Single-Cell Biophysics: Measurement, Modulation, and Modeling

Single-Cell Biophysics: Measurement, Modulation, and Modeling

Poster Abstracts

93-POS Board 47 Real-Time Probing the Binding of Tumor Necrosis Factor-alpha (TNF-α) to TNF Receptor in Single Living Cell Using Biofuntionalized Quantum Dots Que Wan-Shan 1 , Liou Bing-Chun 2 , Lu Long-Sheng 2,3 , Hsu Che-Yu 4 , Yang Tzu-Sen 2 . 2 Taipei Medical University, Taipei, Taiwan, 1 Taipei Medical University, Taipei, Taiwan, 3 Taipei Medical University Hospital, Taipei, Taiwan, 4 National Taiwan University Hospital, Taipei, Taiwan. Tumor necrosis factor (TNF-α) is an inflammatory cytokine produced mainly by macrophages. Binding of TNF-α to TNF receptor (TNFR) activates nuclear factor kappa B (NF-kB) transcription factors, leading to oscillations in nuclear NF-kB and target gene expression by negative feedback loops. However, it is difficult to probe real-time binding of TNF-α to TNFR because TNF-α needs its specific membrane-derived receptors to form a trimeric complex. To this end, we have developed a platform in which the temperature-controlled microfluidic flow cell is incorporated into single-molecule fluorescence microscopy systems. In addition, we utilize the biofunctionalized quntum dots conjugated with TNF-α to monitor the cellular distribution of TNF-α, which can provide a more direct access to probing the spatio-temporal distribution of TNF-α- TNFR complex and the corresponding transport pathway. Furthermore, we applied biotin anti-human TNF-α antibody to bind with TNF-α followed by chemical bonding for streptavidin- quantum dots or labeled biotin-TNF-α with streptavidin conjugated quantum dots to identify the space structure between TNF-α and TNFR. The observation demonstrated that antibody-functionalized quntum dots are more adequate for live monitoring the binding of TNF-α to TNFR in living NIH-3T3 cells, which implied that the choice of marker material may affect the success rate that TNF-α binding to its receptor in space. Furthermore, we also established an optimized method for labeling TNF-α, it can help us to explore its signal transduction pathway and the spatiotemporal interaction between binding of TNF-α to TNFR and the corresponding NF-kB dynamics.

91