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Topic

Investigating macropinocytosis as the mechanism of ATP-induced PANX1 internalization in Neuro2a cells

Division of Medical Sciences

Date & location

• Tuesday, December 17, 2024
• 2:30 P.M.
• Medical Sciences Building, Room 150

Examining Committee

Supervisory Committee

• Dr. Leigh Anne Swayne, Division of Medical Sciences, University of Victoria (Supervisor)
• Dr. Hector Caruncho, Division of Medical Sciences, UVic (Member)
• Dr. Lisa Reynolds, Department of Biochemistry and Microbiology, UVic (Outside Member)

External Examiner

• Dr. Patrick Nahirney, Division of Medical Sciences, UVic

Chair of Oral Examination

• Dr. Stephen Tuffs, Department of Biochemistry and Microbiology, UVic

Abstract

The widely expressed pannexin 1 (PANX1) ion and metabolite channel, facilitates the release of ATP release for downstream signaling, and in turn, is regulated by extracellular ATP concentrations. This thesis focuses on the mechanisms underlying ATP-induced PANX1 internalization. Our previous work demonstrated that PANX1 inhibits neuronal differentiation and neurite outgrowth (Wicki-Stordeur & Swayne, 2013) partly through the sequestration of key cytoskeletal proteins (Xu et al., 2018). PANX1 levels peak in the neonatal mouse brain and drop precipitously between the first and second weeks of life, coinciding with peak synapse stabilization (Sanchez-Arias et al., 2019). These findings suggest that the developmental downregulation of PANX1 levels contributes to proper neuronal development, but the mechanisms regulating PANX1 trafficking in neural cells are still relatively unknown (reviewed in Frederiksen et al., 2023). Our prior work suggests that extracellular ATP concentrations regulate PANX1 surface expression in the murine Neuro-2a (N2a) neuroblastoma cell line (Boyce et al., 2015; Boyce & Swayne, 2017). Our recent findings point towards ATP-induced macropinocytosis (‘cell drinking’) of PANX1, as treatment with the macropinocytosis-inhibitor amiloride prevents ATP-induced PANX1 internalization (Boyce et al., 2020); however further experiments are needed to confirm this finding. My thesis investigates the hypothesis that ATPinduced PANX1 internalization occurs through macropinocytosis. I used the N2a cell line, which allows for high transfection efficiency, well-controlled and reproducible culture conditions, and large cytoplasmic volumes ideal for imaging subcellular compartments (Cibelli et al., 2022). Previous work from the lab showed that ATP-dependent macropinocytosis leads to an increase in cell size, which is disrupted by the PANX1 W74A (ATP-insensitive) mutation (Boyce et al., 2020). By modifying extracellular ATP, I quantified macropinocytosis using the uptake of fluorescent dextran (≥70 kDa) (Commisso et al., 2014) and super-resolution confocal/Stimulated Emission-Depletion (STED) microscopy to measure the size of dextran-containing vesicles. In these experiments, I determined a significant proportion of total PANX1-EGFP localized to dextran-positive vesicles, suggesting that PANX1 is internalized via macropinocytosis in response to extracellular ATP. Additionally, I tested several macropinocytosis inhibitors and observed a significant decrease in the co-localization rate of PANX1-EGFP and dextran. The diameter of PANX1-EGFP/dextran co-positive vesicles was consistent with macropinosome size (0.2 to 8 μm; Swanson & Yoshida, 2019). These results further validate that the endocytosis mechanism involved is macropinocytosis. The outcomes of this work will enhance our understanding of neuronal PANX1 trafficking, with implications for neurodevelopment and disease.