Investigation of Dynamic Regulation of TFEB Nuclear Shuttling by Microfluidics and Quantitative Modelling

Authors

Iacopo Ruolo
Sara Napolitano
Lorena Postiglione
Gennaro Napolitano
Andrea Ballabio
Diego di Bernardo

Publication Date

3-15-2025

Journal

Communications Biology

DOI

10.1038/s42003-025-07870-x

PMID

40089585

PMCID

PMC11910602

PubMedCentral® Full Text Version

Post-print

Published Open-Access

yes

Keywords

Basic Helix-Loop-Helix Leucine Zipper Transcription Factors, Humans, Cell Nucleus, Microfluidics, Phosphorylation, Transient Receptor Potential Channels, Mechanistic Target of Rapamycin Complex 1, Active Transport, Cell Nucleus, Models, Biological

Abstract

Transcription Factor EB (TFEB) controls lysosomal biogenesis and autophagy in response to nutritional status and other stress factors. Although its regulation by nuclear translocation is known to involve a complex network of well-studied regulatory processes, the precise contribution of each of these mechanisms is unclear. Using microfluidics technology and real-time imaging coupled with mathematical modelling, we explored the dynamic regulation of TFEB under different conditions. We found that TFEB nuclear translocation upon nutrient deprivation happens in two phases: a fast one characterised by a transient boost in TFEB dephosphorylation dependent on transient calcium release mediated by mucolipin 1 (MCOLN1) followed by activation of the Calcineurin phosphatase, and a slower one driven by inhibition of mTORC1-dependent phosphorylation of TFEB. Upon refeeding, TFEB cytoplasmic relocalisation kinetics are determined by Exportin 1 (XPO1). Collectively, our results show how different mechanisms interact to regulate TFEB activation and the power of microfluidics and quantitative modelling to elucidate complex biological mechanisms.