A plasma membrane microdomain compartmentalizes ephrin-generated cAMP signals to prune developing retinal axon arbors

Abstract

The development of neuronal circuits is controlled by guidance molecules that are hypothesized to interact with the cholesterol-enriched domains of the plasma membrane termed lipid rafts. Whether such domains enable local intracellular signalling at the submicrometre scale in developing neurons and are required for shaping the nervous system connectivity in vivo remains controversial. Here, we report a role for lipid rafts in generating domains of local cAMP signalling in axonal growth cones downstream of ephrin-A repulsive guidance cues. Ephrin-A-dependent retraction of retinal ganglion cell axons involves cAMP signalling restricted to the vicinity of lipid rafts and is independent of cAMP modulation outside of this microdomain. cAMP modulation near lipid rafts controls the pruning of ectopic axonal branches of retinal ganglion cells in vivo, a process requiring intact ephrin-A signalling. Together, our findings indicate that lipid rafts structure the subcellular organization of intracellular cAMP signalling shaping axonal arbors during the nervous system development.

Primary cilium-dependent cAMP/PKA signaling at the centrosome regulates neuronal migration

“…This "coldspot" was dynamic, disappeared during mitosis and was important for cell cycle progression (Terrin et al, 2012). cAMP microdomains were also described in diverse neuronal subcompartments such as growth cones, axons and dendrites (Averaimo et al, 2016;Calebiro and Maiellaro, 2014;Castro et al, 2014). However, to our knowledge, we are the first to perform this type of analysis in migrating neurons.…”

“…In addition, the use of genetically-encoded cAMP specific FRET (Förster Resonance Energy Transfer)-based biosensors in living cells revealed that cAMP itself can be highly compartmentalized at the subcellular level (Calebiro and Maiellaro, 2014;Castro et al, 2014;Lefkimmiatis and Zaccolo, 2014). In neurons, it was described in microdomains of axons, dendrites or growth cones (Averaimo et al, 2016;Calebiro and Maiellaro, 2014;Castro et al, 2010;Maiellaro et al, 2016). In addition, the use of ciliary-targeted FRET-biosensor in fibroblasts and kidney epithelial cells showed that the PC is a cAMP rich microdomain (Moore et al, 2016;Sherpa et al, 2019).…”

Lipids at membrane contact sites: cell signaling and ion transport

“…This confined localization of cAMP signaling components generates spatially restricted and segregated cAMP signals. Interestingly, the cAMP generated in the raft resides in a region that is depleted for PI(4,5)P 2 [105]. In addition, depletion of ER Ca 2+ that clustered STIM1 at the ER/PM junctions recruited AC3 to the puncta and increased cAMP [106] and STIM1 was shown to activate AC5 [107].…”

Interrogating cyclic AMP signaling using optical approaches

“…Averaimo and colleagues used a combination of targeted cAMP sponge and light-activated adenylyl cyclases to control cAMP levels in the vicinity of lipid rafts in retinal ganglion cells. Their work demonstrated the critical importance of this cAMP signaling microdomain for executing ephrin-dependent retraction of axons and the pruning of ectopic axonal branches during nervous system development [37]. Recently, the Tomes lab reported on refinements to the original cAMP sponge by generating a membrane-permeant version employing a TAT sequence from HIV.…”

“…In addition, spatial differences exist in the localization of the ten distinct isoforms of adenylyl cyclase (the enzymes that generate cAMP) and the 20-plus phosphodiesterases (the enzymes that degrade cAMP). These disparities create local sources and sinks for cAMP that translate into differential signaling efficiencies in discrete microdomains, for example in lipid rafts [37]. It’s also worth noting that cellular structures with a high surface-to-volume ratio, such as neuronal projections will be expected to have more cAMP-generating machinery in the plasma membrane for a given volume of cytosol, thus accounting for the appearance of higher cAMP production in those locales [5, 46].…”