Detection of kinase translocation using microfluidic electroporative flow cytometry
Detection of kinase translocation using microfluidic electroporative flow cytometry
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DOI：
10.1021/ac702065e
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发表时间：
2008-02-15
影响因子：
7.4
通讯作者：
Lu, Chang
中科院分区：
文献类型：
--
作者：
Wang, Jun;Bao, Ning;Lu, Chang
激酶在细胞内的定向定位对于它们的激活和参与信号转导是重要的。这些事件的检测主要基于少量细胞的成像和亚细胞分级分离/Western印迹进行。这些常规技术要么缺乏探测整个细胞群所需的高通量，要么仅提供细胞群的平均行为而没有来自单个细胞的信息。在这里，我们展示了一种新的工具，称为微流控电穿孔流式细胞术，检测易位。在B细胞中，在细胞群体水平上，将EGFP标记的酪氨酸激酶Syk转移至质膜。我们将联合收割机电穿孔与流式细胞术相结合，观察电穿孔过程中细胞内激酶的释放。我们发现，激酶的释放受到其亚细胞定位的强烈影响。通过抗原受体刺激的细胞在质膜上具有一部分激酶，并且在电穿孔后比没有刺激和易位的细胞保留更多的激酶。我们能够区分具有易位的细胞群体与没有易位的细胞群体，并从整个群体的单个细胞中收集信息。Ibis技术潜在地允许在单细胞水平上检测蛋白质易位。由于激酶易位在疾病过程中的频繁参与，如肿瘤发生，我们的方法将有激酶相关的药物发现和肿瘤的诊断和分期的效用。
Directed localization of kinases within cells is important for their activation and involvement in signal transduction. Detection of these events has been largely carried out based on imaging of a low number of cells and subcellular fractionation/Western blotting. These conventional techniques either lack the high throughput desired for probing an entire cell population or provide only the average behaviors of cell populations without information from single cells. Here we demonstrate a new tool, referred to as microfluidic electroporative flow cytometry, to detect the translocation. of an EGFP-tagged tyrosine kinase, Syk, to the plasma membrane in B cells at the level of the cell population. We combine electroporation with flow cytometry and observe the release of intracellular kinase out of the cells during electroporation. We found that the release of the kinase was strongly influenced by its subcellular localization. Cells stimulated through the antigen receptor have a fraction of the kinase at the plasma membrane and retain more kinase after electroporation than do cells without stimulation and translocation. We are able to differentiate a cell population with translocation from one,without it with the information collected from individual cells of the entire population. Ibis technique potentially allows detection of protein translocation at the single-cell level. Due to the frequent involvement of kinase translocations in disease processes such as oncogenesis, our approach will have utility for kinase-related drug discovery and tumor diagnosis and staging.