A stochastic model for copper-induced ATP7B trafficking in polarized epithelial cells

Saha, Baivab Kumar (2025) A stochastic model for copper-induced ATP7B trafficking in polarized epithelial cells. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS Dissertation of Baivab Kumar Saha (20MS035)) 20MS035_Thesis_file.pdf - Submitted Version Restricted to Repository staff only Download (3MB)

Abstract

Copper is a crucial micronutrient in several biochemical reactions that are important for cellular functioning and metabolic regulation. ATP7B, a copper-transporting P-type ATPase, is important in ensuring cellular copper homeostasis. In basal copper conditions, ATP7B is resident in specific domains of the trans-Golgi network (TGN), where it provides copper to different cuproenzymes in TGN lumen. However, upon elevated intracellular copper levels, ATP7B is redistributed to endosomal/lysosomal compartments and the apical plasma membrane, mediating copper excretion. In polarized MDCK cells, ATP7B traffics to the cell surface via endosomal compartments, including common recycling endosomes (CREs), apical recycling endosomes (AREs), and apical sorting endosomes (ASEs), before releasing excess copper at the apical plasma membrane. Despite significant advancements in comprehending ATP7B's role in copper transport, the precise pathways, timing, and dynamics of its trafficking, particularly its interactions with endosomal populations and the influence of cytoskeletal elements, remain unclear. Furthermore, the impact of fluctuating copper levels on the speed and directionality of ATP7B's movement has not been quantitatively assessed. This study addresses these gaps by mapping the spatiotemporal dynamics of ATP7B in polarized MDCK cells under varying copper concentrations. Through image analysis-based quantification, the distances between key subcellular compartments were measured: TGN to CRE (~0.2 μm), CRE to ARE (~0.5 μm), ARE to ASE (~1.2 μm), and ASE to the apical plasma membrane (~0.1 μm). Additionally, the colocalization of ATP7B with these compartments was assessed at multiple time points following 25 μM CuCl2 treatment. Notably, maximum colocalization of ATP7B with both ARE and the apical plasma membrane was observed 15 minutes post-treatment, indicating a rapid trafficking response under copper-loaded conditions. The colocalization dynamics with CRE and ASE remain to be fully characterized. These findings allow us to generate a spatiotemporal distribution map of ATP7B in polarized MDCK cells, providing new insights into its trafficking itinerary under different copper concentrations. Moreover, the quantitative measurements obtained form the basis for a stochastic mathematical model built to predict ATP7B localization with defined confidence intervals based on copper levels. This work not only enhances our understanding of ATP7B-mediated copper export in polarized epithelia but also establishes a framework for predicting protein trafficking behavior in live cell systems.

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