Fig. 5: ER – lysosome contacts are enriched in a somatic pre-axonal region and they require ER tubule formation.

a Schematic representation of Split-APEX system used to visualize ER–lysosome contacts. An ER tubule-contact marker (protrudin) is fused to an AP module and the lysosome adaptor Rab7 is fused to an EX module. Only proximity of the two proteins allows reconstitution of full APEX2. After addition of biotin-phenol (BP) and H2O2, APEX2 biotinylates proteins in close proximity and this can be detected by fluorescently conjugated-streptavidin (Strep; red radius). b Representative images of a magnified region from the soma of a neuron transfected with AP-V5-protrudin (green) and EX-HA-Rab7, treated with biotin-phenol (BP) and H₂O₂, and labeled with an antibody against LAMTOR4 (blue) and Alexa568-conjugated Strep (red). Intensity profile line, bottom. c Average Strep intensity in soma of neurons transfected as in (b) plus control pSuper or shRNAs targeting RTN4 plus DP1. Control neurons were treated with BP in the absence (n = 13) or presence (n = 20) of H₂O₂ and knockdown neurons treated with BP plus H₂O₂ (n = 14) prior labeling with Alexa568-conjugated Strep. d Representative images of a magnified region from the soma of a neuron transfected with AP-V5-protrudin (green) and EX-HA-Rab7 (blue) (left panels) or with EX-HA-Rab7T22N (blue) (right panels), treated with BP and H₂O₂, and labeled with Alexa555-conjugated Strep (red). Intensity profile line to the right of merged images. e–h Representative images of neurons transfected, treated, and labeled as in (b), plus control pSuper plasmid (e) and (f) or shRNAs targeting RTN4 plus DP1 (g) and (h). Higher magnification of dashed orange boxes in (e) and (g) are shown in (f) and (h). i–k Representative image of a neuron transfected and treated as in (e) and labeled for Alexa555-conjugated Strep (red) plus an antibody against TRIM46 (blue). Light blue dashed lines represent the non-pre-axonal region, light green dashed lines represent the pre-axonal region. Average Strep intensity in non-pre-axonal and pre-axonal regions of neurons transfected as in (e) and (g); (j; n = 23) and (k; n = 19); respectively. Scale bars represent 1 µm (b) and (d); 2 µm in (f) and (h); 5 µm in (e), (g), and (i). Data are presented as mean values ± SD in (c) and individual datapoints each represent a neuron in (c); ***p < 0.001 comparing conditions to control (one-way ANOVA test followed by a Sidak’s multiple comparisons test) in (c). Line graphs shows individual datapoints each represent a neuron (depicted with different colors) in (j) and (k); ***p < 0.001 comparing non-pre-axonal region versus pre-axonal region (two-sided Paired t test in (j) and two-sided Wilcoxon test in (k). Source data and exact p values are provided as a Source Data file.