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. 2022 Nov 2;13:6558. doi: 10.1038/s41467-022-34052-8

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a NIH3T3 cells that express vector control or Halo-eIF4E and SNAP_f-eIF4G were treated with DMSO (vehicle) or 250 nM torin-1 for 2 h. Total cell lysates (input) were subjected to cap-pull-down assay (m⁷GTP-pull-down) and analyzed by western blotting. eIF4G and eIF4E antibodies detected both endogenous (endog.) and exogenous (exog.) proteins as indicated by the arrows. b Simultaneous diffusion of _JF585Halo-eIF4E and _JF646SNAP_f-eIF4G analyzed by dual-color fluorescent cross-correlation spectroscopy (FCCS) in cell treated with vehicle (left panel) or 250 nM torin-1 for 2 h (torin-1, right panel) in the cytoplasm (gray) and in the nucleus (red) (N = 10 ± SEM). Cross-correlation was detected in the cytoplasm of translating cells, but not in the nucleus, and abolished 2 h upon mTOR inhibition. c, d Individual _JF585Halo-eIF4E (c) and _JF646SNAP_f-eIF4G (d) averaged autocorrelation curves from (b). e Simultaneous single-particle tracking of _JF549Halo-eIF4E and _JF646SNAP_f-eIF4G was simultaneously recorded in the cytoplasm of an NIH3T3 (dotted line outlined the nucleus). Left: Diffusion properties of 4319 trajectories of _JF549Halo-eIF4E and 4001 trajectories of _JF646SNAP_f-eIF4G are displayed via heat maps. Each point is false‐colored according to the mean square displacement calculated over all displacements originating in a circle (r = 80 nm). f Co-movement analysis of _JF549Halo-eIF4E and _JF646SNAP_f-eIF4G. Maximum intensity projections of 5000 frames of _JF549Halo-eIF4E (in magenta) and 5000 frames of _JF646SNAP_f-eIF4G (in green) were simultaneously acquired at 100 Hz. The co-moving _JF549Halo-eIF4E (bold, magenta) and _JF646SNAP_f-eIF4G (bold, green) trajectories are displayed on top. Scale bar: 10 μm. Inset top: co-moving _JF549Halo-eIF4E/_JF646SNAP_f-eIF4G trajectories with their associated diffusion heat maps displayed at higher magnification (scale bar: 1 μm). g (left) The distribution of apparent diffusion coefficients shifts to a slower population when _JF549Halo-eIF4E is comoving with _JF646SNAP_f-eIF4G (in gray). (right) Violin plots of one-step mean square displacements for eIF4E (355,730, in magenta), for eIF4G (462,072, in green), and for co-moving eIF4E/eIF4G pairs (7947, in gray). Box quartile method: Tukey. The median line is shown, whisker method: min and max data. Two-sided t-test with two mean values of two distributions (*** means p < 0.001, ** means p < 0.01).