Abstract
Tumor necrosis factor-alpha (TNF-α) represents a major pro-inflammatory cytokine associated with chronic inflammation. Enhanced understanding of intracellular trafficking of TNF-α enables the identification of drug targets that modulate TNF-α secretion at the cellular source. However, conventional cytokine assays provide only bulk measurements of secreted TNF-α or static measurements in fixed cells and therefore cannot provide mechanistic information. Here, we report a fluorescence imaging platform to visualize and quantify intracellular TNF-α trafficking in human T cells. Using a Jurkat T cell line stably expressing TNF-α fused to mStayGold as a model system, we revealed through single-particle tracking (SPT) that T cell activation increased the motility of TNF-α vesicles and that microtubule perturbation disrupted this increased motility. 3D structured illumination microscopy uncovered spatial associations between TNF-α vesicles and lysosomes, and SPT further demonstrated their coordinated transport. Using total internal reflection fluorescence microscopy, we captured the sub-second splitting of single TNF-α vesicles into multiple puncta that subsequently separated, suggesting delivery of TNF-α from the vesicle to the plasma membrane. Confocal-based super-resolution microscopy further resolved TNF-α vesicles fusing with the plasma membrane and transmembrane TNF-α. Together, these imaging capabilities for tracking and super-resolving the intracellular transport and membrane delivery of TNF-α in live T cells enable mechanistic studies of TNF-α trafficking and release.