Modulation of Plasma Membrane Composition and Microdomain Organization Impairs Heat Shock Protein Expression in B16-F10 Mouse Melanoma Cells.

Tim Crul,Imre Gombos,Gabor Balogh,Annamaria Marton,Balint Csoboz,Laszlo Vigh,Maria Peter,Lajos Szente,Csaba Vizler

doi:10.3390/cells9040951

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.

Highlights

When exposed to stress, cells induce the heat shock response (HSR), a multi-level signaling network characterized by the accumulation of a conserved set of so-called stress/heat shock proteins (HSPs) [1]
Suggest that altered dynamics of specific cholesterol pools could represent a mechanism to fine tune HSP expression
We aimed to compare the effect of MβCD- and nystatin-induced cholesterol modulations on heat-induced activation of HSP70 and HSP25—both known to be induced upon heat—in mouse B16-F10 melanoma cells

Summary

Introduction

Cells induce the heat shock response (HSR), a multi-level signaling network characterized by the accumulation of a conserved set of so-called stress/heat shock proteins (HSPs) [1]. HSPs recognize and prevent non-native protein conformations from forming deleterious protein aggregates during stress and, once the stressful event passed, assist in refolding or proteasomal degradation, depending on the extent of harmful exposure [2]. Cells 2020, 9, 951 a trimeric conformation, which is able to move into the nucleus while being modulated by multiple posttranslational modifications [4]. Based on this original model, HSP induction was primarily thought to be activated by protein denaturation and aggregation. It is recognized that cells sense heat stress and activate the HSP expression machinery in multiple ways. Exposure to elevated temperatures fluidizes the plasma membrane and alters its physical properties and microdomain organization [5]

Objectives

Methods

Discussion

Conclusion