Cap-dependent translation initiation monitored in living cells

Figures

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  Fig. 1. Binding of exogenous Halo-eIF4E to the 5’cap is detected by FCS. a NIH3T3 cells that express Halo-eIF4E, in which the endogenous counterpart was silenced by shRNA, were treated for 2 h with vehicle (DMSO) or 250 nM torin-1. Averaged autocorrelation curves show temporal diffusion of Halo-eIF4E molecules (ms = milliseconds) in the cytoplasm (black) and in the nucleus (red) in the indicated conditions (N = 10 ± SEM). Halo-eIF4E diffusion is slower in translating cells as compared to cells treated with torin-1. Cytoplasmic autocorrelation best fit with two components (fast – dark yellow dotted curve: τfast = 1.14 ± 0.03 ms, and slow – dash-dotted curve: τslow = 372.69 ± 10.8 ms). Percentages of slow (Dslow = 0.05 μm2/s) and fast (Dfast = 14.78 μm2/s) moving molecules are indicated. While one-component fits nuclear Halo-eIF4E well (see red curves), a one-component fit (see the blue dashed curve, with dotted fit residual) cannot adequately describe the cytoplasmic Halo-eIF4E autocorrelation curve. b Total cell lysates from the cells described in (a) were analyzed by western blotting with the indicated antibodies. 4E-BPs phosphorylation and CyclinD1 expression were significantly reduced…

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  Fig. 2. eIF4E is released from the 5’cap upon binding to 4E-BP1. a Differentiated parental and mESC in which Halo and SNAPf tags were inserted into the EIF4E and 4EBP1 locus, respectively (Halo-4E+/+/SNAP-BP1+/+), were treated with vehicle (DMSO) or 250 nM torin-1 for 1 h and 30 min. Total cell lysates (Input) were subjected to cap-pull-down assay and analyzed by western blotting using the indicated antibodies. The Halo and SNAPf tags do not affect eIF4E:4E-BP1 binding upon mTOR inhibition. b mESC double knock-in described in (a) were treated with vehicle (DMSO) or 250 nM torin-1. Simultaneous diffusion of JF585Halo-eIF4E and JF646SNAPf-4E-BP1 was analyzed by dual color cross-correlation spectroscopy in the indicated conditions. Cross-correlation was detected, in the cytoplasm, 30 to 1 h 20 min upon mTOR inhibition and with differential diffusion speed. The vehicle showed no correlation over time (gray) (N = 10 ± SEM). c mESC double knock-in described in (a) was treated with vehicle (DMSO) or 250 nM torin-1 for 30 or 90 min. Total cell lysates were analyzed by western blotting using the indicated antibodies. 4E-BP1 and rpS6 were used as loading controls. d–f Individual diffusion of…

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  Fig. 3. eIF4E and eIF4G binding to the mRNA is detected by FCS upon mTOR inhibition. a Parental mESC and double knock-in Halo-eIF4E+/+/SNAP-eIF4G+/+ total cell lysates (input) were subjected to cap-pull-down assay (m7GTP-pull-down) and analyzed by western blotting. eIF4G and eIF4E antibodies showed binding of wild-type and tagged proteins to the 5’cap. Tagging of endogenous eIF4E and eIF4G did not affect cap-binding as compared to the parental counterpart. b–e Halo-eF4E+/+/SNAP-eIF4G+/+ cells were treated with vehicle (DMSO) or 250 nM torin-1 for 2 h and 5 h respectively. b Simultaneous diffusion of JF585Halo-eIF4E and JF646SNAPf-eIF4G was analyzed by dual color cross-correlation spectroscopy (FCCS) in the indicated conditions. Cross-correlation was detected in the cytoplasm of control cells (left panel), but not in the nucleus (right panel). Minor residual eIF4E:eIF4G was detected in the cytoplasm upon 2 h mTOR inhibition (left panel). c, d Averaged autocorrelation curves representing individual diffusion of JF585Halo-eIF4E (c) and JF646SNAPf-eIF4G (d) in the indicated conditions (N = 10 ± SEM). In control cells, both JF585Halo-eIF4E (c) and JF646SNAPf-eIF4G (d) autocorrelations showed…

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  Fig. 4. eIF4E and eIF4G binding dynamics detected by FCS and SPT. a NIH3T3 cells that express vector control or Halo-eIF4E and SNAPf-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 JF646SNAPf-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 JF646SNAPf-eIF4G (d) averaged autocorrelation curves from (b). e Simultaneous single-particle tracking of JF549Halo-eIF4E and JF646SNAPf-eIF4G was simultaneously recorded in the cytoplasm of an NIH3T3 (dotted line outlined the nucleus). Left: Diffusion properties of 4319 trajectories of…

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  Fig. 5. Single-particle tracking of Halo-eIF4E in primary hippocampal neurons. Rat hippocampal neurons were activated with TTX withdrawal with or without 250 nM torin-1. a Total cell lysates were analyzed by western blotting with the antibodies indicated next to the corresponding blot (n = 2). b Diffusion heat map of Halo-eIF4E trajectories obtained in inactivated neurons (16 h TTX) at a frame rate of 100 Hz (with a dotted neuronal outline, scale bar: 5 μm. Each point in the image is false‐colored according to the mean square displacement calculated over all displacements originating in a circle (r = 80 nm). Inset: Trajectories and corresponding diffusion heat map (scale bar: 1 μm). c Diffusion map of Halo-eIF4E of activated neurons after TTX withdrawal (scale bar: 5 μm). Inset: Trajectories and corresponding diffusion heat map (scale bar: 1 μm). d Diffusion speed of Halo-eIF4E in neurons treated with 250 nM torin-1 (red colors) (scale bar: 5 μm). e Single particle tracking was recorded at a frame rate of 200 Hz. The cumulative distribution function (CDF) of all single-molecule displacements for Halo-eIF4E is shifted to the left when compared to neurons treated with torin-1, which…

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  Fig. 6. Simultaneous single-particle tracking of endogenous Halo-eIF4E and SNAPf-eIF4G in mESC cells that were differentiated into neurons. a Top left: Diffusion heat map of 18,220 JF585Halo-eIF4E trajectories from eight 10,000 frames 50 Hz movies obtained from a dense network consisting of more than 100 individual dendritic branches (scale bar: 10 μm). Top center: The corresponding heat map of the simultaneously recorded 26,438 JF646SNAPf-eIF4G trajectories. Top right: The distributions of apparent diffusion coefficients of JF585Halo-eIF4E (in green) and JF646SNAPf-eIF4G (in magenta). Two-sided t-test with two mean values of two distributions (*** means p < 0.001). b Cumulative distribution functions (CDF) were obtained from 26,438 eIF4G and 18,220 eIF4E trajectories. While the majority of Halo-eIF4E exhibit slow diffusion (54.3 ± 1.0%), this percentage increases to 73.2 ± 1.1% for SNAPf-eIF4G. c Thick dendritic process of a cortical neuron from ARCP/+;PCP-GFP;v-Glut2Cre mice expressing Halo-eIF4E after TTX withdrawal (dotted outline, scale bar: 10 μm). The median projection of the JF646Halo-eIF4E channel of 5000 frames was recorded at 100 Hz (in magenta). Overlay of two ARC mRNA…