The University of Queensland, Queensland Brain Institute.
In chromaffin and neurosecretory cells, the cortical actin network is emerging as an active player in neuroexocytosis. However, its role in compensatory endocytosis is unclear. In this study, we used time-lapse confocal microscopy of chromaffin cells transfected with Lifeact, a fluorescently labelled actin binding peptide, to investigate the role of the actin cortical network in activity-dependant bulk endocytosis. We first show, using a high molecular weight dextran uptake assay, that bulk endocytosis is the predominant form of compensatory endocytosis in chromaffin cells in response to secretagogue stimulation. Importantly, we then show that in chromaffin cells expressing Lifeact, actin rings form concomitantly with and precisely surround these dextran-positive bulk endosomes. Further analysis revealed that these rings were contractile (maximum diameter ~ 1 μm) and formed through dynamic changes of the cortical actin network, characterized by a reorganization of actin fibers following a phase of depolymerization. FRAP analysis of Dextran-positive compartments at different stages of the actin ring constriction revealed differential fluorescence recovery, suggesting a role for these rings in narrowing the neck of bulk endocytic structures prior to their pinching off from the plasma membrane. The contractile nature of these actin rings points to the involvement of myosin-II in their formation. We confirmed that myosin-II is indeed required for both bulk uptake of dextran and actin ring formation as both these processes are sensitive to blebbistatin treatment or myosin-II shRNA knockdown. Collectively, our results point to a selective role of acto-myosin II in promoting activity-dependent bulk endocytosis in neurosecretory chromaffin cells, which may represent a universal process that present in central neurons and at the neuromuscular junction.