Date of Award

Spring 5-9-2025

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Physics

First Committee Advisor

Samuel T. Hess

Second Committee Member

R. Dean Astumian

Third Committee Member

Neil F. Comins

Additional Committee Members

Julie A. Gosse

Karissa Tilbury

Abstract

Quantification of the nanoscale properties of molecules has pushed the frontier of biological discovery in fields such as virology and toxicology. We are able to use super-resolution microscopy techniques such as fluorescence photoactivation localization microscopy (FPALM) to break the diffraction limits of light and see these processes in live cells and use molecular dynamics (MD) simulations to quantify the mechanisms of these nanoscale interactions.

The components of influenza A virus (IAV), and in particular the spike protein Hemagglutinin (HA), are one such example of molecules which interact with host cell components on a nanoscale. HA, previously seen to interact with phosphatidylinositol 4,5-bisphoaphate (PIP2), is found to cluster at the plasma membrane (PM) before the arrival of PIP2 in some instances. Furthermore, we found that these molecules are not clustered together in all regions, particularly in regions with the highest HA density. Looking for other potential binding partners, we find that HA interacts with phosphatidylinositol 4-phosphate (PI4P) through an electrostatic interaction, and both HA and PI4P are found together from the Golgi to the PM of NIH-3T3 cells transfected with labeled forms of the two species. These interactions appear stronger than those between PIP2 and HA both through MD simulations and through inhibition of the PI4P → PIP2 conversion enzyme, PIP5K, which increased HA cluster density.

Interactions between HA and PIP2 have also been previously seen to be disrupted by low micromolar doses of a common chemical in consumer products, Cetylpyridinium chloride (CPC), studied for potential electrostatic interactions between positively charged CPC and negatively charged PIP2. The ability to disrupt interactions with PIP2, particularly at these non-cytotoxic and exposure relevant doses, raises the question of the effects it has on our cells. Here, we find that PIP2 clusters in RBL-2H3 mast cells, clustering which is disrupted by pretreatment with 5 µM CPC. Furthermore, antigen stimulation of these cells demonstrates a functional change in PIP2 clustering; however this change is inhibited by pretreatment with 5 µM CPC. Beyond simply affecting clustering patterns, the binding protein used to label PIP2 is seen to have increased mobility following 5 µM CPC pretreatment, suggesting that CPC disrupts the binding between the probe and PIP2. This disruption of PIP2 interactions is a likely candidate contributing to inhibition of mast cell degranulation, a key immune response which is also disrupted by CPC.

CPC does not only have effects on immune cell function, however. Primary human keratinocytes and RBL-2H3 cells demonstrated dampened O2 consumption rates following treatment with low micromolar CPC. Furthermore, low µM CPC is seen to have drastic effects on mitochondrial function. Low µM CPC treatments are seen to potently inhibit mitochondrial production of ATP and result in lower mitochondrial Ca2+ levels. We observe that CPC disrupts mitochondrial networks at these dosages in NIH-3T3 cells, causing the networks to break apart and the individual mitochondria to fission into small spheres. Overall, low µM CPC causes a variety of toxicological effects on our cells at levels similar to estimated human exposure.

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Available for download on Wednesday, June 16, 2027

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