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Additional file 4: Figure S4. Set up of optogenetic activation of mTORC1 in HeLa cells. A) CIBN-Rab7 was correctly targeted to LEs in HeLa cells transfected with CIBN-myc-Rab7 and immunolabeled for LAMP1 and myc tag. B) Photoactivation was able to target CRY2-mCherry-Raptor to LEs. HeLa cells were transfected with CIBN-myc-Rab7 and CRYII-mCherry-Raptor; 24 h later cells were either kept in the dark (left panel) or photoactivated for 5 min (30 × 100 ms-pulses of blue light given at 0.1 Hz at 2.42 mW/cm2) (right panel) before being processed for immunofluorescence. C) Photoactivation (as in B) was able to target mTOR with CRY2-mCherry-Raptor to LEs. D) Left, clustering of CRY2-mCherry-Raptor on LEs induced by photoactivation reverses within a few min. Shown is an index of raptor clustering (intensity (max/min)/mean) per pixel obtained on raptor surface area). Right, the same treatment induces effective pS6 phosphorylation only 30 min after it was given. Experiments were performed as in (B) with immunofluorescence done at different times after illumination (PA) as indicated. Cells were incubated in culture medium after illumination (N = 90 images per condition from three independent experiments). E) Rapamycin blocks photoactivation-induced P-pS6 increase (N = 90 images per condition from three independent experiments). F) The effect of photoactivation-induced mTOR recruitment to LEs on mTOR activity depends on FCS but only partially relies on amino acids. Cells were fed ( +) or starved (-) with serum (FCS) or amino acids (AA) for the time indicated in the diagram. In AA-starved cells, photoactivation induces an increase in P-pS6, although significantly reduced in comparison to cells fed with both AA and FCS. FCS starvation suppresses mTORC1 activity even after photoactivation (N = 60 images per condition from two independent experiments).

Additional file 3: Figure S3. TrkB inhibitors ANA12 or cyclotraxin-B reduced the recruitment of mTOR to LEs (IN/OUT ratio, see Fig. 3) induced by NMDAR- activation even in starvation conditions. Neurons were treated as in Fig. 3D in ACSF plus Mg2+ or in Mg2+-free ACSF, as indicated A) Effect of ANA12 (25 µM) on mTOR IN/OUT ratio from 3 independent experiments. B) Effect of Cyclotraxin-B (CtxB) on mTOR IN/OUT ratio from 2 independent experiments. In stimulated neurons treated with the TrkB inhibitors, the IN/OUT mTOR ratio stays significantly higher than the one observed in non-stimulated neurons. C) Bafilomycin-A (BafA 200 nM) prevented the translocation of mTOR to LEs triggered by Mg2+ removal as in Fig. 3D (N = 20 images per condition from 2 independent experiments).

Additional file 2: Figure S2. Increased pS6 phosphorylation 90 min after NMDAR stimulation in soma and proximal dendrites. Neurons were treated in cLTP medium as in Fig. 1B with or without AP5 or rapamycin as indicated, then incubated again in conditioned medium (CM) for 80 min before P-pS6 and MAP2 immunofluorescence. Note that over such extended-duration treatments, ACSF was replaced by CM to preserve neuron health. Individual neurons were imaged and P-pS6 quantified in soma or proximal dendrites (mean length 75 µM) for 40–60 neurons from 3 experiments.