Titre : Subcellular Analytical Strategies.
Pour des raisons en dehors de notre contrôle, la conférence du professeur Edgar Arriaga des départements de chimie et de génie biomédical de l'University of Minnesota doit être annulée. Elle sera reportée en temps et lieu (détails à venir)
Résumé : Chemical cytometry opened up exciting opportunities for monitoring multiple chemical species in single cells and understand cellular heterogeneity. Such heterogeneity results partially from the complex subcellular organization of the cell, which is defined by thousands of organelles and their interactions. Furthermore, organelles are themselves highly heterogeneous, plastic, and typically of sub-micrometer dimensions. These characteristics make organelles difficult to investigate with conventional analytical technologies. This presentation describes our current work aiming at developing and applying bioanalytical methods to analyze and purify organelles. Three strategies will be highlighted: (1) Biological imaging of mitochondrial subpopulations and their carbonylation status, (2) affinity purification of subcellular compartments, and (3) capillary electrophoretic separations of individual organelles. The first strategy focuses on the analysis of organelles in situ. After dual immunolabeling and quantitative imaging of rat skeletal muscle cross-sections, images were analyzed using a semi-automated Image J protocol to determine carbonylation levels in subsarcolemmal and interfibrillar mitochondria. Age-related changes in the carbonylation levels of mitochondria will be discussed. The second strategy is aimed at understanding the biological function of specific organelle types. Mitochondria and peroxisomes usually co-purify. Peroxisomes were magnetically immunopurified and then their ability to process fatty acids and the anti-cancer drug Doxorubicin was investigated. Ongoing efforts to immunopurify mitochondria will also be presented. The third strategy refers to the use of capillary electrophoretic separations of organelles to understand subcellular heterogeneity. Mitochondria utilize cytoskeleton as a scaffold for movement within the cell. Individual organelle analysis by capillary electrophoresis with dual laser-induced fluorescence detection revealed that mitochondria are either cytoskeleton-bound or –free organelles. The relevance of these studies to abnormal mitochondria will be discussed.
