T cell activation is initiated when ligand binding to the T cell receptor (TCR) triggers intracellular phosphorylation of the TCR-CD3 complex. However, it remains unknown how biophysical properties of TCR engagement result in biochemical phosphorylation events. Here, we constructed an optogenetic tool that induces spatial clustering of ζ-chain in a light controlled manner. We showed that spatial clustering of the ζ-chain intracellular tail alone was sufficient to initialize T cell triggering including phosphorylation of ζ-chain, Zap70, PLCγ, ERK and initiated Ca²⁺ flux. In reconstituted COS-7 cells, only Lck expression was required to initiate ζ-chain phosphorylation upon ζ-chain clustering, which leads to the recruitment of tandem SH2 domain of Zap70 from cell cytosol to the newly formed ζ-chain clusters at the plasma membrane. Taken together, our data demonstrated the biophysical relevance of receptor clustering in TCR signaling.

Signal transducer and activator of transcription 3 (STAT3) signaling is a major driver of colorectal cancer (CRC) growth, however therapeutics, which can effectively target this pathway, have so far remained elusive. Here, we performed an extensive screen for STAT3 inhibitors among a library of 1167 FDA-approved agents, identifying Ponatinib as a lead candidate. We found that Ponatinib inhibits STAT3 activity driven by EGF/EGFR, IL-6/IL-6R and IL-11/IL-11R, three major ligand/receptor systems involved in CRC development and progression. Ponatinib was able to inhibit CRC migration and tumor growth in vivo. In addition, Ponatinib displayed a greater ability to inhibit STAT3 activity and mediated superior anti-proliferative efficacy compared to five FDA approved SRC and Janus Kinase (JAK) inhibitors. Finally, long-term exposure of CRC cells to Ponatinib, Dasatinib and Bosutinib resulted in acquired resistance to Dasatinib and Bosutinib occurring within six weeks. However, acquired resistance to Ponatinib was observed after long-term exposure of >4 months. Overall, our results identify a novel anti-STAT3 property of Ponatinib and thus, Ponatinib offers a potential therapeutic strategy for CRC.

A DNA double-strand break (DSB) takes place in the context of chromatin, and there is increasing evidence for chromatin structure to play a functional role in DSB signaling and repair. Thus, there is an emerging need for quantitative microscopy methods that can directly measure chromatin network architecture and detect changes in this structural framework upon DSB induction within an intact nucleus. To address this demand, here we present the phasor approach to fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) between fluorescently labeled histones in the DSB inducible via AsiSI cell system (DIvA), which has sufficient spatial resolution to map nuclear-wide chromatin compaction at the level of nucleosome proximity with respect to multiple site-specific DSBs. We also demonstrate that when phasor histone FLIM-FRET is coupled with immunofluorescence, this technology has the unique advantage of enabling exploration of any heterogeneity that exists in chromatin structure at the spatially distinct and genetically induced DSBs.

Linker for activation of T cells (LAT) is an essential adaptor protein in early T cell receptor (TCR) signaling that propagates multiple signaling pathways. However, how LAT spatial organization facilitates signal initiation and propagation after TCR triggering is not clear. To differentiate de novo assembly in the plasma membrane from pre-existing LAT vesicles and clusters, we developed imaging protocols and analyses to capture the organization and dynamics of single LAT molecules immediately after TCR engagement. We could observe individual LAT molecules in the plasma membrane that assembled into immobile signaling entities requiring LAT phosphorylation. This step-wise assembly process was temporally highly coordinated via the zeta-chain-associated protein kinase 70 (Zap70)-LAT-growth factor receptor-bound protein 2 (Grb2) pathway. While multiple spatial organization co-existed even within the plasma membrane, our data suggest that de novo plasma membrane assemblies facilitated signal propagation.