# Biophysical Mechanisms of Cholesterol Homeostasis

> **NIH NIH R01** · RUSH UNIVERSITY MEDICAL CENTER · 2021 · $353,972

## Abstract

Abstract
Understanding mechanisms cells use to maintain cholesterol homeostasis are critical in cell biology and many
diseases. To achieve this, the chemical activity of cholesterol in cell plasma membranes must be measured
because activity controls cholesterol’s effects on cellular processes. To date, plasma membrane cholesterol
concentration has been used to quantify cholesterol activity. But the activity of cholesterol is determined by its
chemical potential; concentration contributes to, but does not accurately reflect membrane activity. Because a
method to measure cholesterol chemical potential had not been available, it was not possible to properly
evaluate many of cholesterol’s effects, including those on cellular signaling. We have now developed methods
to do so. These methods and a new perfusion fluorimetry apparatus we have devised allow us to follow the
chemical potential of cholesterol of plasma membranes in real time. We have discovered that cells quickly
respond to changes in extracellular cholesterol by adjusting the cholesterol chemical potential of their plasma
membranes without changing the total content of cellular cholesterol. This finding reveals a previously unknown
mechanism to maintain cholesterol homeostasis: quick adjustment of plasma membrane chemical potentials
to control cholesterol influx and efflux. We have identified protein scaffolded domains, as typified by caveolae,
as sites at which cells sense and rapidly respond to external cholesterol. The abundance and total amount of
cholesterol that resides in caveolae are determined by the extent of phosphorylation at position Ser80 of
caveolin-1, the foundational protein of the domain. The shuttling of cholesterol between scaffolded domains
and the surround which must result upon Ser80 phosphorylation alters cholesterol chemical potential. We
therefore hypothesize that signaling cascades initiated within scaffolded domains are responsible for
maintaining cholesterol homeostasis when cells are subjected to changes in external cholesterol and to growth
factors. We further posit that these activated signaling cascades feed back to the plasma membrane to maintain
chemical potentials. Cells will be stimulated with growth factors and relevant signaling cascades will be
identified. The abundance of caveolae will be assessed by measuring the FRET (fluorescence resonance energy
transfer) signals between caveolins. Our preliminary evidence strongly implicates that growth factors and/or
changes in the level of external cholesterol stimulate the PI3K/Akt/mTOR signaling pathway that feeds back to
achieve cholesterol homeostasis. Optogenetic techniques will be used to determine whether it and/or others are
indeed responsible for control of cholesterol. Parallel experiments using the same strategies will determine if
flotillins, analogous to caveolin, also serve as sensors/regulators of cholesterol chemical potentials.

## Key facts

- **NIH application ID:** 10117604
- **Project number:** 1R01GM136777-01A1
- **Recipient organization:** RUSH UNIVERSITY MEDICAL CENTER
- **Principal Investigator:** FREDRIC S COHEN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $353,972
- **Award type:** 1
- **Project period:** 2021-08-01 → 2025-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10117604

## Citation

> US National Institutes of Health, RePORTER application 10117604, Biophysical Mechanisms of Cholesterol Homeostasis (1R01GM136777-01A1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10117604. Licensed CC0.

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