Real Time Analysis of System xc- trafficking in U138MG cells
Dr. Leah Chase, Hope College
The membrane transporter, System xc-, catalyzes the exchange of cystine and glutamate across the membrane of many cell types. As such, this transporter plays significant roles in controlling intracellular cysteine levels and ultimately, the synthesis of glutathione, and regulating extracellular levels of glutamate, the primary excitatory neurotransmitter in various areas of the brain. Previous studies in our lab have demonstrated that hydrogen peroxide (whether added exogenously or produced endogenously) regulates the trafficking of System xc- to the plasma in cultured human glioma cells (U138MG) or PC 2 cells, and that this process is responsible for maintenance of glutathione levels under conditions of oxidative stress. The objective of this study was to develop an inherently fluorescent form of System xc- (GFP-xCT chimera), so that we can study the trafficking of the transporter in response to oxidants in real time. The xCT-GFP construct was prepared previously by Matthew Wixson in the lab, and we developed the transfection protocol to express this protein in U138MG cells. Using this technique, we were able to achieve a 10-15% transfection rate. We are currently in the process of developing a stable cell line expressing the GFP-xCT construct and completing the necessary controls to confirm that the chimeric protein traffics to the plasma membrane in response to hydrogen peroxide like the wild-type transporter. Once these controls have been completed, we will begin our real-time analysis of xCT trafficking.
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