Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Tuesday Speaker Abstracts

PROTEIN REGULATION OF EXOSOME SECRETION Michelle K. Knowles 1,2 ; 1 University of Denver, Chemistry and Biochemistry, Denver, Colorado, USA 2 University of Denver, Molecular and Cellular Biophysics Program, Denver, Colorado, USA Exosomes are small vesicles (diameter < 200 nm) that are secreted by most types of cells for intercellular communication. Exosome biogenesis is initiated in multivesicular bodies (MVBs) and their secretion into the extracellular fluid is driven by the fusion of MVBs with the plasma membrane. Once in the extracellular fluid, exosomes can be taken up by surrounding cells, and can, thus, be used to transfer important biomolecules such as nucleic acids, lipids and proteins between cells. This mode of communication has recently gained interest because it is exploited by diseased cells. For instance, it is well documented that exosome secretion is upregulated in cancer cells where exosomes carry nucleic acids with oncogenic mutations that have the potential to alter gene expression in recipient cells, leading to the progression of disease. Despite the recent growth of interest in exosomes and their use in early screening assays, little is known about the protein regulators of fusion. Past work suggests a role for VAMP7, SNAP23, and actin in the process. In this work, we have investigated the role of SNAREs and SNARE interacting proteins in the regulation of exosome fusion. Cells (A549, PC12) expressed a pHluorin or pHuji labeled CD63 on exosomes as a marker of MVB fusion events and the presence of proteins in another color channel was concurrently measured using TIRF microscopy methods. The timing of protein arrival and loss from exosome fusion sites was measured for a variety of proteins involved with the membrane fusion process with the goal of identifying targets for therapeutic intervention in the future.

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