Fig. 3. pMHC:TCR binding dwell time modulates probability of LAT condensate formation.

a Top: time series images illustrating the temporal partitioning of the total pMHC:TCR dwell time into a delay time (pink) and a remaining productive dwell time (green). Bottom: A bar plot of a collection of 1071 pMHC:TCR binding events from 12 cells across 3 mice. Each bar represents a fully tracked pMHC:TCR binding event. The moment of condensation (if any) is indicated by the bar transitioning from pink (unproductive dwell time) to green (productive dwell time). The gray lines demarcate the bins used in the histograms for panels b and c. For b and c: Linearly increasing bin widths were used to improve the sampling rate of rare long-binding events. b MCC pMHC dwell time segments were aggregated according to the indicated bin widths. The number of productive dwell time segments (red) for a particular time bin was the number of binding events within that dwell time window which had at some prior time produced a LAT condensate. The total population of dwell time segments (orange) was fit with an exponential decay rate of $k_{obs}=k_{off}+k_{bleach}=\frac{1}{23.8\mathrm{s}}$. The fit distribution was integrated for each bin and plotted as a black circle. c T102S pMHC dwell time segments were aggregated according to the indicated bin widths. The number of productive dwell time segments (green) for a particular time bin was the of number binding events within that dwell time window which had at some prior time produced a LAT condensate. The total population of dwell time segments (aquamarine) was fit with an exponential decay rate of $k_{obs}=k_{off}+k_{bleach}=\frac{1}{9.1\mathrm{s}}$. The fit distribution was integrated for each bin and plotted as a black circle. d Bottom: The probability of a pMHC:TCR binding event producing a localized LAT condensate as a function of dwell time. For each bin of the histograms in panels b and c, the fraction of dwell time segments that were productive is plotted as a point. The error bars were computed as the standard error of the mean for a binomial variable ($n$, the total number of binding events in the bin, and $p$ the productive fraction). The interpolating gray line was a fitted regularized gamma function with a maximal amplitude parameter. Top: The effective rate of LAT production $k_c\left(t\right)$, which is derived from the gray interpolating line in the bottom panel, is plotted along the same time axis. e Nucleation rates for various hypothetical simple kinetic proofreading schemes (N = 1, 2, or 3 steps) are plotted to illustrate key differences compared with the observed effective nucleation rate (see “$k_c\left(t\right)$ as a propensity function” in Methods for more details). Source data are provided as a Source data file.