Cyclic AMP (cAMP) and calcium are ubiquitous, interdependent second messengers that regulate a wide range of cellular processes. During development of neuronal networks they are critical for the first step of circuit formation, transducing signals required for axon pathfinding. Surprisingly, the spatial and temporal cAMP and calcium codes used by axon guidance molecules are unknown. Here, we identify characteristics of cAMP and calcium transients generated in growth cones during Netrin-1-dependent axon guidance. In filopodia, Netrin-1-dependent Deleted in Colorectal Cancer (DCC) receptor activation induces a transient increase in cAMP that causes a brief increase in calcium transient frequency. In contrast, activation of DCC in growth cone centers leads to a transient calciumdependent cAMP increase and a sustained increase in frequency of calcium transients. We show that filopodial cAMP transients regulate spinal axon guidance in vitro and commissural axon pathfinding in vivo. These growth cone codes provide a basis for selective activation of specific downstream effectors.cyclic AMP dynamics | calcium dynamics | compartmentalization | photoactivated adenylyl cyclase alpha | cyclic AMP oscillations C yclic AMP (cAMP) is a major cellular second messenger that activates and integrates multiple intracellular signaling pathways. Microdomains of cAMP are generated in cardiac myocytes (1), regional domains of cAMP are present in neurons (2, 3), and cAMP diffusion is restricted by rapid degradation by phosphodiesterases (4) that limit the duration of cAMP signaling (5, 6). However, little is known about spatial compartmentalization and temporal dynamics of cAMP in neuronal growth cones, despite the importance of cAMP for axon pathfinding in response to a wide range of molecular guidance cues, including Netrin-1 (7-11), semaphorins (12), Slits, and ephrins (13).Calcium is another ubiquitous second messenger involved in axon pathfinding, mediating responses to axon guidance molecules. In addition to axon steering, calcium modulates axon outgrowth and retraction (14,15). A sustained gradient of calcium across the growth cone is thought to be generated by asymmetric activation of axon guidance receptors, and to be the relevant calcium signal for axon pathfinding (16). Spontaneous fast and spatially restricted filopodial calcium transients are also sufficient to steer axons (17, 18). The frequency of slower transients in the entire growth cone controls the rate of axon extension in vivo and in vitro (19). The regulation of these transients by axon guidance cues has not been investigated.Filopodia are critical for axon pathfinding (20), and clues to their operation are provided by subcellular localization of signaling components. Filopodial enrichment of regulatory subunit II of protein kinase A, a major effector of cAMP, is required for growth cone attraction mediated by intracellular gradients of cAMP or PACAP (21). Modulation of spontaneous calcium transients in filopodia regulates axon turning (17). Temporal regulation o...
BackgroundcAMP is a ubiquitous second messenger involved in a wide spectrum of cellular processes including gene transcription, cell proliferation, and axonal pathfinding. Precise spatiotemporal manipulation and monitoring in live cells are crucial for investigation of cAMP-dependent pathways, but existing tools have several limitations.FindingsWe have improved the suitability of cAMP manipulating and monitoring tools for live cell imaging. We attached a red fluorescent tag to photoactivated adenylyl cyclase (PACα) that enables reliable visualization of this optogenetic tool for cAMP manipulation in target cells independently of its photoactivation. We show that replacement of CFP/YFP FRET pair with GFP/mCherry in the Epac2-camps FRET probe reduces photobleaching and stabilizes the noise level during imaging experiments.ConclusionsThe modifications of PACα and Epac2-camps enhance these tools for in vitro cAMP studies in cultured living cells and in vivo studies in live animals in a wide range of experiments, and particularly for long term time-lapse imaging.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.