Sealing the autophagosome for cellular self-consumption (AutoSeal)

Intracellular degradation of cytoplasm by a process known as autophagy plays a key role in human physiology, and impairment of this process is associated with aging and diseases ranging from neurodegenerative diseases through infections, cancer, muscle dystrophies, heart disease and autoimmunity. Pharmacological up- or downregulation of autophagy is therefore likely to have an important role in future therapies. However, in order to target autophagy pharmacologically we need to understand the detailed biology of this process. In this project we have addressed one of the outstanding questions in the autophagy field, namely how the "waste container" that encloses the material to be degraded, called autophagosome, is sealed. Knowing this will equip us with novel strategies for up- or downregulation of autophagy for therapeutic purposes. Based on recent data from our Laboratory we hypothesized that a protein machinery called "ESCRT" mediates sealng of the autophagosome, and the present proposal was designed to test this hypothesis and exploit the data for manipulatng autophagy in vitro and in vivo. By using advanced live-cell microscopy we have indeed observed recruitment of ESCRT proteins to sealing autophagosomes, and we have also obtained evidence that ESCRT proteins are required for sealing of the autophagosome. As an example we showed that ESCRT-mediated sealing of autophagosomes is required for cellular degradation of damaged mitochondria, a degradation that is known to protect our organism from cancer development. The results from the project have been published in prestigious journals such as Autophagy, EMBO Journal and Nature Reviews Molecular Cell Biology.

The project has led to a molecular understanding of how autophagosomes are sealed and shown that such sealing is essential for autophagy. Given the important function of autophagy-mediated degradation of cytoplasm and potentially harmful objects such as damaged organelles, microorganisms, viruses and protein aggregates, this new mechanistic insight provides an important advance in in physiology and medicine.

Intracellular degradation of cytoplasm by autophagy plays a key role in human physiology, and impairment of this process is associated with aging and diseases ranging from neurodegenerative diseases through infections, cancer, muscle dystrophies, heart disease and autoimmunity. Pharmacological up- or downregulation of autophagy is therefore likely to have an important role in future therapies. However, in order to target autophagy pharmacologically we need to understand the detailed biology of this process. Previous work has established that autophagy is initiated when a double membrane, called isolation membrane or phagophore, sequesters portions of cytoplasm to form an autophagosome. When the autophagosome fuses with a lysosome to form an autolysosome, the sequestered material will be degraded by lysosomal hydrolases. A number of molecular components that mediate autophagosome biogenesis and autophagosome-lysosome fusion have been identified, but one key mechanistic question remains unsolved: How is the autophagosome sealed so that its content is separated from cytosol? We have very recently found that components of the endosomal sorting complex required for transport (ESCRT) machinery mediate sealing of holes in the nuclear envelope during mitotic exit (Vietri et al., Nature, 2015), and we now propose that the ESCRT machinery also mediates sealing of autophagosomes given the topological similarities between closing of these double membrane structures. The aim of the present project is to test this provocative hypothesis and to use the obtained information to gain substantial new insight into the molecular mechanisms and physiological functions of autophagy. If successful, this project will not only provide novel mechanistic insight into a cellular process of high importance but also open new strategies for pharmacological up-or downregulation of autophagy for therapeutic purposes.