PSILAC-SP3 of RGC axonal growth cones

ABSTRACT: To form operative neuronal networks neurons connect to their synaptic partners by axonal outgrowth guided by the movement of the axon tip, a specialized structure known as the growth cone (GC), which travels along stereotypical paths by sensing extracellular guidance cues. Interestingly, GCs contain complex transcriptomes and different guidance cues can rapidly regulate the translation of specific mRNAs which help to remodel the GC cytoskeleton thus steering axon navigation. However, the full landscape of proteomic changes elicited by different cues is not known and the precise protein synthesis (PS)-dependent molecular mechanisms underlying GC repulsive and attractive turning remain largely elusive. Here we have investigated the cue-induced changes in the nascent proteome by pulse Stable Isotope Labeling by Amino acids in Cell culture (pSILAC) in somaless developing retinal axons derived from Xenopus laevis primary cultures. The experimental approach presented major challenges due to the limited quantity of axonal material (~2 μg per condition) and to the desired rapid (5-15 min) time-scale (no previous work has accomplished SILAC with a pulse less than 20 min). To overcome these difficulties we used a superior protocol, termed Single-Pot Solid- Phase-enhanced Sample Preparation, based on ultrasensitive paramagnetic bead technology which allows high proteomic sample recovery from sub-microgram amounts of material. By employing this method we were able to identify up to 500 newly synthesized proteins after only 5 min of pSILAC. Subsequently, we successfully validated the purity and viability of the axonal samples and the reliability of the method. Finally, to characterize and quantify the global patterns of the axonal translational changes in response to specific guidance cues and to unravel the switch between repulsive and attractive responses, we carried out pSILAC in the presence of different repulsive cues and pharmacological reagents that convert repulsion into attraction. Our results uncovered a surprisingly high level of translational activity within the axonal compartment, even in basal conditions. Remarkably, comparative analysis revealed many changes in the nascent proteome in response to different cues, some distinct and some common. Interestingly, several proteins showed cue-specific downregulation. Taken together, our findings suggest a comprehensive GC PS-dependent chemotropic model and novel cue-specific molecular mechanisms possibly underlying the integration of chemotropic responses.

INSTRUMENT(S): LTQ Orbitrap Velos, Orbitrap Fusion, Q Exactive

ORGANISM(S): Xenopus Laevis (african Clawed Frog)

SUBMITTER: Christian Frese  

LAB HEAD: Jeroen Krijgsveld

PROVIDER: PXD005469 | Pride | 2018-08-10

REPOSITORIES: Pride