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The Regulation of Macropinocytosis

巨胞饮作用的调节

基本信息

批准号：
10784227
负责人：
JOEL A SWANSON
金额：
$ 8.72万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-01 至 2025-03-31
项目状态：
未结题

项目摘要

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.

摘要该提案要求提供资金，用于从以下公司购买IncuCyte SX 1微孔板系统： Sartorius，Inc.，这将用于分析大胞饮在氨基酸- 巨噬细胞和其他细胞的依赖性生长。由父母R35支持的研究该奖项集中在大胞饮的机制和调节，巨噬细胞其他研究小组的研究确定，一些癌细胞通过清除细胞外蛋白如白蛋白。相比之下，我们最近对T细胞的合作研究表明激活蛋白质合成所必需的游离胞外氨基酸进入T 巨胞饮作用。因此，我们假设巨胞饮作用是必要的，将细胞外氨基酸输送到细胞质中并随后增加细胞蛋白质内容为了测量在具有各种浓度的培养基的不同配方中的生长，亮氨酸和其他氨基酸，我们需要的精度，吞吐量和时间分辨率 IncuCyte SX 1提供的。该仪器是一个成像酶标仪（显微镜）其位于组织培养箱内。收集细胞培养物的图像在24孔或96孔培养皿中的精确位置以规则的间隔进行。后续图像分析允许量化细胞的生长和增殖，使得每个细胞的时间序列成像场产生细胞生长对时间图。我们将测定CSF 1依赖性细胞当亮氨酸浓度有限时以及在不同浓度的亮氨酸下生长，支持增长。如果亮氨酸依赖性生长需要内吞作用，那么抑制巨胞饮作用和其他种类的内吞作用应该移动浓度曲线，亮氨酸依赖性生长。这些研究将确定巨胞饮在细胞内的作用，获得生长所需的氨基酸。