毫秒单分子定位显微镜结合卷积分析和自动图像分割，以确定结构复杂的功能细胞中的蛋白质浓度，一次一个细胞
Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time
摘要：
我们提出了一种称为CoPro（蛋白质浓度）方法的单分子工具，该方法使用具有卷积分析的毫秒成像，自动图像分割和超分辨率定位显微镜来生成单个活细胞不同隔室中蛋白质浓度的稳健估计，并使用复杂的多隔室细胞类型的真实模拟进行验证。我们证明了它的效用实验模型大肠杆菌细菌和酿酒酵母芽殖酵母细胞，并用它来解决信号如何在细胞中转导的生物学问题。在生命的所有领域中，细胞通过信号转导机制动态地感知它们的环境，其中许多涉及基因调控。对S.酿酒酵母是研究基因调控信号转导的模型系统。它使用GAL基因家族的多拷贝表达抑制剂Mig1，在细胞外葡萄糖浓度升高的情况下抑制不需要的基因。我们用绿色荧光蛋白（GFP）荧光标记Mig1分子，通过染色体整合在活的S.酿酒酵母细胞，除了RNA聚合酶蛋白Nrd1与荧光蛋白报告mCherry。使用CoPro，我们定量估计Mig1和Nrd1蛋白浓度在细胞质和细胞核室的细胞的基础上，在生理条件下。这些估计表明，如果外部葡萄糖浓度升高，细胞核中扩散性Mig1浓度向更高值移动4倍，而细胞质中的等效水平向更小值移动，相对变化小一个数量级。这与Nrd1相比，Nrd1不直接参与葡萄糖传感，它几乎完全位于细胞核中，在高浓度和低浓度下。
英文摘要：
We present a single-molecule tool called the CoPro (Concentration of Proteins) method that uses millisecond imaging with convolution analysis, automated image segmentation and super-resolution localization microscopy to generate robust estimates for protein concentration in different compartments of single living cells, validated using realistic simulations of complex multiple compartment cell types. We demonstrates its utility experimentally on model Escherichia coli bacteria and Saccharomyces cerevisiae budding yeast cells, and use it to address the biological question of how signals are transduced in cells. Cells in all domains of life dynamically sense their environment through signal transduction mechanisms, many involving gene regulation. The glucose sensing mechanism of S. cerevisiae is a model system for studying gene regulatory signal transduction. It uses the multi-copy expression inhibitor of the GAL gene family, Mig1, to repress unwanted genes in the presence of elevated extracellular glucose concentrations. We fluorescently labelled Mig1 molecules with green fluorescent protein (GFP) via chromosomal integration at physiological expression levels in living S. cerevisiae cells, in addition to the RNA polymerase protein Nrd1 with the fluorescent protein reporter mCherry. Using CoPro we make quantitative estimates of Mig1 and Nrd1 protein concentrations in the cytoplasm and nucleus compartments on a cell-by-cell basis under physiological conditions. These estimates indicate a 4-fold shift towards higher values in concentration of diffusive Mig1 in the nucleus if the external glucose concentration is raised, whereas equivalent levels in the cytoplasm shift to smaller values with a relative change an order of magnitude smaller. This compares with Nrd1 which is not involved directly in glucose sensing, which is almost exclusively localized in the nucleus under high and...
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