Calcium and Protein Kinase C Regulate the Actin Cytoskeleton in the Synaptic Terminal of Retinal Bipolar Cells

Abstract

The organization of filamentous actin (F-actin) in the synaptic pedicle of depolarizing bipolar cells from the goldfish retina was studied using fluorescently labeled phalloidin. The amount of F-actin in the synaptic pedicle relative to the cell body increased from a ratio of 1.6 ± 0.1 in the dark to 2.1 ± 0.1 after exposure to light. Light also caused the retraction of spinules and processes elaborated by the synaptic pedicle in the dark.Isolated bipolar cells were used to characterize the factors affecting the actin cytoskeleton. When the electrical effect of light was mimicked by depolarization in 50 mM K+, the actin network in the synaptic pedicle extended up to 2.5 μm from the plasma membrane. Formation of F-actin occurred on the time scale of minutes and required Ca2+ influx through L-type Ca2+ channels. Phorbol esters that activate protein kinase C (PKC) accelerated growth of F-actin. Agents that inhibit PKC hindered F-actin growth in response to Ca2+ influx and accelerated F-actin breakdown on removal of Ca2+.To test whether activity-dependent changes in the organization of F-actin might regulate exocytosis or endocytosis, vesicles were labeled with the fluorescent membrane marker FM1-43. Disruption of F-actin with cytochalasin D did not affect the continuous cycle of exocytosis and endocytosis that was stimulated by maintained depolarization, nor the spatial distribution of recycled vesicles within the synaptic terminal. We suggest that the actions of Ca2+ and PKC on the organization of F-actin regulate the morphology of the synaptic pedicle under varying light conditions.