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(623h) Nucleocytoplasmic Translocation of Proteins Detected By a Novel Flow Cytometry Assay Without Imaging

Authors: 
Cao, Z., Virginia Tech
Lu, C., Virginia Tech



Nucleocytoplasmic transport (the protein translocation between the cytosol and the nucleus) is often regulated via upstream signal transduction pathways that lead to protein modifications.  As expected, alterations in any of the regulatory steps can result in the mislocalization of a protein that may lead to cancers. [1] Most traditional research on nucleocytoplasmic translocation focuses on population-average assays (e.g. immunoblotting), which mask the distinct responses by cellular subpopulations. Recent studies with live cell imaging can achieve single cell resolution and track signal dynamics for a period of time with fused fluorescent protein markers. However, intracellular signaling can be changed by the fusion of fluorescent protein markers [2]. Furthermore, the low number of cells interrogated by imaging-based analysis does efficiently reflect population statistics [3, 4]. In contrast, flow cytometry provides both single cell resolution and high throughput. However, traditional protocol with flow cytometry lacks subcellular resolution (i.e. only the fluorescence from the entire cell is quantified) and does not detect nuclear translocation [3].

In this work, we demonstrate a novel and simple protocol for studying nucleocytoplasmic transport based on preferential release using classic flow cytometry combined with immunofluorescence staining. We demonstrate the proof-of-principle using the translocation of a transcription factor NF-κB. It is known that NF-κB translocates from the cytosol to the nuclear under stimulation by reagents such as TNF a. In our experiment, we permeabilized the plasma membrane using saponin which dissolved its cholesterol content. Such permeabilization preferentially allowed NF-κB 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-κB, 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 method provides 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 predominant localization of NF-κB being found in breast, ovary, colon, pancreas and thyroid tumor cells5], we envision that our method will provide an effective approach for a range of clinical investigations.