Fig. 5: The temporal order of nuclear import in cell-free droplets observed via imaging recapitulates the nuclear entry order observed in the embryo proteomic assay.

a Left: Imaging of nuclear import in cell droplets. Xenopus egg extract doped with sperm DNA, which initiates the formation of nuclei, and GFP-tagged protein of interest (here Phf5a) and mCherry-NLS were encapsulated in oil droplets with a microfluidic device. Right: We monitored nuclear import kinetics via fluorescence microscopy. b We quantified the relative fluorescent signal intensity I in the nucleus and cytoplasm and fit the data with a sigmoid to extract the time (T_droplet1/2) at which the relative intensity reaches half of its max value. To overcome extract variability, we calculate the import time difference (∆T_droplet1/2) between mCherry-NLS and the protein of interest. Markers represent the raw measurements. The different symbols represent different droplets; lines are sigmoid fits of corresponding droplets. From these experiments, we extract the median ∆T_droplet1/2. c Scatter plot of the order in nuclear import time (∆T_droplet1/2) from the cell-free assay and the order in T_embryo1/2 for the nine TFs show strong agreement (Spearman correlation of 0.82, p-value = 0.007). d Imaging results of the nuclear protein concentration [nuclear]. The early titrator nuclear protein Yy1 (blue) is high at the early stage and decreases over time, followed by Gtf2e2 (brown) and Gtf2b (orange). e In our import model, we predict that nuclear concentration of high-affinity proteins (blue) is high in the early nuclei and decreases with the continuous import of additional nuclear proteins and the increasing nuclear volume. Nuclear proteins with lower affinities (brown then orange) will reach their highest nuclear concentration at some later times and in a sequence corresponding to their interaction strengths to importin. The imaging results are consistent with the model (visualized by colors corresponding to (d)).