Instrumentation for probing how oxygen damage alters the properties of neuron cell membranes
Abstract
We propose performing significant modifications of our microscopy infrastructure that will allow us to probe key biophysical properties of cell membranes under the conditions of hyperbaric oxygen (HBO2) toxicity. HBO2 toxicity represents a persistent health problem for Navy divers, leading to acute neurological and pulmonary symptoms and putting divers at great risk. A keybarrier to alleviating this problem is our almost complete lack of understanding regarding its cellular and molecular origins. The PI~s laboratory is studying these origins by probing one major potential mode of HBO2 action: direct damage to the lipid bilayer comprising the plasma membrane of human cells. Major parts of this work include investigations of how HBO2 can alter the mechanical properties of the lipid bilayers and the function of neurotransmitter receptors thatreside in these bilayers. To study these, we use model cell membranes in giant unilamellar lipid vesicles (GUVs). Modifying our microscopy infrastructure with two new functionalities will allow us to probe key properties of both the bilayer and receptors that extend our current studies in valuable directions. To investigate mechanical properties, we will add optical trapping capabilityto our microscope. This will give us the capacity to probe bilayer mechanics and determine exactly how the ability of the cell membrane to bend changes as it oxidizes. Having an optical trapping system in place will allow us to definitively resolve ambiguities in mechanical measurements and resolve important questions about the degree of oxidation of lipid bilayers and their mechanicalstability, pushing us closer faster to a complete understanding of how HBO2 damages cell membranes. Since the ability of the membrane to bend is central to neuronal communication, this study will provide direct insights into molecular oxygen damage and changes in cellular function. We will also add total internal reflection fluorescence (TIRF) capability to our microscopy infrastructure, allowing us to observe directly neurotransmitter receptor clustering andoligomerization. While we have promising preliminary data showing that membrane oxidation changes the function of these important receptor proteins, we have little insight into the mechanism of this change. One likely potential mechanism is that oxidation changes interactions between the lipid bilayer and receptors such that they no longer form multi-protein oligomers in a mannernecessary for proper physiological function. TIRF microscopy will allow us to see the formation of oligomers in membranes of varying oxidation states at a single-molecule level. Both of these new experimental capabilities are important to understanding how neuronal function is altered by oxidative processes, and are therefore essential to understanding the connection between molecularHBO2 damage and the neurobiological symptoms of HBO2 toxicity.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jul 10, 2018
- Source ID
- N000141812288
Entities
People
- Noah Malmstadt
Organizations
- Office of Naval Research
- United States Navy
- University of Southern California