Extended Data Fig. 2: Modeling pulse-chase experiments in vivo, constrained by dye-ligand clearance measurements.

a, Model compartments, molecules, and reactions for modeling protein turnover measurements. A pulse or chase dye-ligand is added to the cytosol compartment and subsequently: (1) cleared; (2) can move to a lipid compartment (representing the slow dye-ligand clearance component); or (3) irreversibly binds to the protein-HT. Here, the protein synthesis rate is equal to its degradation rate, with dye-bound proteins degrading at the same rate. This maintains total protein amount constant regardless of dye-ligand binding. See Supplementary Text for more information. b, Fitting of the dye-ligand clearance data (Extended Data Fig. 1) to generate model variants. Twelve experiments (data in blue circles) and the fitted response (blue lines) are shown. The parameters fit were dye-ligand clearance rate, cytosol-to-lipid rate constant, and volume of the lipid compartment. The cytosol compartment was fixed to having a volume of 1 ml. c-e, Example model simulations with a selection of 7 HT-protein mean lifetimes (c: 10-640 h; Legend as in Fig. 1a). The correlation between estimated and true protein lifetime is excellent (d). The mean error (e, top) decreases with increasing lifetime, whereas the standard error between model variants (simulating variability in dye-ligand clearance) peaks around the mean dye-ligand clearance rate (e, bottom). f, Simulation of error in the lifetime estimate as a function of the pulse-chase interval (∆T). The error (blue line) decreases faster than the Pulse concentration (black line). This shows that the error with DELTA is dominated by dye-ligand clearance, with overall low error rates. g, Error in lifetime estimation using pulse-only measurements, given ideal dye-ligand injection delivery. Here, variability of the measurement across animals (CV y-axis) could be countered only by averaging across animals (number of animals x-axis). Error rates (color-coded) are higher than in DELTA, where the chase dye-ligand and the Fraction Pulse calculation normalizes for inter-animal variability under ideal dye-ligand injection conditions. h, Modeling to compare errors due to shot noise with DELTA (left panel) vs. pulse-only labeling (right panel). In both cases three measurement times (white dashed lines) were used to estimate a known decay (x-axis: 2-200 days). Increasing the signal to background ratio (y- axis) and adding a chase dye-ligand (as in DELTA; left panel) reduced shot-noise induced errors. i, Modeling the effects of a change in total protein on the error in lifetime estimation (% error in colormap) as a function of % change in protein levels (x-axis) and the measured fraction pulse (y-axis). Error increases with longer intervals (smaller fraction pulse) and larger changes in protein.