Department of Biological, Chemical, and Physical Sciences, Illinois Institute of Technology, Chicago, Illinois 60616, USA.
J Phys Chem B. 2010 Mar 4;114(8):2894-900. doi: 10.1021/jp906737q.
The local expression and distribution pattern of protein on a cell play essential roles in signal transduction within a cell or between cells. Here we report on the development of a spatially resolved quantification method, which was applied in the study of E-cadherin local expression in identified undifferentiated and differentiated human embryonic stem (hES) cells in their native cellular environment. This was achieved by a novel immunofluorescence assisted affinity mapping (IF-AM) method, in which immunofluorescence provides the guidance to locate a desired type of cell in a cell community for performing affinity mapping to quantify the local protein density. The results unveiled the crucial role of E-cadherin in mediating hES cell proliferation and differentiation: the expression of E-cadherin is markedly higher on undifferentiated cells, and the growth of hES cells in unique colonies is contingent on the homogeneous distribution of E-cadherin. Due to the ability of directly assessing individual proteins of a cell, the IF-AM method is shown to be a sensitive tool for resolving subtle differences in the local expression of membrane proteins even at low abundance.
蛋白质在细胞中的局部表达和分布模式在细胞内或细胞间的信号转导中起着至关重要的作用。在这里，我们报告了一种空间分辨定量方法的发展，该方法应用于研究其天然细胞环境中的鉴定未分化和分化的人胚胎干细胞（hES）中 E-钙黏蛋白的局部表达。这是通过一种新的免疫荧光辅助亲和作图（IF-AM）方法实现的，其中免疫荧光提供了指导，以定位细胞群体中所需类型的细胞，以便进行亲和作图以定量局部蛋白质密度。结果揭示了 E-钙黏蛋白在介导 hES 细胞增殖和分化中的关键作用：E-钙黏蛋白在未分化细胞上的表达明显更高，hES 细胞在独特集落中的生长取决于 E-钙黏蛋白的均匀分布。由于该方法能够直接评估细胞的单个蛋白质，因此 IF-AM 方法被证明是一种敏感的工具，即使在低丰度下，也可以解决膜蛋白局部表达的细微差异。
