lapse movies, the ratio of nuclear-to-cytoplasmic fluorescence is plotted as function of time. The response of cells bearing the functional sensor and stimulated with 1 μM α-factor (Nc = 170, same curve on panels c to f) is compared to unstimulated cells (Nc = 550, c) or to cells deficient for signal transduction (ste11∆, Nc = 360, d), cells bearing a non-docking variant of the sensor (Ste7ND, Nc = 300, e), or a non-phosphorylatable (NLS-4A, Nc = 360, f) or phospho-mimicking variant of the sensor (NLS-4E, Nc = 750, f). Unitless measurements, such as the Nucl/Cyto ratio, are denoted by the symbol [–] in the axis legend. For all similar graphs and unless stated otherwise, the solid lines represent the median of the cell population and the shaded area the 25 and 75 percentile of the population. Nc represents the number of single cells measured