Figure 4. The shaft of the growing axon is scarcely populated by ribosomes.

(A) Examples of ribosomes observed in cryo-tomograms of axon shafts from ALI-COs, of other cellular processes from ALI-COs, and of HeLa cells. The bottom panel shows 0.05 µm³ cryo-ET volumes, corresponding to the area shown in the upper panel. Positions of all ribosome-like particles observed in that volume are shown as orange spheres. (B) Comparison of the numbers of ribosome-like particles, normalized to the tomographic volume, observed in axon shafts, other processes and HeLa cells. Individual data points represent individual cryo-tomograms (30, 4, and 5 tomograms, respectively). Mann-Whitney tests were employed for statistical analysis: p < 0.0001 (****); p < 0.05(*). (C) Immunofluorescence images of dissociated neurons from organoids reveal low signal for the ribosomal 60 S component RPL8 in axon processes (identified by SMI312⁺/MAP2^- labeling) in comparison to dendrites (identified by SMI312^-/MAP2⁺ labeling). The yellow box outlines the area magnified in the right panel. The top image of the right panel shows the immunofluorescence signal for the ribosomal subunit RPL8. The bottom image shows the SMI312/MAP2/RPL8 composite. The white dashed line depicts the outline of axons and dendrites and was traced based on the MAP2 and SMI312 signal. The image shown is representative of the data used for quantifications shown in D. (D) Quantification of immunofluorescence images of ALI-CO derived dissociated neurons labeled for five distinct ribosomal proteins. The bars report the mean pixel grey value along axons and dendrites (mean ± SD). Each data point represents a different axon or dendrite. With the exception of quantifications done on the ribosomal protein S6, axons were identified as SMI312⁺/MAP2^- neuronal processes, while dendrites were identified as SMI312^-/MAP2⁺ neuronal processes. Due to antibody incompatibility, in the case of S6, dendrites were identified as MAP2⁺ processes while axons were identified as GFP⁺/MAP2^- processes. The data pertains to one biological replicate. Mann-Whitney tests were employed for statistical analysis: RPL8, N = 10, p < 0.0001 (****); RPS10, N = 12, p < 0.0001 (****); RPS16, N = 12, p < 0.0001 (****); RPS26, N = 12, p < 0.0001 (****); S6, N = 12, p = 0.0001 (***). Scale bars: 50 nm in A., 20 µm and 2 µm in C.

Figure 4—figure supplement 1. Ribosome-like particles in developing human axon shafts.

(A) Gallery of ribosome-like particles seen in tomogram slices of developing human axons. Yellow arrows highlight ribosome-like densities. (B) Diameter of ribosome-like particles measured from tomographic slices seen in A. (mean = 26.8 nm, SD = 3.7, N = 27). (C) Quantification of the association of ribosome-like particles with the ER. Scale bars: 25 nm in A.

Figure 4—figure supplement 2. Immunofluorescence analysis of axons and dendrites supports ribosomal depletion observed by cryo-ET in the axon shaft.

(A) Representative immunofluorescence images of dissociated organoid-derived neurons stained for the dendritic marker MAP2, the pan-axonal marker SMI312 and the ribosomal components RPS10, RPS16, and RPS26 used for the quantifications reported in Figure 4D. Boxed in yellow is the region shown at higher magnification in the right panel as the individual ribosomal stain and the composite image. Dashed yellow is the outline of the processes reported on the single channel image of the ribosomal stain. The data highlights how dendrites (MAP2⁺/SMI312^-) are enriched in ribosomal components compared to axons (MAP2^-/SMI312⁺) of comparable thickness. (B) Representative immunofluorescence images of dissociated organoid-derived neurons transduced with Sendai EmGFP and stained for the dendritic marker MAP2 and for the ribosomal components S6. Due to antibody incompatibility, in this case dendrites were identified as MAP2⁺ processes while axons were identified as GFP⁺/MAP2^- processes. These images were used for the quantifications reported in Figure 4D. Boxed in yellow is the region shown at higher magnification in the bottom panel as the S6 stain (right) and the MAP2-GFP composite stain (left). Dashed in yellow is the outline of the processes reported on the single channel image of the S6 stain. The data highlights how dendrites (MAP2⁺) are enriched in ribosomal components compared to axons (GFP⁺/MAP2^-) of similar thickness. The data shown in A and B pertains to one biological replicate. Scale bars: 20 µm in the overview images in A. and B., 2 µm in the high-magnification inserts.