(A) Kymographs of single molecule nucleation on NPF surfaces (mCherry-WAVE1ΔN, magenta) by spike-in of a small fraction of Alexa647-Arp2/3 (green, c=20 pM) into the overall Arp2/3 pool (100 nM) at indicated applied stress (lower panel) or in an adjacent unloaded control network (upper panel). (B) Mean nucleation rates determined by single molecule imaging normalized to the nucleation rate in an adjacent unloaded control network at indicated growth stress from N=3 independent experiments. Pairs of control and loaded network are measured in the same flow chamber in a large field of view as illustrated by the lines linking two data points. Error bars are SEM. p-values were derived from paired Wilcoxon signed rank tests. (C) Scheme of single-molecule Arp2/3 dynamics followed by TIRF microscopy. Exponential decay of Arp2/3 fluorescence reflects movement of the fluorophore away from the coverslip driven by actin filament assembly. Some Arp2/3 molecules remain attached to the growing actin network for their entire transit through the TIRF illumination field (blue) whereas others detach from the network while still detectable by TIRF Illumination (red). Individual intensity trajectories, therefore, reflect either the transit time through the evanescent field or the time to detachment (dwell time). (D) Normalized frequency of fluorescence transit times of single Alexa 647-Arp2/3 complexes (n=1109, 594, and 318 Arp2/3 molecules at 0, 255, and 1020 Pa growth stress from N=3 independent experiments) in networks assembled at indicated stress. (E) Double-logarithmic plot of the mean fluorescence transit time (+/-SD) as a function of network growth velocity. The dashed line show perfect reciprocal correlation (slope = –1). (F) Normalized frequency of fluorescence dwell times of single Alexa 647-Arp2/3 complexes (n=1109, 594, and 318 Arp2/3 complexes at 0, 255, and 1020 Pa growth stress from N=3 independent experiments) in networks assembled at indicated stress. (G) Examples of kymographs from TIRF microscopy of individual Arp2/3 complexes in networks under high load (1020 pN/m2). Individual complexes are either continuously moving towards the rear of the evanescent field (continuous, green arrows) or dissociating prematurely (abortive, red arrows). (H) Representative time courses of fluorescence intensity for individual Arp2/3 complexes as a function of number of imaging frames categorized as either continuous (top panel) or abortive (bottom panel). (I) Relative frequency of dwell times for Arp2/3 complexes in dendritic networks at high load (1020pN/m2, red, n=318 Arp2/3 molecules from N=3 independent experiments) compared to the bleaching and loss of tracking control for surface-immobilized Arp2/3 complexes (blue, see Figure 3—figure supplement 1, n=274 Arp2/3 complexes from N=3 independent experiments). Note that the frequency of early loss events is exceeding the combined bleaching and tracking loss frequency.