Author ORCID Identifier

0000-0003-2719-6309

Date of Graduation

8-2022

Document Type

Dissertation (PhD)

Program Affiliation

Biochemistry and Molecular Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Ilya Levental, PhD

Committee Member

Vasanthi Jayaraman, PhD

Committee Member

Guangwei Du, PhD

Committee Member

Alemayehu Gorfe, PhD

Abstract

In the 1970s, it was established that phospholipid (PL) classes are asymmetrically distributed across the (PM) leaflets. Recapitulating strategies coupled with newly developed, high-resolution shotgun lipidomics, our lab confirmed PL class asymmetry and surprisingly measured the endoplasmic leaflet to contain double the number of PLs vs. the exoplasmic leaflet. My re-analysis of classical studies show that PM PL abundance imbalance has been a consistent finding across decades of literature. This is surprising, because bilayers do not tolerate interleaflet area imbalances to the degree that this imbalance would suggest, which prompted us to consider the existence of an elusive membrane resident with opposing asymmetry. Cholesterol, the most abundant PM lipid, has wildly conflicting reports of PM distribution, likely due to its unique structure that allows for rapid interleaflet flipping. We hypothesize that the PM exoplasmic leaflet is highly enriched in cholesterol to oppose its relative depletion of PLs, yielding unique biophysical properties of PM at steady state. To experimentally assess transbilayer cholesterol distribution and confirm PL abundance imbalance, we used a combination of in vitro, in vivo and in silico experiments. To investigate PM biophysical properties, we measure differences in PM permeability and leaflet-specific lipid packing during scrambling (loss of asymmetry) to reveal the consequential biophysical properties of the steady state PM bolster PM fundamental function. Together, our observations suggest the steady state exoplasmic leaflet to be enriched in cholesterol and contain fewer PLs than the endoplasmic leaflet. Further, the consequential biophysical properties of this PM configuration bolster its fundamental function as a signal transducer and structural barrier. Finally, this prompts an update to our current model of PM lipid and biophysical asymmetry—a lipid asymmetry which is fundamental to PM steady state function and thereby, fundamental to cellular life.

Keywords

membrane asymmetry, lipidomics, cholesterol, phospholipid, lipid packing, permeability, area per lipid, erythrocytes

Available for download on Thursday, August 10, 2023

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