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bioRxiv · 10.64898/2026.10.07.757379

Direct visualization of lysosome-mediated intracellular transport and membrane anchoring of TNF-α in human T cells

Burgess, A.·Gunasekara, H.·Xie, J.·Hu, Y. S.
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Abstract

Tumor necrosis factor-alpha (TNF-) represents a major pro-inflammatory cytokine associated with chronic inflammation. Enhanced understanding of intracellular trafficking of TNF- enables the identification of drug targets that modulate TNF- secretion at the cellular source. However, conventional cytokine assays provide only bulk measurements of secreted TNF- 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- trafficking in human T cells. Using a Jurkat T cell line stably expressing TNF- fused to mStayGold as a model system, we revealed through single-particle tracking (SPT) that T cell activation increased the motility of TNF- vesicles and that microtubule perturbation disrupted this increased motility. 3D structured illumination microscopy uncovered spatial associations between TNF- 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- vesicles into multiple puncta that subsequently separated, suggesting delivery of TNF- from the vesicle to the plasma membrane. Confocal-based super-resolution microscopy further resolved TNF- vesicles fusing with the plasma membrane and transmembrane TNF-. Together, these imaging capabilities for tracking and super-resolving the intracellular transport and membrane delivery of TNF- in live T cells enable mechanistic studies of TNF- trafficking and release.

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Burgess, A., Gunasekara, H., Xie, J., Hu, Y. S.. 2026-10-09. Direct visualization of lysosome-mediated intracellular transport and membrane anchoring of TNF-α in human T cells. https://doi.org/10.64898/2026.10.07.757379

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