Macroautophagy is a cellular degradation process responsible for the clearance of excess intracellular cargo. Existing methods for bulk quantification of autophagy rely on organelle markers that bind to multiple autophagy organelle types, making it difficult to tease apart the subcellular mechanisms implicated in autophagy dysfunction in liver and other pathologies. To address this issue, methods based on individual organelle measurements are needed. Capillary electrophoresis with laser-induced fluorescent detection (CE-XLIF) was previously used to count and determine properties of individual autophagy organelles isolated from an LC3-GFP expressing cell line, but has never been used on autophagy organelles originating from a tissue sample. Here, we used DyLight488-labeled anti-LC3 antibodies to label endogenous LC3 present on organelles isolated from murine liver tissue prior to CE-LIF analysis. We evaluated the ability of this method to detect changes in a known model system of altered autophagy, as well as confirmed the specificity and reproducibility of the antibody in the labeling of autophagy organelles from liver tissue. This is both the first demonstration of CE-LIF to analyze individual organelles labeled with fluorophore-conjugated antibodies, and the first application of individual organelle CE-LIF to measure the properties of autophagy organelles isolated from tissue. The observations described here demonstrate that CE-LIF of immunolabeled autophagy organelles is a powerful technique useful to investigate the complexity of autophagy in any tissue sample of interest.
Bibliographical noteFunding Information:
We thank Dr. M. Kyba from the Department of Pediatrics and Dr. D. Lowe from the Department of Physiology at the University of Minnesota for providing mice for the autophagy organelle isolation. We thank Dr. N. Mizushima from the University of Tokyo for the generous gift of ATG5(−/−) and ATG5(+/+) MEF cells, provided to us by Dr. D. Kim from the Department of Biochemistry, Molecular Biology, and Biophysics at the University of Minnesota. K.A.M., H.M.G., and E.A.A. thank the National Institutes of Health (Grants RO1-AG020866, T32-GM008700, and T32-AG029796).