Rapid Cue-Specific Remodeling of the Nascent Axonal Proteome
Roberta Cagnetta, Christian K. Frese, Toshiaki Shigeoka, Jeroen Krijgsveld, Christine E. Holt
Neuron 99, 29–46. July 11, 2018 a 2018 The Author(s). Published by Elsevier Inc. https://doi.org/10.1016/j.neuron.2018.06.004.
- pSILAC-SP3 reveals the newly synthesized axonal proteome within minutes
- Extrinsic cues rapidly up- and down-regulate large subsets of nascent proteins
- Different repulsive cues generate distinct proteomic signatures
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Axonal protein synthesis and degradation are rapidly regulated by extrinsic signals during neural wiring, but the full landscape of proteomic changes remains unknown due to limitations in axon sampling and sensitivity. By combining pulsed stable isotope label- ing of amino acids in cell culture with single-pot solid- phase-enhanced sample preparation, we character- ized the nascent proteome of isolated retinal axons on an unparalleled rapid timescale (5 min). Our anal- ysis detects 350 basally translated axonal proteins on average, including several linked to neurological disease. Axons stimulated by different cues (Netrin-1, BDNF, Sema3A) show distinct signatures with more than 100 different nascent protein species up- or downregulated within the first 5 min followed by further dynamic remodeling. Switching repulsion to attraction triggers opposite regulation of a subset of common nascent proteins. Our findings thus reveal the rapid remodeling of the axonal proteomic land- scape by extrinsic cues and uncover a logic underly- ing attraction versus repulsion.
Figure 1. pSILAC-SP3 Detects the Axonal Newly Synthesized Proteome within 5 min
(A) Intact whole-eye primordia were cultured in compartmentalized Boyden chambers. Only the axons of RGCs exit the eye—via optic nerve head (ONH)—and extend through the 1 mm pores to grow on the laminin-coated underside of the transfilter. Cell bodies and dendrites remain in the eye on the upper surface and are removed immediately prior the experiment, leaving pure somaless axons.
(B) RT-PCR confirms the purity of the axonal compartment. Actb (positive control; Leung et al., 2006), but not Actg or Brn3 (negative controls; Willis and Twiss, 2011; Yoon et al., 2012), mRNAs were detected.
(C) Schematic representation of the pSILAC-SP3 methodology applied to somaless retinal axons. Axons are exposed to vehicle or cue in pSILAC medium containing either ‘‘medium’’ or ‘‘heavy’’ isotope-coded Arg and Lys.
(D) Proteins identified in axons derived from 100 eye explants and NSPs identified in axons after 5 min of pSILAC. Error bars, SEM.
Figure 3. Validation of the pSILAC-SP3 Approach in Axonal Growth Cones
(A) Images of puro-PLA for negative controls (Figure 1B).
(B and C) Puro-PLA quantification (B) and representative images (C) to validate NSPs across different functional categories, conditions, and time points (Table S1; Mann-Whitney test and one-way ANOVA with Bonferroni’s multiple comparisons test).
(D and E) IF representative images (D) and quantification (E) to validate NSPs across different functional categories, conditions, and time points (Table S1; Mann-Whitney test and one-way ANOVA with Bonferroni’s multiple comparisons test).
(F) Direct comparison of pSILAC and IF-derived detection of NSPs reveals excellent correlation with r = 0.81 (rpuro-PLA = 0.87; rqIF = 0.41).
Error bars, SEM. Scale bars, 5 mm. See also Figure S3.
Figure 4. Nascent Axonal Proteome Changes Dynamically over the Duration of Cue Stimulation
(A) Overlap of the NSP changes among different times of stimulation in response to each cue. Both common (i.e., the same NSP undergoes similar directional change with log2(H/M) ratio > j0.3j) and different (i.e., a new NSP exhibits change with log2(H/M) ratio > j0.3j or the same NSP undergoes opposite directional change) NSP changes were detected among different cue stimulation periods. Rectangles with solid lines outline KEGG pathway analysis for the NSP changes unique for each time point, and rectangles with dashed lines outline KEGG pathway analysis for the NSP changes constant among different time points (cutoff R 3 proteins per pathway). Red indicates upregulated pathways, and blue indicates downregulated pathways.
(B) Enriched GO terms in the biological process, molecular function, and cellular composition categories of selected categories (category count > 15; for complete table, see Figure S4). Rectangles indicate significantly enriched GO terms (p < 0.05).
See also Figure S4.
Adapted with permission from Cell Press on behalf of Neuron: Cagnetta et al. (2018). Rapid Cue-Specific Remodeling of the Nascent Axonal Proteome. Neuron 99, 29–46. July 11, 2018 a 2018 The Author(s). Published by Elsevier Inc. https://doi.org/10.1016/j.neuron.2018.06.004. Copyright (2018).
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