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

, Liou Bing-Chun

, Lu Long-Sheng

2,3

, Hsu Che-Yu

, Yang Tzu-Sen

Taipei Medical University, Taipei, Taiwan,

Taipei

Medical University Hospital, Taipei, Taiwan,

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.