Journal: bioRxiv
Article Title: Active transport of tRNAs facilitates distributed protein synthesis
doi: 10.64898/2026.01.26.698744
Figure Lengend Snippet: (A) Schematic of Cy5-tRNA labeling strategy. (B) Representative snapshot of Cy5-tRNA (white puncta with white circles) in C2C12 cells overlaid with tracks color coded by distance traveled (dark blue, <1 µm; dark red ≥ 9 µm), and zoom in (white boxes) and representative kymographs of tRNAs undergoing long range, short or static movements (purple, static; light blue, short; orange, long). (C) Frequency distributions depicting the maximum distance traveled, average tRNA puncta speed, and confinement ratio per punctum, where 0 is confined to an area, and 1 indicates linear forward movement ( n = 5 myotubes, with approximately 2000 puncta/video analyzed). Heatmaps below each respective graph denote the color coding of maximum and minimum values. (D) Representative video snapshots of C2C12 myotubes with ELVs labeled with Lyostracker (green) and transfected with Cy5-tRNAs (pink), with a white box indicating the track (yellow arrow) zoomed in for the kymograph. (E) Representative Cy5-tRNA and Lysotracker tracks (colored based on maximum distance traveled per punctum, dark blue, <1 µm; dark red ≥ 9 µm) and kymographs from zoomed in black box in C2C12s treated with DMSO (top panel) or treated with 500nM nocodazole for 1h (bottom panel). Pink kymograph indicates tRNA track, and green indicates an ELV track. (F) Representative images of endogenous LamTOR4-labeled ELVs (green), microtubules (grey), and pan tRNAs (pink) in ARVMS and quantification of the percentage of ELVs colocalized with tRNAs (Obs = observed, bright pink) compared to that expected from random chance using randomly distributed puncta of the same size and shape (grey, R.D.), as well as, quantification of tRNA puncta size and circularity plotted on the log10 scale (with each individual graphical dot indicating an individual puncta measurement and white dots indicating means for each individual cell). From non-transformed circularity measurements, frequency distribution of observed tRNA circularity when tRNA are off (light pink) or on (bright pink) ELVs, with 1 indicating a perfect circle and decreasing values from 1 indicating more elongated morphology. All experiments were done in biological triplicate ( N = 3), with 10 cells imaged per biological replicate ( n = 10), unless noted. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Two-sided Student’s t-test in f.
Article Snippet: Myoblast C2C12 cells (ATCC) were cultured in standard culture medium (20% fetal bovine serum, high-glucose DMEM, 1% Pen-Strep) and differentiated in differentiation medium with daily media changes to promote formation of myotubes at 70% confluence (1%horse serum (Gibco), DMEM high-glucose (Gibco), 1% Pen-Strep (Invitrogen), Insulin-Transferrin-Selenium Supplement (ITS, Gibco, 100x stock)).
Techniques: Labeling, Transfection, Transformation Assay