Study of Protein Translocation Using Microfluidic Electroporative Flow Cytometry

Virginia Polytechnic Institute And State University, Blacksburg VA

Investigators

Abstract

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.