Induction of filopodia by direct local elevation of intracellular calcium ion concentration

In neuronal growth cones, cycles of filopodial protrusion and retraction ar e important in growth cone translocation and steering. Alteration in intrac ellular calcium ion concentration has been shown by several indirect method s 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 signa ling pathways, can initiate filopodial protrusion. We raised [Ca2+](i) leve l transiently in small areas of nascent axons near growth cones in situ by localized photolysis of caged Ca2+ compounds. After photolysis, [Ca2+](i) i ncreased from similar to 60 nM to similar to 1 mu M within the illuminated zone, and then returned to resting level in similar to 10-15 s, New filopod ia arose in this area within 1-5 min, and persisted for similar to 15 min. Elevation of calcium concentration within a single filopodium induced new b ranch filopodia. In neurons coinjected with rhodamine-phalloidin, F-actin w as observed in dynamic cortical patches along nascent axons; after photolys is new filopodia often emerged from these patches. These results indicate t hat local transient [Ca2+](i) elevation is sufficient to induce new filopod ia from nascent axons or from existing filopodia.