Elucidation of TFEB nuclear translocation dynamics in human cells by means of Quantitative Modelling and Microfluidics

The Transcription Factor EB (TFEB) plays a pivotal role in the transcriptional regulation of lysosomal biogenesis and autophagy in response to starvation. TFEB activity is regulated by the kinase mTOR. In nutrient-rich conditions, mTOR phosphorylates TFEB by sequestering it in the cytoplasm. During starvation, mTOR is inhibited and unphosphorylated TFEB translocates into the nucleus, where it regulates the expression of its target genes. We performed an experiment monitoring in real-time the nuclear localization of TFEB in individual cells growing in a microfluidics device following alternating pulses of starvation and refeeding. The results revealed the presence of an “overshoot” dynamics with TFEB translocating to the nucleus upon starvation but then partially retranslocating to the cytoplasm. To elucidate the mechanism behind its nuclear shuttling dynamics, we first developed a two-compartment dynamical model (nucleus and cytoplasm) with two different species (dephosphorylated and phosphorylated TFEB) for each of the two compartments. The transport and de/phosphorylation kinetics are described with first order kinetics whose parameters depend on the nutrient status, while the total TFEB is kept constant. This model was not capable to recapitulate the observed overshoot dynamics. Consequently, we investigated two alternative models, hypothesizing that upon starvation, a nuclear export channel is activated either by TFEB itself (negative feedback loop - NFL) or as a direct consequence of mTOR inhibition (incoherent feedforward loop -IFFL). The new models were both able to qualitatively recapitulate the observed dynamics. Finally, we propose an experiment able to distinguish between the two alternative models.