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.
Intellectual merit We developed and optimized an unique technique, referred to as "electroporative flow cytometry" for studying translocation of intracellular proteins which are tagged by fluorescent protein markers (e.g. EGFP). We found that the release of a protein from cells into surrounding solution during electroporation is dependent on its subcellular location. We tested two important proteins that are critical for cellular functions and development: a kinase Syk which translocated from the cytoplasm to the plasma membrane and a transcription factor NF-kappaB which transported from the cytoplasm to the nucleus upon stimulation. The Syk binding to the surface receptors on the plasma membrane and the deep location of NF-kappaB in the nucleus after translocation prevented the release of the protein due to electroporation. By detecting the loss of the protein due to electroporation at the single cell level using laser-induced fluorescence, we were able to evaluate the activation state of cells (whether translocation occurred) at the single cell level. We also demonstrated a simple protocol for studying nucleocytoplasmic transport based on preferential release using classic flow cytometry combined with immunofluorescence staining. This approach complements electroporative flow cytometry because it allows detection of translocation of native intracellular proteins without tagging by fluorescent protein markers. We demonstrate the proof-of-principle using the translocation of a transcription factor NF-kappaB. It is known that NF-kappaB translocates from the cytosol to the nuclear under stimulation by reagents such as TNF alpha. In our experiment, we permeabilized the plasma membrane using saponin which dissolved its cholesterol content. Such permeabilization preferentially allowed NF-kappaB in the cytosol to release out of the cells while keeping the nuclear fraction of the same protein intact. After fluorescent labeling using an antibody that was specific to NF-kappaB, the fluorescence intensity of cells was screened by a classic flow cytometer and the fluorescence intensity detected can be correlated with the original subcellular localization of the protein. Our methods provide a simple solution to study protein translocation at the single cell level without imaging at high speed. This approach is ideal for studying a large population of cells. With aberrant protein localizations frequently involved in diseases such as cancer and inflammation, we envision that our methods will provide an effective approach for a range of clinical investigations. Broader impacts Our methods are universally applicable to a wide range of cell and protein types. They will find applications in basic molecular biology studies, drug discovery, and patient diagnosis/prognosis. We trained a number of undergraduate and graduate students on this project. We also disseminated the knowledge on microfluidic biotechnology to high school students via organized summer camps at VT campus. A large number of students from underrepresented minorities were involved in our project.
