Regulation of Tumor Necrosis Factor Receptor-1 Endocytosis

Fernandez, Diana Denzil (2025) Regulation of Tumor Necrosis Factor Receptor-1 Endocytosis. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS Dissertation of Diana Denzil Fernandez (20MS211)) 20MS211_Thesis_file.pdf - Submitted Version Restricted to Repository staff only Download (9MB)

Abstract

Tumor necrosis factor receptor 1 (TNFR1) is a key mediator of TNF-α signalling, regulating apoptosis, inflammation, and immune responses. TNFR1 plays a critical role in mediating diverse cellular responses, including inflammation, survival, and apoptosis, through its activation by TNF-α. A key component of TNFR1 function is its trafficking within the cell, particularly its internalization via endocytosis, which has been closely associated with apoptotic signalling. While the importance of ligand-induced endocytosis is recognized, the behavior and regulation of basal (ligand-independent) endocytosis of TNFR1 remain less understood. In this study, I investigate the basal internalization of endogenous TNFR1 and its regulation under various biochemical and mechanical stress conditions using a combination of high-resolution imaging methods, including TIRF and STED. Initial imaging studies revealed that TNFR1 undergoes basal endocytosis even in unstimulated conditions, with low but detectable colocalization with Rab5 and transferrin-positive compartments, suggesting partial engagement with early endocytic machinery. Upon TNF-α stimulation, TNFR1 clustering increases and is accompanied by a marked enhancement in colocalization with transferrin, a marker of clathrin-mediated endocytosis (CME), and to a lesser extent with Rab5, a marker of early endosomes. This indicates that TNF-α signalling activates surface clustering and drives efficient entry into the CME pathway. In contrast, Zafirlukast, a TNFR1 signalling inhibitor, significantly reduces clustering and colocalization, implying that receptor internalization strongly depends on signalling activation. IFN-γ pre-treatment, which primes cells for apoptosis, leads to a substantial reduction in TNFR1–Rab5 colocalization, suggesting that membrane tension or altered cytoskeletal dynamics restrict internalization or reroute trafficking away from canonical Rab5 compartments. Interestingly, the combined treatment of IFN-γ and TNF-α, which induces apoptosis, shows a similar but slightly less severe reduction in Rab5 colocalization, possibly reflecting residual internalization necessary for apoptotic complex formation. Mechanical stress induced by hypo-osmotic shock results in significant s

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