Non-canonical regulation of the plasma membrane copper transporter CTR1 through modulation of membrane mechanical properties

Dash, Tisha (2025) Non-canonical regulation of the plasma membrane copper transporter CTR1 through modulation of membrane mechanical properties. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS Dissertation of Tisha Dash (20MS111)) 20MS111_Thesis_file.pdf - Submitted Version Restricted to Repository staff only Download (8MB)

Abstract

Copper homeostasis in mammalian cells is tightly regulated by the high-affinity trimeric transporter CTR1, which internalizes via endocytosis under elevated extracellular copper conditions to limit excess uptake. In this study, we uncover a non-canonical, receptor-like regulatory mechanism wherein human CTR1 undergoes ligand-induced clustering at the plasma membrane prior to its endocytosis. Using live-cell TIRF and IRM imaging in HEK293T cells, complemented by coarse-grained molecular dynamics simulations, we demonstrate that CTR1 clustering leads to localized increases in membrane tension, reduced membrane fluctuation, and the induction of positive curvature - biophysical changes that facilitate endocytic pit formation. This clustering is mediated by the methionine-rich extracellular N-terminus of CTR1 and is enhanced by membrane cholesterol, whose depletion impairs the internalization process. We further show that CTR1 clustering precedes the recruitment of the adaptor protein AP-2, linking it to Clathrin-mediated endocytosis. Together, our findings reveal a previously unrecognized pre-endocytic regulatory mechanism, positioning CTR1 as a metal channel that mimics membrane receptor behaviour to orchestrate its own trafficking and maintain copper balance.

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