Detection of kinase translocation using microfluidic electroporative flow cytometry.
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. This 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.
D015153:Blotting, Western; D018274:Electroporation / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D007425:Intracellular Membranes / Q000378:metabolism; D044085:Microfluidics / Q000379:methods*; D008856:Microscopy, Fluorescence; D010770:Phosphotransferases / Q000032:analysis* / Q000378:metabolism*; D013997:Time Factors
D010770:Phosphotransferases