Calcium influx alters actin bundle dynamics and retrograde flow in Helisoma growth cones

The ability of calcium (Ca(2+)) to effect changes in growth cone motility requires remodeling of the actin cytoskeleton. To understand the mechanisms involved, we evaluated the effect of elevated intracellular calcium ([Ca(2+)](i)) on actin bundle dynamics, organization, and retrograde flow in the large growth cones of identified Helisoma neurons. Depolarization with 15 mM KCl (high K(+)) for 30 min caused a rapid and sustained increase in [Ca(2+)](i) and resulted in longer filopodia, shorter actin ribs, and a decrease in lamellipodia width. Time-lapse microscopy revealed that increasing [Ca(2+)](i) affected actin bundle dynamics differently at the proximal and distal ends. Filopodial lengthening resulted from assembly-driven elongation of actin bundles whereas actin rib shortening resulted from a distal shift in the location of breakage. Buckling of ribs occurred before breakage, suggesting nonuniform forces were applied to ribs before shortening. Calcium (Ca(2+)) influx also resulted in a decrease in density of F-actin in bundles, as determined by contrast changes in ribs imaged by differential interference contrast microscopy and fluorescent intensity changes in rhodamine-labeled ribs. The velocity of retrograde flow decreased by 50% after elevation of [Ca(2+)](i). However, no significant change in retrograde flow occurred when the majority of changes in actin bundles were blocked by phalloidin. This suggests that inhibition of retrograde flow resulted from Ca(2+)-induced changes in the actin cytoskeleton. These results implicate Ca(2+) as a regulator of actin dynamics and, as such, provide a mechanism by which Ca(2+) can influence growth cone motility and behavior.