Autophagosome Formation
Autophagosomes are organelles that capture cellular components (including proteins, membrane fragments and whole organelles) and deliver them to lysosomes. Autophagosomal membranes are formed and mature by recruiting a well-conserved set of cytosolic proteins that modify cellular membranes. PI3 kinase activity is required on progenitor membranes. These membranes mature into double or multilamellar structures which trap substrates before fusing with lysosomes. The substrates are then digested by lysosomal proteases. Currently, it is unclear where the initial autophagic membranes derive from, how these membranes mature into a fully enclosed autophagic vesicles, and whether autophagosomes have specificity for particular substrates. It is also unclear whether there is only one or diverse pathways that induce autophagosome formation. We are investigating these questions using live-cell imaging techniques.
The classic way to induce autophagosomes is through amino acid starvation. We have identified and characterized other induction conditions. Using pulse-chase experiments in living cells with photoactivatable-GFP fusions, we observe stabilization of key autophagy proteins and dramatic formation of autophagic vacuoles when 26S proteasome acitivity is inhibited. We also find that treatments that affect the protein folding environment of the ER lead to the accumulation of autophagosomes.
Using different induction conditions we are testing whether there are multiple membrane sources for autophagosomes and whether autophagosomes formed under different cellular stresses have distinct behaviors and functions. For example, in conditions of protein folding stress, autophagosome formation may serve primarily to isolate misfolded proteins from the functional cell environment, whereas autophagosomes formed during starvation may serve principally to retrieve amino acids and energy.
To investigate the characteristics of autophagosomes formed under different induction conditions, we are utilizing photoactivatable GFP technology to perform pulse-chase experiments of whole autophagosomes in live cells. We are also using the fluorescent protease protection (FPP) assay to identify subpopulations of substrates captured by autophagosomes under different induction conditions, and to determine whether specific motifs on the substrates are important for this process. 
Figure: Cell line stably expressing LC3 (the rat homolog of S. cerevisiae ATG8) fused to the cyan fluorescent protein. CFP-LC3 present in the cytosol (top panel) is rapidly recruited to membrane structures in response to serum starvation (bottom panel). The cytosolic CFP-LC3 is conjugated to phosphotidyl ethanolamine in target membranes of autophagosomes. It persists on those membranes through their fusion with lysosomes.
For more information, contact Dale Hailey.
