Biomembranes are ubiquitous lipid structures that delimit the cell surface and organelles and operate as platforms for a multitude of biomolecular processes. Tension is a major property of membranes. It contributes significantly to the regulation of membrane deformation and topological alteration during fusion and fission events. Therefore, membrane tension probably regulates all membrane dynamics in cells to some extent. Unfortunately, limited data are available on intracellular membrane tension and its regulation due to lack of established tools to measure it. This precludes an understanding of major regulatory mechanisms of organelle dynamics. The objective of this proposal is to comprehensively reveal the molecular mechanisms regulating intracellular membrane tension during autophagy, a conserved autodigestive pathway that is characterized by major endomembrane deformations. Particularly, we will focus on membrane tension regulation by actin-dependent motors of the myosin family and their control of membrane dynamics during autophagy. Our hypothesis is that unconventional myosins, Myo1B and Myo1C, known to regulate autophagy, potentially helped by Myo7A found at lysosomes, exert their distinct functions through control of lysosomal membrane tension. Here we will perform 3D resolved measurements of lysosomal membrane tension through the development of fast and high resolved FLIM microscopy in cells during autophagy (WP1). We will combine this approach with mechanistic studies using giant unilamellar vesicles (GUVs)- and organelle-based in vitro reconstitution assays (WP2) and structural analysis of the motors (WP3) to provide unprecedented detail on how cytoskeletal motors regulate membrane tension. We believe that our multi-scale study will allow to fully grasp the molecular mechanisms of intracellular membrane tension control by unconventional myosins 1 and 7A during autophagy.