Fig. 1: Experimental measurements of TFEB nuclear shuttling dynamics in response to mTOR inhibition and refeeding in single cells by microfluidics and microscopic imaging.

A Schematic representation of the molecular reactions involved in the regulation of TFEB nucleus-cytoplasm shuttling. In nutrient-rich medium, the mTORC1 complex phosphorylates TFEB at rate k₁ leading to its retention in the cytoplasm. At the same time, nuclear TFEB is phosphorylated at rate k₂ and is exported to the cytoplasm by XPO1 at a rate β₂. In the absence of nutrients, mTORC1 is inhibited and TFEB is no longer phosphorylated, while the MCOLN1-mediated calcium release subsequent to mTORC1 inhibition contributes to dephosphorylate TFEB via Calcineurin (CaN) at a rate of k₋₁. Dephosphorylated TFEB can translocate to the nucleus at a rate of β₁. The pharmacological treatments employed in the study and their associated effects are denoted in red. Experiments were performed in a monoclonal HeLa cell line stably expressing the indicated constructs (i.e. nuclear mCherry and TFEB fused to the EGFP). B–G Representative microscopic images of HeLa cells in phase contrast and fluorescence at the indicated conditions. RPMI: nutrient-rich cell culture medium. HBSS: nutrient-deprived Hank’s Balanced Salt Solution. H Experimental platform to measure TFEB-GFP localisation in real-time. A microfluidic chip for cell culture²³ is hosted under a time-lapse inverted epifluorescence microscope (Nikon Eclipes Ti-e) to image HeLa cells. The chip is connected to two syringes filled with two different media whose position is controlled by a computer^22,48. I Starvation ‘step’ experiment: HeLa cells were grown overnight in the microfluidics chip in RPMI. At the beginning of the experiment, the chip was placed under the microscope and connected to two syringes, one filled with RPMI medium, and the other with HBSS medium. Cells were kept for 30 min in RPMI before being switched to HBSS medium for 6 hours, as indicated by the input (orange). Nuclear TFEB (%): the GFP fluorescence in the nucleus as a percentage of the cell’s total GFP fluorescence. Nuc/Cyto TFEB: the ratio of the GFP fluorescence in the nucleus to that in the cytoplasm. Single-cell traces are shown as thin solid green lines, whereas the average trace is represented by the thick solid green line. J Starvation ‘pulse’ experiment: Cells were kept for 180 min in RPMI, then switched to HBSS medium for 180 min, and switched back to RPMI for 180 min as indicated by the input (orange). K Pharmacological mTOR inhibition ‘step’ experiment: Cells were kept for 30 min in RPMI before being switched to RPMI medium supplemented with AZD8055 (AZD) for 6 hours as indicated by the input (orange). L Pharmacological mTOR inhibition ‘pulse’ experiment: Cells were kept for 180 min in RPMI, then switched to RPMI medium supplemented with AZD8055 (AZD) for 180 min, and switched back to RPMI for 180 min as indicated by the input (orange). A, H were created using BioRender.com.