Fig 1: DCC and Nrp1 are in close proximity to ribosomes in axonal growth cones in a cue-dependent manner.(A) Expansion imaging shows partial co-localization of DCC and (B) Nrp1 with ribosomal proteins (Scale bars, 5 µm). (C) Representative proximity ligation assay signal in axonal growth cones between DCC and RPL5/uL18, RPS4X/eS4 or IgG control (Scale bars, 5 µm). (D) Representative proximity ligation assay signal in axonal growth cones between Nrp1 and RPS3A/eS1, RPS23/uS12 or IgG control (Scale bars, 5 µm). (E) Representative PLA signal in axonal growth cones between EphB2 and RPL5/uL18 (left) and quantification of PLA signal in axonal growth cones compared to DCC-RPL5/uL18 or Nrp1-RPS23/uS12 (right) (Mann-Whitney test; three biological replicates; bars indicate mean, error bars indicate SEM, ***p<0.0001; Scale bars, 5 µm). (F) EM image of an unstimulated axonal growth cone showing ribosomes aligned in a row (red arrows) under plasma membrane (PM). Inset shows the growth cone at lower magnification; the red box indicates the area shown in higher magnification. The section glances through the extreme surface of growth cone, where it attaches to the culture dish, giving rise to areas that lack subcellular structure. (G) Distribution frequency of the inter-ribosome distance in nm of ribosomes in axonal growth cones (n = 20) or in RGC soma (n = 5). All distances larger than 100 nm were pooled together. (H, I, J, K) Quantification of PLA signal in cue-stimulated axonal growth cones relative to control (unpaired two-tailed t-test; bars indicate mean, error bars indicate SEM; ***p<0.0001; *p=0.0423; for n.s. in J p=0.3522; for n.s. in K, p=0.885). (L) Relative PLA quantification of DCC and RPL5/uL18 compared to control after Dynasore pre-treatment (50 µM for 20 min), Netrin-1, or Netrin-1 + Dynasore (one-way ANOVA with Bonferroni’s multiple comparisons test; bars indicate mean, error bars indicate SEM; p=0.001027 for Control vs. Netrin-1, p=0.000402 for Netrin-1 vs Netrin-1 + Dynasore, p=0.590377 for Control vs. Dynasore, p=0.384848 for Control vs Netrin + Dynasore). For all PLA experiments, numbers in bars indicate total number of growth cones quantified from at least three independent experiments.Figure 3—source data 1.Spreadsheet containing PLA counts and relative comparisons from each axonal growth cone in Figure 3E, all inter-ribosome distances and distribution shown in Figure 3G, and all normalized PLA count values for each axonal growth cone in Figure 3H–L.
Fig 2: Ribosomes, RNA-Binding Proteins, and mRNAs Localize to Endosomes in Axons(A) Colocalization between endosomal markers and RNA-binding proteins (RBPs) or ribosomal proteins.(B) Pearson’s coefficient between endosomal markers and RBPs or ribosomal proteins. n, number of axon segments.(C) Time-lapse images and kymographs (1 min) illustrating RPS3A-GFP association with RFP-Rab5a or Rab7a endosomes (yellow arrowheads) in axons.(D) Motion types of RFP-tagged Rab5a (n = 27 axons), Rab7a (n = 29 axons), or LAMP-1 (n = 22 axons) endosomes associated with the RPS3A-GFP signal.(E) Time-lapse images and kymographs illustrating GFP-Vg1RBP association with RFP-Rab5a or Rab7a endosomes (yellow arrowheads) in axons.(F and G) Frequency (F) and motion types (G) of GFP-Vg1RBP associated with RFP-tagged Rab5a (n = 15 axons), Rab7a (n = 13 axons), or LAMP-1 (n = 12 axons) endosomes.(H and I) Frequency (H) and motion types (I) of ß-actin molecular beacon (MB) signals associated with GFP-Rab5a (n = 22 axons) or GFP-Rab7a (n = 20 axons) endosomes.(J) Representative images showing ß-actin MBs associated with GFP-Rab5a or GFP-Rab7a endosomes (white arrowheads) in axons.Mean ± SEM. Scale bars: 2.5 µm in (A), 2 µm in (C) and (E), and 5 µm in (J). Time stamps are in the format of seconds:milliseconds. See also Figure S3 and Videos S2 and S3.
Fig 3: Ribosomes Associate with GFP-Rab5a and GFP-Rab7a Endosomes in Axons, Related to Figure 2(A) Representative Western Blot of Vg1RBP and ribosomal proteins RPL10A and RPS3A co-immunoprecipitated with GFP-Rab5a and GFP-Rab7a in brain lysates.(B) qRT-PCR of cDNA synthesized from RNAs co-precipitated with GFP, GFP-Rab5a and GFP-Rab7a in brain lysates revealing a significant enrichment of ß-actin mRNA (n=3 biological replicates).(C) OMX Super-resolution microscopy revealing the presence of RPL10A and RPS3A puncta on GFP-Rab5a or GFP-Rab7a endosomes in axons (left panels, white arrows). Orthogonal views of the indicated areas confirming the contact (yellow arrows) between the ribosomal proteins and endosomes (right panels).(D) Time-lapse images and kymographs illustrating RPS4X-GFP association with RFP-Rab5a or Rab7a endosomes (white arrowheads) in axons. Mean±s.e.m.; **P<0.01, Mann-Whitney test. Scale bars: 1µm in left panels in C and D; 200nm in right panels in C.
Fig 4: mRNAs Essential for Mitochondrial Integrity Are Translated on Rab7a Endosomes in Axons(A) Co-immunoprecipitation (coIP) between Rab7 and Vg1RBP, FXR, SFPQ, RPS3A, and RPL10A in brain lysates.(B) CoIP between GFP-Rab7a and SFPQ in brain lysates.(C) Colocalization of GFP-Rab7a with SFPQ in axons (white arrowheads).(D) RGC axon segment analyzed by live imaging and its corresponding kymograph (1 min), showing the association between Cy5-labeled laminb2 mRNA (lmnb2) and GFP-Rab7a endosomes.(E) Schematic of SunTag system-based nascent polypeptide labeling to visualize Lamin B2 (LB2) synthesis.(F) Representative images showing the nascent SunTag-LB2 protein (green), the SunTag-lmnb2 mRNA (blue), and the RFP-Rab7a endosome (red) in live axons (indicated by white arrows).(G) Percentage of SunTag-lmnb2 mRNA associated with nascent SunTag-LB2 protein (translating) per 50-µm axon segment.(H) Percentage of SunTag-lmnb2 mRNA associated with RFP-Rab7a endosomes colocalized with nascent SunTag-LB2 protein (translating).(I) Percentage of RFP-Rab7a endosomes associated with SunTag-lmnb2 mRNA.(J) Quantitative analysis of lmnb2 mRNA by qRT-PCR of cDNA synthesized from RNAs co-precipitated with GFP and GFP-Rab7a from brain extracts (n = 3 biological replicates).(K) Example of LB2 Puro-PLA signals in proximity to GFP-Rab7a endosomes (white arrowheads) in axons.(L) Super-resolution microscopy analysis of LB2 Puro-PLA signals next to a GFP-Rab7a endosome.(M) Quantification of LB2 Puro-PLA signals in treated RGC axons.(N) Quantitative analysis of vdac2 mRNA by qRT-PCR of cDNA synthesized from RNAs co-precipitated with GFP and GFP-Rab7a from brain extracts (n = 3 biological replicates).(O) Quantification of VDAC2 Puro-PLA signals in treated RGC axons.N, number of axon segments in (G)–(I), (M), and (O). Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, Mann-Whitney test. Scale bars: 5 µm in (C), (D), and (K); 1 µm in (F); and 500 nm in (L). Time stamps are in the format of seconds:milliseconds. See also Video S4.
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