Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - Autophagy in vitro (Reconstituting Autophagosome Biogenesis in vitro)

Teaser

Autophagy is a catabolic pathway that delivers cytoplasmic material to lysosomes for degradation. Under vegetative conditions, the pathway serves as quality control system, specifically targeting damaged or superfluous organelles and protein-aggregates. Selective autophagy is...

Summary

Autophagy is a catabolic pathway that delivers cytoplasmic material to lysosomes for degradation. Under vegetative conditions, the pathway serves as quality control system, specifically targeting damaged or superfluous organelles and protein-aggregates. Selective autophagy is also involved in regulating cellular responses related to immunity, inflammation, and antimicrobial defense. Central to all these responses is the autophagic core machinery that comprises two interconnected Ubiquitin (Ub)-like conjugation systems. The conjugation machinery targets cellular components to the lysosome for degradation by inducing complex membrane remodeling events. The underlying molecular mechanism is, however, poorly understood. Obtaining insights into the function of the conjugation machinery is needed to unravel how impaired autophagy contributes to human diseases including cancer, neurodegeneration, and autoimmune diseases.
We take an interdisciplinary and highly innovative approach to reveal the molecular mechanisms of autophagy. We will reconstitute the human Ub-like conjugation systems on model membranes in vitro to uncover how this machinery regulates diverse aspects of cellular responses. We will combine in vitro reconstitutions with cryo-electron microscopy and atomic force microscopy to gain insights into membrane remodeling events with high spatial resolution. Super-resolution microscopy will complement structural data with single molecule dynamics. Finally, we will correlate mechanisms that we identified from reconstitutions in vivo by interfering with specific autophagic functions in cultured cells. Innovative genome editing techniques will allow us to specifically manipulate defined autophagic activities. Our novel approach will provide essential insights into the pathophysiology of cancer, neurodegenerative, and autoimmune diseases.

Work performed

AIM1: We reconstituted the human ATG8-conjugation system on membranes and studied the assembly using atomic force microscopy and high resolution microscopy. We observed so that all of the human ATG8-homologs can form protein-lattices on supported lipid bilayers.

AIM2: We compared the lipidation efficiency of ATG8-homologs in the presence of TECPR1 and ATG16L1. We observed that whereas ATG16L1 promotes lipidation of all ATG8-homologs in vitro, TECPR1 appears to be selective. Deletion of TECPR1 (using CRISPR-Cas9 technology) did not affect conjugation efficiencies in vivo.

Final results

We extended our studies using not only conventional cell culture models (HeLa and HEK 293), but also neural progenitor cells (NPC cells). These new direction has outmost relevance for human health as autophagy and the degradation of protein aggregates is closely related to the onset of human neurodegenerative diseases.