Self-assembling branched actin networks autonomously adapt to increasing loads, ensuring the robustness of critical cellular processes such as clathrin-mediated endocytosis (CME). A detailed understanding of the mechanisms governing this load adaptation remains lacking. Our experimentally grounded model of endocytic actin networks previously predicted increased actin filament nucleation under elevated plasma membrane tension. Given that the apical and basal surfaces of polarized cells differ in membrane tension, we investigated how this prediction of actin load adaptation materialized on each cellular surface. With 3D live-cell lattice light-sheet microscopy, we imaged human-induced pluripotent stem cells endogenously expressing fluorescently tagged markers for CME (AP2-RFP) and branched actin networks (ARPC3-Halo). Quantifying endocytic dynamics by surface, we found similar endocytic lifetimes on apical and basal membranes, consistent with results in other cell types. We increased membrane tension with hypo-osmotic treatment and observed a modest increase in lifetimes, which was more pronounced on the basal surface. Quantifying ARPC3-GFP intensities following osmotic shock showed a negligible change on the apical surface but a ∼40% increase in intensity on the basal surface relative to control. These results indicate that osmotic shock differentially increases membrane tension on the basal surface of hiPSCs, stalling endocytosis and triggering responsive actin accumulation at those sites. From published in vitro work, we hypothesized that the load-dependent reduction of actin filament capping (force-attenuated capping) contributes to increased filament nucleation and force production. We tested this in our model of endocytosis by adding a force-attenuated capping term, constrained by published measurements. Implementing force-attenuated capping led to a ∼30% increase in the number of actin filaments under elevated tension, closely matching our experimental measurements. These results implicate force-attenuated capping in the general principles for load-adaptation by branched actin networks.
Raghunathan et al. (Sun,) studied this question.