Tri-dimensional exploration of membrane assembly during autophagosome biogenesis

Summary

Autophagy is an intracellular catabolic process conserved among eukaryotes involved in numerous physiological functions and multiple pathological situations. Structures targeted for destruction are sequestered into large double-membrane vesicles called autophagosomes, and transported to the lysosome/vacuole where they are degraded. The mechanism that leads to autophagosome formation remains largely unknown despite the identification and partial characterization of the Atg proteins, the factors specifically involved in autophagy. One of the obstacles that has limited progress has been the absence of an experimental setup visualizing membranes, which would allow to directly assess the function of the Atg proteins during autophagosome biogenesis.

The goal of this project is to unveil how membranes are assembled into autophagosomes. We will uniquely combine yeast Saccharomyces cerevisiae genetics and immuno-electron tomography to reach this objective. At first, we will devise a novel electron tomography protocol to preserve yeast autophagosomal membranes near their native state and be able immunolabel them. Subsequently, we will exploit this procedure to develop a model about how autophagosomal membranes are assembled into autophagosomes by analysing the precursor structures accumulated in yeast autophagy mutants. This unique experimental system will allow us visualizing the formation of autophagosomes through 3-dimentional images with ultrastructural resolution, and determine the distribution of Atg proteins, thus acquitting insights into their function.

With the realization of this project, we expect to acquire new knowledge on the mechanism of autophagy, which is essential for the development of autophagy-modulating therapies for human diseases. Moreover, our technological advances will be beneficial for the large scientific community studying autophagy and yeast cell biology.

Details

Project number

ALWOP.355

Main applicant

Dr. M.C. Mari

Affiliated with

Rijksuniversiteit Groningen, Universitair Medisch Centrum Groningen, Celbiologie

Duration

01/05/2018 to 15/01/2021

Budget

EUR265,722