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Modulation of Plasma Membrane Composition and Microdomain Organization Impairs Heat Shock Protein Expression in
Tim Crul1, Balint Csoboz1,2, Imre Gombos1
1Institute of Biochemistry, Biological Research Centre, Szeged 6726, Hungary.
Abstract:
The heat shock response (HSR) regulates induction of stress/heat shock proteins (HSPs) to preserve proteostasis during cellular stress. Earlier, our group established that the plasma membrane (PM) acts as a sensor and regulator of HSR through changes in its microdomain organization. PM microdomains such as lipid rafts, dynamic nanoscale assemblies enriched in cholesterol and sphingomyelin, and caveolae, cholesterol-rich PM invaginations, constitute clustering platforms for proteins functional in signaling cascades. Here, we aimed to compare the effect of cyclodextrin (MβCD)- and nystatin-induced cholesterol modulations on stress-activated expression of the representative HSPs, HSP70, and HSP25 in mouse B16-F10 melanoma cells. Depletion of cholesterol levels with MβCD impaired the heat-inducibility of both HSP70 and HSP25. Sequestration of cholesterol with nystatin impaired the heat-inducibility of HSP25 but not of HSP70. Imaging fluorescent correlation spectroscopy marked a modulated lateral diffusion constant of fluorescently labelled cholesterol in PM during cholesterol deprived conditions. Lipidomics analysis upon MβCD treatment revealed, next to cholesterol reductions, decreased lysophosphatidylcholine and phosphatidic acid levels. These data not only highlight the involvement of PM integrity in HSR but also suggest that altered dynamics of specific cholesterol pools could represent a mechanism to fine tune HSP expression.
Insights
Cholesterol modulation in the plasma membrane impacts heat shock protein (HSP) expression. Altered cholesterol dynamics in cellular membranes fine-tune the heat shock response (HSR).
Area of Science:
- Cellular Biology
- Membrane Biophysics
- Stress Response
Background:
- The heat shock response (HSR) is crucial for maintaining proteostasis under cellular stress.
- Plasma membrane (PM) microdomains, including lipid rafts and caveolae, are key regulators of HSR.
- These microdomains organize proteins involved in cellular signaling cascades.
Purpose of the Study:
- To investigate the impact of cholesterol modulation on the stress-induced expression of HSP70 and HSP25.
- To compare the effects of MβCD and nystatin on cholesterol levels and HSR in B16-F10 melanoma cells.
Main Methods:
- Cholesterol depletion using methyl-β-cyclodextrin (MβCD) and sequestration with nystatin.
- Assessment of heat shock protein (HSP70 and HSP25) expression.
- Fluorescent correlation spectroscopy to analyze cholesterol diffusion in the PM.
- Lipidomics analysis to determine changes in membrane lipid composition.
Main Results:
- MβCD-induced cholesterol depletion impaired heat-inducibility of both HSP70 and HSP25.
- Nystatin-induced cholesterol sequestration affected HSP25 but not HSP70 heat-inducibility.
- Cholesterol-deprived conditions altered cholesterol lateral diffusion in the PM.
- MβCD treatment reduced lysophosphatidylcholine and phosphatidic acid levels.
Conclusions:
- Plasma membrane integrity plays a significant role in the HSR.
- Specific cholesterol pool dynamics may act as a regulatory mechanism for HSP expression.
- Understanding PM cholesterol's role in HSR offers insights into cellular stress adaptation.
