AMPK Regulates Phagophore-to-Autophagosome Maturation

Abstract

Autophagy is an important metabolic pathway that can non-selectively recycle cellular material or lead to targeted degradation of protein aggregates or damaged organelles. Autophagosome formation starts with autophagy factors accumulating on lipid vesicles containing ATG9. These phagophores attach to donor membranes, expand via ATG2-mediated lipid transfer, capture cargo, and mature into autophagosomes, ultimately fusing with lysosomes for their degradation. Autophagy can be activated by nutrient stress, for example by a reduction in the cellular levels of amino acids. In contrast, how autophagy is regulated by low cellular ATP levels via the AMP-activated protein kinase (AMPK), an important therapeutic target, is less clear. Using live-cell imaging and an automated image analysis pipeline, we systematically dissect how nutrient starvation regulates autophagosome biogenesis. We demonstrate that glucose starvation downregulates autophagosome maturation by AMPK mediated inhibition of phagophores tethering to donor membranes. Our results clarify AMPK’s regulatory role in autophagy and highlight its potential as a therapeutic target to reduce autophagy.

The formation of ubiquitin rich condensates triggers recruitment of the ATG9A lipid transfer complex to initiate basal autophagy

“…We recently developed a panel of endogenously tagged autophagy proteins to quantitatively measure the formation of autophagy foci by live cell imaging (Barnaba et al, 2023;Broadbent et al, 2023). Our experiments with these cell lines suggest that the autophagy machinery rapidly assembles and disassembles into foci, but only a fraction of these foci ultimately mature into LC3 containing autophagosomes (Broadbent et al, 2023).…”

“…Live-cell imaging was performed in standard conditions of 5% CO2 and 37 C. (description of microscope) Images were acquired at 1 frame every second for 8 minutes. For image analysis feasibility, images were scaled down and analyzed using a previously established pipeline called K-FOCUS (Barnaba et al, 2023). Statistical differences were evaluated by two-tail t-test.…”