"Early and Late Stage Mechanisms of Autophagosome Formation" by Taryn J. Olivas

Date of Award

Fall 2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cell Biology

First Advisor

Melia, Thomas

Abstract

During autophagosome biogenesis, the incorporation of transmembrane proteins into the expanding phagophore is not readily observed. In addition, the membrane surface area of the organelle expands rapidly, while the volume of the autophagosome is kept low. This means that canonical methods for membrane biogenesis do not apply to autophagosomes. Exciting developments in recent years support a new mechanism that could explain these unique attributes of this double-membrane organelle. Several recent studies have suggested the autophagosome expands predominantly through the direct protein-mediated transfer of lipids through the lipid transfer protein ATG2.As these lipids are only introduced into the cytoplasmic-facing leaflet of the expanding phagophore, full membrane growth also requires lipid scramblase activity. ATG9 has been demonstrated to harbor scramblase activity and is essential to autophagosome formation, however if and when it is integrated into mammalian autophagosomes remains unclear. This work shows that in the absence of lipid transport, ATG9 vesicles are already fully competent to collect proteins normally found on mature autophagosomes, including LC3-II. Further, through the novel use of styrene-maleic acid lipid particles as a nanoscale interrogation of protein organization on intact membranes, I show that ATG9 is fully integrated in the same membranes as LC3-II, even on maturing autophagosomes. The ratios of these two proteins at different stages of autophagosome biogenesis demonstrates that ATG9 is not continuously integrated, but rather present on the seed vesicles only and become diluted in the rapidly expanding phagophore membrane. Thus, ATG9 vesicles are the seed membrane from which mammalian autophagosomes form. In addition, here I also present work exploring cells missing all four ATG4 genes that can be used in the future for experiments with autophagosome precursor membranes stalled at a stage between pre-ATG2 membranes and mature autophagosomes. LC3/GABARAP-II decorates the inner and outer membrane of the expanding phagophore, but is removed from the outer membrane of the mature autophagosome by the ATG4 family proteases. When delipidation is blocked, we found GABARAPL1-positive structures accumulate with WIPI2, which is generally lost from fully formed autophagosomes. The accumulation of autophagy proteins with WIPI2 might be evidence for intermediate structures, or it may represent stalled autophagosomes that are never processed. ATG4A could not resolve WIPI2 accumulations in ATG4 QKO cells alone to restore macroautophagy. Intriguingly, new evidence published after this story now suggests that not only is ATG4A actually involved in mitophagy, but it is also a direct interacting partner with ATG9. Future work on the redundancy of ATG4 family proteins should therefore focus on selective autophagy pathways.

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