LAMININ DIRECTS GROWTH CONE NAVIGATION VIA 2 TEMPORALLY AND FUNCTIONALLY DISTINCT CALCIUM SIGNALS

During development, growth cones navigate to their targets via numerou s interactions with molecular guidance cues, yet the mechanisms of how growth cones translate guidance information into navigational decisio ns are poorly understood. We have examined the role of intracellular C a2+ in laminin (LN)-mediated growth cone navigation in vitro, using ch ick dorsal root ganglion neurons. Subsequent to contacting LN-coated b eads with filopodia, growth cones displayed a series of stereotypic ch anges in behavior, including turning toward LN-coated beads and a phas e of increased rates of outgrowth after a pause at LN-coated beads. A pharmacological approach indicated that LN-mediated growth cone turnin g required an influx of extracellular Ca2+, likely in filopodia with L N contact, and activation of calmodulin (CaM). Surprisingly, fluoresce nt Ca2+ imaging revealed no LN-induced rise in intracellular Ca2+ in f ilopodia attached to their parent growth cone. However, isolation of f ilopodia by laser-assisted transection unmasked a rapid, LN-specific r ise in intracellular Ca2+ (+73 +/- 11 nM). Additionally, a second, sus tained rise in intracellular Ca2+ (+62 +/- 8 nM) occurred in growth co nes, with a distinct delay 28 +/- 3 min after growth cone filopodia co ntacted LN-coated beads, This delayed, sustained Ca2+ signal parallele d the phase of increased rates of outgrowth, and both events were sens itive to the inhibition of Ca2+/CaM-dependent protein kinase II (CaM-k inase II) with 2 mu M KN-62. We propose that LN-mediated growth cone g uidance can be attributed, in part, to two temporally and functionally distinct Ca2+ signals linked by a signaling cascade composed of CaM a nd CaM-kinase II.