Article Title: Axonal ER tubules regulate local translation via P180/RRBP1-mediated ribosome interactions
Figure Lengend Snippet: (A, D, G) Representative STED (A), TREx (D) and dual-color SMLM (G) images of the ER and ribosomes in axons from neurons expressing GFP-Sec61β and stained for RpS12. (B, E, H) Magnifications and intensity profile lines from merged images for each microscopy method. (C, F, I) Quantification of RpS12 intensity in ER mask, enlarged ER mask, and one-color flipped images, for each microscopy method. (J) Representative STED images and intensity profile line for an axon segment of a neuron transfected as in (A) and co-labeled for puromycilated peptides. (K) Quantification of RpS12 intensity in ER mask and enlarged ER mask with or without high puromycin treatment using dual-color SMLM. (L-N) Schematic representation of split APEX system used to detect ER-ribosome contacts. When the ER protein Sec61β fused to AP module and the ribosomal protein RpL10A fused to EX module interact with each other, APEX is reconstituted and contact sites can be visualized as a biotinylation radius around the interactions (L). Representative images of split APEX assay in distal axons from neurons expressing RpL10A-3xHA-EX and V5-AP-Sec61β (left), or V5-AP-RTN4A as a negative control (right). Expression of constructs are visualized with V5 and HA antibodies, and biotinylation is detected with conjugated Strep-555 (M). Quantification of Strep signal in distal axons from neurons as in (M), and without H2O2 as a negative control for the biotinylation reaction (N). (O) Quantification of axonal ER-bound ribosomes using split APEX assay with RpL10A-3xHA-EX and V5-AP-Sec61β, in neurons stimulated for 30-minutes with BSA (control), BDNF, NT-3 or NGF. Individual data points each represent a neuron in (C, F, I, K, N and O). Boxplots show 25/75-percentiles, the median, and whiskers represent min to max in (C, F, I and K). Data are presented as mean values ± SEM in (N, O). ns = not significant, *p < 0.05, **p < 0.01, ***p<0.001 comparing conditions to control using unpaired t-tests or ordinary one-way ANOVA tests. Scale bars represent 1μm (A, D, G, J) and 5μm (M).
Article Snippet: Following vectors were used: pSuper , pGW1-mCherry and pGW1-BFP , RTN4A-GFP (a gift from Dr. Gia Voeltz, Addgene plasmid #61807), pEGFP(A206K)-N1 and pEGFP(A206K)-C1 (a gift from Dr. Jennifer Lippincott-Schwartz), GFP-Sec61β (a gift from Dr. Tom Rapoport, Addgene # 15108), RpL10A-tagRFP (a gift from dr. Robert Singer, Addgene #74172) and TOM20-V5-FKBP-AP (a gift from Alice Ting, Addgene#120914).
Techniques: Expressing, Staining, Microscopy, Transfection, Labeling, Negative Control, Construct