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.

抽象的该提案要求资金用于购买 IncuCyte SX1 微孔板系统 Sartorius, Inc.，将用于分析巨胞饮作用在氨基酸- 巨噬细胞和其他细胞的依赖性生长。该研究由母公司 R35 支持该奖项集中于巨胞饮作用的机制和调节巨噬细胞。其他研究小组的研究确定，巨胞饮作用增加一些癌细胞通过清除营养物质使其能够在营养贫乏的环境中生长细胞外蛋白质，例如白蛋白。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需的游离胞外氨基酸进入 T 细胞通过巨胞饮作用。因此，我们假设巨胞饮作用对于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。测量不同浓度的培养基中的生长情况亮氨酸和其他氨基酸，我们需要精度、通量和时间分辨率由 IncuCyte SX1 提供。该仪器是成像酶标仪（显微镜）它位于组织培养箱内。收集细胞生长培养物的图像定期从 24 孔或 96 孔培养皿中的精确位置进行检测。后续图像分析允许量化细胞的生长和增殖，这样每个细胞的时间序列图像场产生细胞生长与时间的关系图。我们将确定 CSF1 依赖性细胞的比率当亮氨酸浓度受到限制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，则抑制巨胞饮作用和其他类型的内吞作用应该使浓度曲线移动亮氨酸依赖性生长。这些研究将定义巨胞饮作用在细胞中的作用获取生长所需的氨基酸。