Direct measurement of local raised subplasmalemmal calcium concentrations in growth cones advancing on an N‐cadherin substrate

Abstract

We have used the membrane-localized calcium probe fura-piperazine-C12H25 (FFP-18) to examine cytosolic calcium concentrations in a volume close to the plasmalemma. Although promotion of axon outgrowth by cell adhesion molecules requires extracellular calcium and is correlated with an opening of plasmalemmal channels, conventional indicators cannot detect a change in the calcium concentration in such stimulated growth cones. We have examined calcium signalling in chick retinal ganglion cell growth cones extending along stripes of N-cadherin. Subplasmalemmal calcium concentrations, reported by FFP-18, were significantly higher in these growth cones than in neighbouring growth cones on either fibronectin or polylysine. In contrast, the bulk cytosolic calcium concentration throughout the growth cone, as measured by Fura-2, was identical in growth cones on and off the N-cadherin stripes. Our results suggest that guidance cues can use extremely local calcium signals to control pathfinding decisions.

Regulation of Axonal Extension and Growth Cone Motility by Calmodulin-Dependent Protein Kinase I

“…CaMKI and the regulation of growth cone motility Transient increases of Ca 2ϩ that are spatially restricted within the growth cone can rapidly alter its morphology and motility, presumably by regulating actin dynamics (Lau et al, 1999;Zheng, 2000;Chadborn et al, 2002;Cheng et al, 2002;Meyer and Feldman, 2002). Our results indicate that CaMKK via its downstream target CaMKI mediates these effects on axonal growth cone motility.…”

Subcellular ROS imaging methods: Relevance for the study of calcium signaling

“…Wherever gradients of Ca 2+ and redox exist local elevations and signaling should also. The plasma membrane divides a high Ca 2+ and oxidized cell exterior from the low Ca 2+ and reducing cytosol, as such Ca 2+ entry drives local elevations in neuronal models [101] which may be detected with probes anchored to the membrane but not the diffuse cytosol [102]. Local elevations of Ca 2+ restricted to the nm range below the plasma membrane have been shown to modulate neighboring plasma membrane Ca 2+ pumps [103] which are further regulated by local mitochondria [104].…”

Cell adhesion and intracellular calcium signaling in neurons

“…Increased steady state levels of intracellular Ca 2+ were also recorded in growth cones of chick retinal ganglion cells grown on N-cadherin and loaded with a membrane targeted Ca 2+ indicator, FFP-18AM [40]. Interestingly, when the Fura-2AM reporter was used, steady state levels of intracellular Ca 2+ were found to be not affected by substrate coated recombinant N-cadherin suggesting that N-cadherin influences predominantly submembrane Ca 2+ levels [40].…”

“…Increased steady state levels of intracellular Ca 2+ were also recorded in growth cones of chick retinal ganglion cells grown on N-cadherin and loaded with a membrane targeted Ca 2+ indicator, FFP-18AM [40]. Interestingly, when the Fura-2AM reporter was used, steady state levels of intracellular Ca 2+ were found to be not affected by substrate coated recombinant N-cadherin suggesting that N-cadherin influences predominantly submembrane Ca 2+ levels [40]. Whole cell voltage clamp recordings also showed that homophilic binding of N-cadherin on neuronal membranes to soluble N-cadherin or N-cadherin overexpressed in Chinese hamster ovary (CHO) cells increases amplitudes of Ca 2+ currents in ciliary ganglion neurons indicating that homophilic interactions of N-cadherin are sufficient to activate a cellular mechanism that regulates Ca 2+ influx [41].…”