Capillary Electrophoresis of Magnetically Enriched Autophagosomes

Autophagy is one of the main cellular degradation processes. This process involves autophagosomes that are organelles credited with accumulating subcellular components marked for degradation. These organelles then fuse with lysosomes to complete degradation.  It has been suggested that the lipid composition of autophagosomes play an important role in autophagy, fusion with other organelles, and signaling pathways but these ideas have not been thoroughly explored.  It is also unknown how changes in lipid content or proteome impact autophagosome function in lysosome storage and neurodegenerative disorders.  The ability to separate and characterize an enriched autophagosome fraction will increase the ability to study these research needs of autophagosomes.

This presentation will report on recent advances to analyze individual autophagosomes by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF).  Whole cell homogenate from EGFP-LC3 (enhanced green fluorescent protein-LC3 protein) transfected cells is prepared and treated with anti-LC3 labeled magnetic microparticles.  Microparticles and bound organelles are separated from the rest of the biological contents with a custom-built on-line magnetic separator.  This fraction is then analyzed for EGFP fluorescence using CE-LIF.