The Regulation of Macropinocytosis

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Abstract This proposal requests funds for the purchase of an IncuCyte SX1 microplate system from Sartorius, Inc., which will be used to analyze the role of macropinocytosis in amino acid- dependent growth of macrophages and other cells. The research supported by the parent R35 award has concentrated on the mechanisms and regulation of macropinocytosis in macrophages. Studies in other research groups determined that increased macropinocytosis in some cancer cells allows them to grow in nutrient-poor environments by scavenging extracellular proteins such as albumin. In contrast, our recent collaborative studies of T cells indicated that free extracellular amino acids necessary for activation of protein synthesis enter T cells by macropinocytosis. Thus, we hypothesize that macropinocytosis is necessary for the delivery of extracellular amino acids into cytoplasm and for subsequent increases in cell protein content. To measure growth in different formulations of media with various concentrations of leucine and other amino acids, we require the precision, throughput and temporal resolution offered by the IncuCyte SX1. The instrument is an imaging microplate reader (microscope) which resides inside a tissue culture incubator. Images of growing cultures of cells are collected at regular intervals from precise locations in 24- or 96-well dishes. Subsequent image analysis allows the growth and proliferation of cells to be quantified, such that the time series of each image field yields plots of cell growth vs. time. We will determine rates of CSF1-dependent cell growth when leucine concentrations are limiting and at different concentrations of leucine that support growth. If leucine-dependent growth requires endocytosis, then inhibiting macropinocytosis and other kinds of endocytosis should shift the concentration curve for leucine-dependent growth. These studies will define the role of macropinocytosis in the cellular acquisition of amino acids for growth.