Advancing organelle analysis: Development of techniques for the enrichment of endocytic organelles and to determine autophagosome properties.

摘要

Lysosomes and endocytic organelles are intracellular bodies present in eukaryotic cells responsible for the degradation of endocytosed extracellular targets. Autophagosomes traffic proteins, organelles, and other intracellular components to lysosomes to facilitate degradation during the degradative process of autophagy. Multiple disorders have been connected to malfunctions in lysosomes (Nieman pick, galactosialidosis, Danon disease) and autophagosomes (Alzheimers, Parkinson's, Huntington's).;Methods are needed to enrich organelles in order to study their properties without contamination from unwanted organelles. Current methods to enrich endocytic organelles do not result in highly enriched organelles (differential centrifugation), are time consuming and tedious (density gradient centrifugation), and can damage membranes. Methods are also needed to determine endocytic and autophagy organelle properties such as organelle molecular composition, organelle-specific biotransformation of anti-cancer drugs, individual organelle surface properties and marker protein levels, and pH. The work described in this thesis develops new techniques to improve our ability to enrich endocytic organelles and determine their properties. This work includes: (1) the magnetic enrichment of endocytic organelles and determination of pH by capillary cytometry, (2) the determination of the biotransformation of N-L-leucyldoxorubicin to doxorubicin, (3) the development of a workflow to determine preliminary identifications of enriched autophagosome samples and (4) determine temporal changes in individual autophagy organelle numbers, surface charge, and LC3-II levels from basal and rapamycin enhanced autophagy levels. These methods will improve our understanding of how lysosomes and autophagosomes contribute to disease, leading to better therapeutic strategies that may improve and lengthen people's lives.;Endocytic organelle enrichment was done by trafficking dextran coated magnetic iron oxide nanoparticles to lysosomes and endocytic organelles prior to magnetic separation. No detectable enzymatic activity from mitochondria and peroxisomes were observed in the enriched endocytic organelle fractions suggesting that the enriched lysosomes were in high enrichment. A majority of enriched, individual endocytic organelles had an acidic pH as determined by capillary cytometry suggesting the enriched endocytic organelle fraction had intact membranes. Enriched endocytic organelle fractions were then used to determine the biotransformation of N-L-leucyldoxorubicin to doxorubicin. Previous reports had suggested endocytic organelles may be important for intracellular biotransformation. About 45% of the biotransformation from uterine sarcoma cell post nuclear fraction occurred in the enriched endocytic organelle fraction suggesting intracellular biotransformation may be more critical to prodrug activation than previously believed.;Ultra high performance liquid chromatography coupled to near-simultaneous low- and high-collision energy mass spectrometry was used to determine preliminary identifications of compounds enriched or unique to enriched autophagosome fractions. A workflow was developed to detect and confirm features (unidentified compounds with a characteristic chromatographic tR and m/z value) in the enriched sample as well as making and confirming identifications from online databases. Multiple high-relevancy preliminary identifications were made that are relevant to autophagy as supported by literature searches. Following validation, these preliminary identifications could prove to be important to maintain autophagosome function and autophagy.;Capillary electrophoresis coupled to laser induced fluorescence detection (CE-LIF) was used to determine temporal changes in the detected number of individual autophagy organelle events (phagophores, autophagosomes, amphisomes, and autolysosomes), of GFP-LC3-II levels, and of surface charge by CE-LIF. Pharmacological treatment with vinblastine was used to accumulate autophagosomes and phagophores from basal and rapamycin enhanced autophagy do detect temporal changes in autophagy organelles characteristic of the autophagy level and its autophagy flux. The dramatic contrast between time dependent changes in individual organelle properties between basal and rapamycin enhanced autophagy conditions demonstrates an anticipated complexity of autophagy flux which likely plays critical role in response to drug treatments, aging, and disease.