Differential interference with actin-binding protein function by acute cytochalasin B

Dynamic actin filament remodeling is crucial for a plethora of fundamental cell biological processes, ranging from cell division and migration to cell communication, intracellular trafficking, or tissue development. Cytochalasin B (CB) and D (CD) are fungal secondary metabolites frequently used for interference with such processes. Although they are generally assumed to block actin filament polymerization at their rapidly growing barbed ends and compete with regulators at these sites, precise molecular understanding of their effects in dynamic actin structures requires further study. Here, we combine live cell imaging and analysis of fluorescent actin-binding protein dynamics with acute treatment of lamellipodia in migrating cells with CB. Our results show that in spite of an abrupt halt of lamellipodium protrusion, CB affects various actin filament barbed end-binding proteins in a differential fashion. CB enhances, instead of diminishes, the accumulation of prominent barbed end-binding factors such as Ena/VASP family proteins and heterodimeric capping protein (CP) in the lamellipodium. Similar results were obtained with CD. These effects are highly specific, as cytochalasin-induced VASP accumulation requires the presence of CP, but not vice versa, and coincides with abrogation of both actin and VASP turnover. We also reconstituted CB-induced VASP accumulation on Arp2/3 complex-dependent actin networks in vitro, which we found to derive from an increase in VASP dwell time on individual filament barbed ends. In conclusion, our results reveal a new spectrum of cytochalasin activities on barbed end-binding factors, with important implications for the interpretation of their effects on dynamic actin structures in situ.