Fig. 1: Light-gated control of nuclear YAP levels mimics endogenous YAP dynamics.

a Left: Hourglass-shaped topology of the YAP signaling network. Right: YAP signaling specificity could be achieved through decoding of YAP concentrations and dynamics controlling downstream effector strength and/or identity to direct cellular decisions (e.g., pluripotency, proliferation, differentiation). b Cellular differentiation progresses from a naive stem cell to a differentiation-competent primed stage, and into committed germ layers (ectoderm, mesendoderm). The process is accompanied by progressive loss of proliferative capacity and acquisition of differentiation competence. c Pulsatile nuclear dynamics of endogenously tagged SNAP-YAP in interphase mESCs at 36 h post directed mesoderm induction. Shown is the quantification of nuclear YAP levels of a representative cell from N = 3 independent experiments. Indicated time points are shown as image on top. The nucleus is outlined (magenta). Scale bar: 10 µm. d Representative single-cell quantification of nuclear YAP levels in the same culture condition as c. Traces are classified as sustained or dynamic using an automated peak detection algorithm (see Supplementary Fig. 2). Peak width and height are indicated by horizontal and vertical red lines. e, f Quantification of the percentage of cells exhibiting YAP pulses (e) and the average YAP pulse duration (f) in mESCs directed along the ectoderm or mesoderm lineages (1.5d post induction). Cells were classified by the peak detection strategy shown in d and Supplementary Fig. 2. Shown are mean ± SEM, N = 3 independent experiments (e), and the Box and Whiskers with median + 5–95 Percentile (f). In f, data was pooled from N = 3 independent experiments. p values from two-sided unpaired Student’s t test (e). g Optogenetic strategy to control nuclear YAP levels by light using the LEXY-tag: the engineered LOV2 protein domain unfolds a nuclear export sequence (NES) upon blue light illumination, causing reversible nuclear LEXY-YAP export. h Representative images from one experiment of mESCs expressing LEXY-YAP in the dark and following 10 min light illumination. Nuclei are– outlined (yellow). Scale bar: 10 µm. i Quantification of nuclear LEXY-YAP levels from microscopy time course demonstrating light-gated induction of consecutive nuclear YAP import/export cycles ranging from the minutes to hours. Blue shading indicate illumination phases, N = 1 experiment.