Feedback Interactions between Actin and its Regulators in Endocytic Protein Patches

Abstract

Clathrin-mediated endocytosis (CME) has multiple functions in cells, including regulation of surface expression of proteins, sampling of the cells environment, and ingestion of essential nutrients. Actin polymerization is involved in CME in varying degrees depending on the cell type. CME in yeast requires actin polymerization to bend the cell membrane. Actin polymerization is driven by a network of regulators that activate the Arp2/3 complex. To better understand the interactions between actin, Arp2/3 regulators, and the cell membrane, we perform a joint modeling/experimental study of actin, regulator and cell membrane during endocytosis in budding yeast. We treat actin and Arp2/3 regulators with a stochastic model that grows an explicit three-dimensional actin network, and incorporates a negative-feedback interaction between actin and the regulators. The mechanical interaction between the actin network and the deformed cell membrane is calculated using the Canham-Helfrich free energy, based on a Gaussian membrane shape. The model provides a microscopic view of the endocytic patch and explains key previously observed phenotypes, including the surprising increase in the peak F-actin count occurring when the branching activity of regulators is reduced, the small increase in F-actin resulting from cofilin mutation, the long-lived actin comets seen in Sla2 deletion mutants, and the long regulator lifetime resulting from latrunculin treatment. In addition, the model predicts that reductions in the regulator branching activity enhance regulator assembly. We confirm this prediction by measuring time-dependent regulator counts in cells with mutations in the regulators Arp2/3-binding regions, using quantitative fluorescence microscopy.