Induction of Filopodia by Direct Local Elevation of Intracellular Calcium Ion Concentration

In neuronal growth cones, cycles of filopodial protrusion and retraction are important in growth cone translocation and steering. Alteration in intracellular calcium ion concentration has been shown by several indirect methods to be critically involved in the regulation of filopodial activity. Here, we investigate whether direct elevation of [Ca²⁺]_i, which is restricted in time and space and is isolated from earlier steps in intracellular signaling pathways, can initiate filopodial protrusion. We raised [Ca²⁺]_i level transiently in small areas of nascent axons near growth cones in situ by localized photolysis of caged Ca²⁺ compounds. After photolysis, [Ca²⁺]_i increased from ∼60 nM to ∼1 μM within the illuminated zone, and then returned to resting level in ∼10–15 s. New filopodia arose in this area within 1–5 min, and persisted for ∼15 min. Elevation of calcium concentration within a single filopodium induced new branch filopodia. In neurons coinjected with rhodamine-phalloidin, F-actin was observed in dynamic cortical patches along nascent axons; after photolysis, new filopodia often emerged from these patches. These results indicate that local transient [Ca²⁺]_i elevation is sufficient to induce new filopodia from nascent axons or from existing filopodia.