(179d) Subcellular Complexity, An Electrophoretic Perspective

Cell function relies on the coordinated function of thousands of subcellular compartments (organelles). To name a few, mitochondria produce chemical energy in the form of ATP, autophagosomes encapsulate organelles tagged for degradation, and lysosomes degrade and recycle subcellular material. These nanometer-micrometer size organelles have an external membrane made of phospholipids and proteins that are electrically charged at biological pH (7.4), thereby presenting a measurable electrophoretic mobility under biological conditions. Due to the high heterogeneity in size and membrane composition, each organelle type is expected to have a unique electrophoretic profile. As such, each profile would be indicative of organelle subpopulations or would reveal changes that occur in the cell with aging and disease. In order to explore this hypothesis our program has been developing techniques to analyze individual organelles by capillary electrophoresis with laser-induced fluorescence detection (individual organelle CE-LIF).

This presentation will highlight the origins, evolution, current status, and the future of individual organelle CE-LIF in our laboratory. Initial developments based on liposomes demonstrated that under biological conditions electrophoretic mobilities of these organelle mimics are strongly dependent on surface composition. Subsequent developments in individual organelle CE-LIF of mitochondria and lysosomes revealed a high degree of heterogeneity and suggested the existence of electrophoretically heterogeneous subpopulations of these organelles. Individual organelle CE-LIF has also been adequate for measuring individual organelle properties such anti-cancer drug contents, pH, subcellular interactions and production of reactive oxygen species. Ongoing projects include the development of new electrophoretic modes such as isoelectric focusing, immunoanalysis of individual organelles, and theoretical predictions of fundamental properties based on experimental measurements. Long-term, extension of these fundamental principles could lead to novel purification strategies of biological and synthetic particles, adaptation to medical diagnostics, or new tools to explore molecular mechanisms in biomedicine.