Single-Cell, Single-Molecule Analysis of TNF-alpha Signaling with Quantum Dots

Unlike traditional molecular biology assays that provide an averaged measurement across millions of cells, single-cell imaging allows analysis of individual cells to assess cell-to-cell heterogeneity underlying averages. Here we apply quantum dots (QDs) as readouts for single-cell molecular biology assays. QDs are fluorescent nanocrystals that are 1000 times brighter and more photostable than organic dyes, and are widely used for single-molecule imaging. We attached QDs to tumor necrosis factor alpha (TNF-α), a cytokine responsible for inflammatory diseases, including rheumatoid arthritis and psoriasis. Binding of TNF-α to the TNF receptor stimulates translocation of the transcription factor NF-kB from the cytoplasm to the nucleus, where it modulates the expression of over 500 genes. The correlation between TNF-α stimulation and NF-kB has not been established. In this study, we measure NF-kB translocation following stimulation with TNF-α bound to QDs, providing an analysis of TNF-α signaling sensitivity at the level of single cells. QDs with a core/shell HgxCd1-xSe/CdyZn1-yS structure were coated with an azide-functional polymer, polyacrylamido(histamine-co-triethyleneglycol-co-azido-triethyleneglycol), and conjugated to TNF-α through a streptavidin (SA) linker. SA was conjugated QDs using copper-free click chemistry by activation of SA with dibenzylcyclooctyne (DBCO) using DBCO-NHS-ester. Azide-functional QDs were mixed with SA-DBCO to yield covalent QD-SA conjugates. QD-SA was then conjugated to biotinylated TNF-α to yield QD-SA-biotin-TNF-α. Specific binding of QD-SA to biotin-TNF-α was verified by mixing at different ratios followed by agarose gel electrophoresis (AGE). HCT116 cells expressing HaloTag® protein fused to the NF-kB p65 subunit were exposed to fluorescent chloroalkane HaloTag ligands (HaloTag Oregon Green® Ligand, Promega) to label the NF-kB for fluorescence microscopy. The number of QDs per cell was quantified from images using the Multiple Target Tracing (MTT) algorithm [1]. Aggregation of the conjugates at biotin-TNF-α : QD-SA ratios of 1:1 and 2:1 were due to crosslinking between biotin-TNF-α and multiple QD-SAs. At ratios of 4:1 and higher, biotins saturated the SA binding sites without cross-linking. No migration shifts occurred when SA was absent or when excess free biotin was added to the reaction mixture, confirming conjugation specificity. QD-SA alone exhibited negligible nonspecific binding to cells (Figure 1b), with < 1 QD per cell at concentrations below 100 nM. We verified that TNF-α induces translocation of NF-kB from the cytosol to the nucleus. QD-SA alone does not induce NF-kB translocation (Figure 1e). The optimal ratio of biotin-TNF-α to QD-SA is 4:1, the minimum amount needed to avoid aggregation due to crosslinking of TNF-α due to multiple biotins per TNF-α and multiple binding sites per QD-SA. QD-SA alone does not induce NF-kB translocation, and QD-SA has very low nonspecific binding across a wide range of concentrations. The Discoveries in Bioimaging REU is funded by the National Science Foundation (EEC 14-61038) with additional support from the University of Illinois at Urbana-Champaign Summer Research Opportunities Program. We thank Pablo Perez-Pinera for generating the transgenic HaloTag-expressing cells. References: [1] Sergé, Arnauld. et al. "Dynamic Multiple-target Tracing to Probe Spatiotemporal Cartography of Cell Membranes." Nat. Meth. 5.8 (2008): 687-94.National Science Foundation (EEC 14-61038)Ope