Study of Protein Translocation Using Microfluidic Electroporative Flow Cytometry

基于大量群体中单细胞数据的技术，尤其是流式细胞术，已成功地应用于研究与蛋白质表达水平上的动态相关的生物学问题。相比之下，蛋白质在细胞内的移位(即蛋白质在细胞质和质膜之间或细胞核和细胞质之间的移动)的确定仅通过大样本方法进行，如分离/Western blotting或对少量细胞进行成像。蛋白质易位在生物学和医学中具有重要意义。虽然绝大多数蛋白质是在细胞质中合成的，但20%的蛋白质位于非细胞质的水腔中，另外25%-30%的蛋白质位于膜内。研究蛋白质转位对于了解细胞内的信号转导和调控途径以及它们所参与的疾病过程至关重要。易位不涉及蛋白表达水平的变化，因此用传统的流式细胞术是检测不到的。因此，检测单细胞中蛋白质转位的高通量技术是非常需要的，以产生对许多重要生物过程的机械性见解。在这个项目中，我们的总体目标是开发一种新的高通量技术，我们称之为微流控电穿孔流式细胞术(EFC)，以检测单细胞水平的蛋白质转位并研究转位过程的动力学。微流控EFC结合了电穿孔(应用外部电场突破细胞膜屏障)和流式细胞术。我们的初步数据表明，在电穿孔过程中，蛋白质从细胞释放到周围溶液中的情况取决于它的亚细胞位置。通过记录单个细胞由于电穿孔而损失的蛋白质数量，我们将能够确定是否发生了易位。使用这种方法，我们将测试两个具有重要生物学意义的模型蛋白：一个是从细胞质转移到质膜的激酶Syk，另一个是在刺激下从细胞质转移到细胞核的转录因子NF-kappaB。我们将在单细胞水平上演示对这两种不同类型易位的检测。此外，我们将使用微流体EFC来生成这些过程在不同刺激条件下的动力学数据。我们将通过将结果与传统方法(如分离/Western blotting和共聚焦荧光显微镜)获得的结果进行比较，来提供该技术的跨平台验证。作为综合教育活动，我们将在跨学科背景下培训本科生和研究生，重点是女性和代表性不足的少数民族学生，并向高中生和普通民众传播知识。

Technologies based on data from single cells within a large population, most notably flow cytometry, have been successfully applied to study biological problems associated with dynamics in the protein expression level. In comparison, the determination of protein translocation within cells (i.e. movement of a protein between the cytoplasm and plasma membrane, or between the nucleus and cytoplasm) has been only carried out by bulk sample methods such as fractionation/Western blotting or imaging of a low number of cells. Protein translocation has fundamental importance in biology and medicine. Although the vast majority of proteins are synthesized in the cytoplasm, 20% proteins are located in non-cytoplasmic aqueous spaces and additional 25-30% of the proteins are located within a membrane. Studying protein translocation is critical for understanding signal transduction and regulation pathways in cells and the disease processes that they are involved in. Translocation does not involve change in the protein expression level therefore is undetectable by conventional flow cytometry. Thus high throughput techniques for detecting protein translocation in single cells are in great demand for generating mechanistic insights into a lot of important biological processes. In this project, our overall goal is to develop a new high throughput technique, which we refer to as microfluidic electroporative flow cytometry (EFC), to detect the protein translocation at the single cell level and study the kinetics of translocation processes. Microfluidic EFC combines electroporation (the application of an external electrical field to breach the cell membrane barrier) with flow cytometry. Our preliminary data indicate that the release of a protein from cells into surrounding solution during electroporation is dependent on its subcellular location. By recording the loss in the protein amount due to electroporation for a single cell, we will be able to determine whether translocation occurs. Using this approach we will test two model proteins that are biologically important: a kinase Syk which translocates from the cytoplasm to the plasma membrane and a transcription factor NF-kappaB which transports from the cytoplasm to the nucleus upon stimulation. We will demonstrate detection of these two different types of translocations at the single cell level. Furthermore we will use microfluidic EFC to generate data on the kinetics of these processes under different stimulation conditions. We will provide cross-platform validation of the technology by comparing the results to those obtained using traditional methods such as fractionation/western blotting and confocal fluorescence microscopy. As the integrated educational activities, we will train undergraduate and graduate students in interdisciplinary settings with emphasis on women and underrepresented minority students and disseminate the knowledge to high school students and the general public.