Periodic Reporting for period 5 - AutoClean (Cell-free reconstitution of autophagy to dissect molecular mechanisms)
Période du rapport: 2024-06-01 au 2025-05-31
Cells constantly recycle damaged or unneeded components through a process called autophagy, which protects against stress and contributes to health and longevity. Central to autophagy is the formation of a double-membrane compartment—the autophagosome—which engulfs cargo and fuses with the cell’s recycling center, the vacuole (in yeast) or lysosome (in humans). Despite its importance, the precise molecular events driving autophagy remain incompletely understood, in part because many of its steps are difficult to observe and dissect in intact cells.
The goal of this project was to rebuild autophagy from the bottom up, using purified components, cell fractions and synthetic systems. By combining cell biology, biochemistry, and synthetic biology, we aimed to reveal how the key kinase Atg1/ULK1 controls autophagy, how autophagosomes form and fuse, and how proteins are organized in space and time to ensure successful recycling.
The goal of this project was to rebuild autophagy from the bottom up, using purified components, cell fractions and synthetic systems. By combining cell biology, biochemistry, and synthetic biology, we aimed to reveal how the key kinase Atg1/ULK1 controls autophagy, how autophagosomes form and fuse, and how proteins are organized in space and time to ensure successful recycling.
The project established new synthetic reconstitution systems that allowed us to observe key steps of autophagy, such as autophagosome-vacuole fusion and early cargo recognition, in real time. We showed that the kinase Atg1 not only initiates autophagy but also controls the timing of membrane fusion by phosphorylating the fusion protein Ykt6. Importantly, this regulation is conserved across species, including humans.
We also identified Vac8 as the anchor that tethers the forming autophagosome to the vacuole, enabling the recycling process. A key conceptual breakthrough was the discovery of “initiation hubs”, small protein clusters that form on cargo and act as launchpads for autophagosome formation. These hubs rely on phase separation, a process similar to oil droplets forming in water, to bring together the required components. Our work also demonstrated that clustering activates the ULK1 complex in mammalian cells and initiates selective recycling of damaged mitochondria (mitophagy).
These findings were made possible by developing versatile in vitro and synthetic in vivo approaches for reconstructing autophagy step-by-step. The results have been published in leading journals and have sparked collaborations focused on diseases where autophagy is disrupted.
We also identified Vac8 as the anchor that tethers the forming autophagosome to the vacuole, enabling the recycling process. A key conceptual breakthrough was the discovery of “initiation hubs”, small protein clusters that form on cargo and act as launchpads for autophagosome formation. These hubs rely on phase separation, a process similar to oil droplets forming in water, to bring together the required components. Our work also demonstrated that clustering activates the ULK1 complex in mammalian cells and initiates selective recycling of damaged mitochondria (mitophagy).
These findings were made possible by developing versatile in vitro and synthetic in vivo approaches for reconstructing autophagy step-by-step. The results have been published in leading journals and have sparked collaborations focused on diseases where autophagy is disrupted.
The project has moved the autophagy field significantly beyond the state of the art by providing: i) The reconstitution of autophagosome-vacuole/lysosome fusion in vitro using purified components and cell fractions, ii) Evidence that Atg1/ULK1 regulates fusion and assembly via phosphorylation and clustering, connecting distinct stages of the pathway, iii) Discovery of initiation hubs that assemble through weak interactions and phase separation, offering a new framework for understanding selectivity and spatial control in autophagy, iv) Identification of Vac8 as a platform organizing these early steps at the vacuole surface.
These breakthroughs redefine how we understand autophagy regulation and open new paths for manipulating the process in neurodegeneration, infection, and cancer. Follow-up work will use these insights to explore therapeutic applications and refine molecular reconstitution of membrane expansion.
These breakthroughs redefine how we understand autophagy regulation and open new paths for manipulating the process in neurodegeneration, infection, and cancer. Follow-up work will use these insights to explore therapeutic applications and refine molecular reconstitution of membrane expansion.