Dataset Information

Cue-induced modulation of eIF2α-eIF2Bε underlies axonal translation up-regulation in neural wiring

ABSTRACT: Local protein synthesis is rapidly regulated by extrinsic signals during neural wiring but the translational control mechanisms remain elusive. Here, we show that the guidance cue Sema3A, but not Slit1, elicits precise spatiotemporal control of the phosphorylation of the translation initiation factor, eIF2α, in axonal growth cones via the Unfolded Protein Response (UPR) kinase PERK. Strikingly, in contrast to the canonical UPR signaling, we find that the Sema3A-induced eIF2α phosphorylation bypasses the conventional global translational repression and underlies a burst in axonal protein synthesis, mediated by differential eIF2B activity. Ultrasensitive proteomics on somaless axons reveals a subset of 75 proteins translationally regulated via Sema3A-p-eIF2α, including several proteostasis- and actin cytoskeleton-related nascent proteins. Finally, in vivo experiments provide evidence that eIF2α phosphorylation drives the formation of the retinotectal axon projection. Thus, specific extracellular cues can trigger non-canonical PERK-eIF2α-eIF2B signaling leading to subcellular dynamic remodeling of the nascent proteome required for neural wiring.

INSTRUMENT(S): LTQ Orbitrap Velos

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

SUBMITTER: Christian Frese

LAB HEAD: Jeroen Krijgsveld

PROVIDER: PXD009250 | Pride | 2019-01-02

REPOSITORIES: Pride

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Publications

Noncanonical Modulation of the eIF2 Pathway Controls an Increase in Local Translation during Neural Wiring.

Cagnetta Roberta R Wong Hovy Ho-Wai HH Frese Christian K CK Mallucci Giovanna R GR Krijgsveld Jeroen J Holt Christine E CE

Molecular cell 20181227 3

Local translation is rapidly regulated by extrinsic signals during neural wiring, but its control mechanisms remain elusive. Here we show that the extracellular cue Sema3A induces an initial burst in local translation that precisely controls phosphorylation of the translation initiation factor eIF2α via the unfolded protein response (UPR) kinase PERK. Strikingly, in contrast to canonical UPR signaling, Sema3A-induced eIF2α phosphorylation bypasses global translational repression and underlies an ...[more]

PMID: 30595434

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Project description: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.

Project description:A data-entrained computational model for testing the regulatory logic of the vertebrate unfolded protein response This model is described in the article: A data-entrained computational model for testing the regulatory logic of the vertebrate unfolded protein response. Diedrichs DR, Gomez JA, Huang CS, Rutkowski DT, Curtu R. Mol. Biol. Cell 2018 Apr; : mbcE17090565 Abstract: The vertebrate unfolded protein response (UPR) is characterized by multiple interacting nodes among its three pathways, yet the logic underlying this regulatory complexity is unclear. To begin to address this issue, we created a computational model of the vertebrate UPR that was entrained upon and then validated against experimental data. As part of this validation, the model successfully predicted the phenotypes of cells with lesions in UPR signaling, including a surprising and previously unreported differential role for the eIF2? phosphatase GADD34 in exacerbating severe stress but ameliorating mild stress. We then used the model to test the functional importance of a feed-forward circuit within the PERK/CHOP axis, and of cross-regulatory control of BiP and CHOP expression. We found that the wiring structure of the UPR appears to balance the ability of the response to remain sensitive to ER stress yet also to be rapidly deactivated by improved protein folding conditions. This model should serve as a valuable resource for further exploring the regulatory logic of the UPR. This model is hosted on BioModels Database and identified by: MODEL1803300000. To cite BioModels Database, please use: Chelliah V et al. BioModels: ten-year anniversary. Nucl. Acids Res. 2015, 43(Database issue):D542-8. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

Project description:Disruptions of protein homeostasis in the endoplasmic reticulum (ER) elicit activation of the unfolded protein response (UPR), a translation- and transcription-coupled proteostatic stress response pathway. The primary translational control arm of the UPR is initiated by the PERK-dependent phosphorylation of eIF2Î±, leading to a large-scale reprogramming of translation and subsequent activation of an ATF4-mediated transcriptional program. It has remained challenging, however, to accurately evaluate the contribution of each component of the eIF2Î±/ATF4 pathway to the remodelling of transcription and translation. Here, we have used mouse embryonic fibroblasts containing either a knock-in of the non-phosphorylatable eIF2Î± S51A mutant or knock-out for ATF4 by ribosome profiling and mRNA-seq to define the specific contributions of eIF2Î± phosphoryation and ATF4 in controlling the translational and transcriptional components of the UPR. These studies show that the transcriptional and translational targets of each P-eIF2Î±, ATF4, and the other UPR gene expression programs overlapped extensively, leading to a core set of UPR genes whose robust expression in response to ER stress is driven by multiple mechanisms. The identification of other, more factor-specific targets illustrated the potential for functional specialization of the UPR. As the UPR progressed temporally, cells employed distinct combinations of transcriptional and translational mechanisms, initiated by different factors, to adapt to ongoing stress. These effects were accompanied by a buffering effect where changes in mRNA levels were coupled to opposing changes in ribosome loading, a property which makes cooperation between transcription and translation essential to confer robust protein expression. Translational analysis by ribosome profiling and mRNA-seq of PERK pathways mutants during the UPR. Mouse embryonic fibroblasts (MEFs) lacking components of the PERK pathway (eIF2a phosphorylation and ATF4) were subjected to ER stress and analyzed by ribosome profiling.

Dataset Information

Cue-induced modulation of eIF2α-eIF2Bε underlies axonal translation up-regulation in neural wiring

Publications

Noncanonical Modulation of the eIF2 Pathway Controls an Increase in Local Translation during Neural Wiring.

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