Distinct Cellular Tools of Mild Hyperthermia-Induced Acquired Stress Tolerance in Chinese Hamster Ovary Cells.

Mild stress could help cells to survive more severe environmental or pathophysiological conditions. In the current study, we investigated the cellular mechanisms which contribute to the development of stress tolerance upon a prolonged (0-12 h) fever-like (40 °C) or a moderate (42.5 °C) hyperthermia in mammalian Chinese Hamster Ovary (CHO) cells. Our results indicate that mild heat triggers a distinct, dose-dependent remodeling of the cellular lipidome followed by the expression of heat shock proteins only at higher heat dosages. A significant elevation in the relative concentration of saturated membrane lipid species and specific lysophosphatidylinositol and sphingolipid species suggests prompt membrane microdomain reorganization and an overall membrane rigidification in response to the fluidizing heat in a time-dependent manner. RNAseq experiments reveal that mild heat initiates endoplasmic reticulum stress-related signaling cascades resulting in lipid rearrangement and ultimately in an elevated resistance against membrane fluidization by benzyl alcohol. To protect cells against lethal, protein-denaturing high temperatures, the classical heat shock protein response was required. The different layers of stress response elicited by different heat dosages highlight the capability of cells to utilize multiple tools to gain resistance against or to survive lethal stress conditions.

Mild heat induces a distinct "eustress" response in Chinese Hamster Ovary cells but does not induce heat shock protein synthesis.

The current research on cellular heat stress management focuses on the roles of heat shock proteins (HSPs) and the proteostasis network under severe stress conditions. The mild, fever-type stress and the maintenance of membrane homeostasis are less well understood. Herein, we characterized the acute effect of mild, fever-range heat shock on membrane organization, and HSP synthesis and localization in two mammalian cell lines, to delineate the role of membranes in the sensing and adaptation to heat. A multidisciplinary approach combining ultrasensitive fluorescence microscopy and lipidomics revealed the molecular details of novel cellular "eustress", when cells adapt to mild heat by maintaining membrane homeostasis, activating lipid remodeling, and redistributing chaperone proteins. Notably, this leads to acquired thermotolerance in the complete absence of the induction of HSPs. At higher temperatures, additional defense mechanisms are activated, including elevated expression of molecular chaperones, contributing to an extended stress memory and acquired thermotolerance.

Oxidized Phospholipids Inhibit the Formation of Cholesterol-Dependent Plasma Membrane Nanoplatforms.

We previously developed a single-molecule microscopy method termed TOCCSL (thinning out clusters while conserving stoichiometry of labeling), which allows for direct imaging of stable nanoscopic platforms with raft-like properties diffusing in the plasma membrane. As a consensus raft marker, we chose monomeric GFP linked via a glycosylphosphatidylinositol (GPI) anchor to the cell membrane (mGFP-GPI). With this probe, we previously observed cholesterol-dependent homo-association to nanoplatforms diffusing in the plasma membrane of live CHO cells. Here, we report the release of this homo-association upon addition of 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) or 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine, two oxidized phospholipids (oxPLs) that are typically present in oxidatively modified low-density lipoprotein. We found a dose-response relationship for mGFP-GPI nanoplatform disintegration upon addition of POVPC, correlating with the signal of the apoptosis marker Annexin V-Cy3. Similar concentrations of lysolipid showed no effect, indicating that the observed phenomena were not linked to properties of the lipid bilayer itself. Inhibition of acid sphingomyelinase by NB-19 before addition of POVPC completely abolished nanoplatform disintegration by oxPLs. In conclusion, we were able to determine how oxidized lipid species disrupt mGFP-GPI nanoplatforms in the plasma membrane. Our results favor an indirect mechanism involving acid sphingomyelinase activity rather than a direct interaction of oxPLs with nanoplatform constituents.

Plasma membranes as heat stress sensors: from lipid-controlled molecular switches to therapeutic applications.

The classic heat shock (stress) response (HSR) was originally attributed to protein denaturation. However, heat shock protein (Hsp) induction occurs in many circumstances where no protein denaturation is observed. Recently considerable evidence has been accumulated to the favor of the "Membrane Sensor Hypothesis" which predicts that the level of Hsps can be changed as a result of alterations to the plasma membrane. This is especially pertinent to mild heat shock, such as occurs in fever. In this condition the sensitivity of many transient receptor potential (TRP) channels is particularly notable. Small temperature stresses can modulate TRP gating significantly and this is influenced by lipids. In addition, stress hormones often modify plasma membrane structure and function and thus initiate a cascade of events, which may affect HSR. The major transactivator heat shock factor-1 integrates the signals originating from the plasma membrane and orchestrates the expression of individual heat shock genes. We describe how these observations can be tested at the molecular level, for example, with the use of membrane perturbers and through computational calculations. An important fact which now starts to be addressed is that membranes are not homogeneous nor do all cells react identically. Lipidomics and cell profiling are beginning to address the above two points. Finally, we observe that a deregulated HSR is found in a large number of important diseases where more detailed knowledge of the molecular mechanisms involved may offer timely opportunities for clinical interventions and new, innovative drug treatments. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.

Combination of small molecule microarray and confocal microscopy techniques for live cell staining fluorescent dye discovery.

Discovering new fluorochromes is significantly advanced by high-throughput screening (HTS) methods. In the present study a combination of small molecule microarray (SMM) prescreening and confocal laser scanning microscopy (CLSM) was developed in order to discover novel cell staining fluorescent dyes. Compounds with high native fluorescence were selected from a 14,585-member library and further tested on living cells under the microscope. Eleven compartment-specific, cell-permeable (or plasma membrane-targeted) fluorochromes were identified. Their cytotoxicity was tested and found that between 1-10 micromolar range, they were non-toxic even during long-term incubations.

Glycerophospholipid profile in oncogene-induced senescence.

Alterations in lipid metabolism and in the lipid composition of cellular membranes are linked to the pathology of numerous diseases including cancer. However, the influence of oncogene expression on cellular lipid profile is currently unknown. In this work we analyzed changes in lipid profiles that are induced in the course of ERBB2-expression mediated premature senescence. As a model system we used MCF-7 breast cancer cells with doxycycline-inducible expression of NeuT, an oncogenic ERBB2 variant. Affymetrix gene array data showed NeuT-induced alterations in the transcription of many enzymes involved in lipid metabolism, several of which (ACSL3, CHPT1, PLD1, LIPG, MGLL, LDL and NPC1) could be confirmed by quantitative realtime PCR. A study of the glycerophospholipid and lyso-glycerophospholipid profiles, obtained by high performance liquid chromatography coupled to Fourier-transform ion cyclotron resonance-mass spectrometry revealed senescence-associated changes in numerous lipid species, including mitochondrial lipids. The most prominent changes were found in PG(34:1), PG(36:1) (increased) and LPE(18:1), PG(40:7) and PI(36:1) (decreased). Statistical analysis revealed a general trend towards shortened phospholipid acyl chains in senescence and a significant trend to more saturated acyl chains in the class of phosphatidylglycerol. Additionally, the cellular cholesterol content was elevated and accumulated in vacuoles in senescent cells. These changes were accompanied by increased membrane fluidity. In mitochondria, loss of membrane potential along with altered intracellular distribution was observed. In conclusion, we present a comprehensive overview of altered cholesterol and glycerophospholipid patterns in senescence, showing that predominantly mitochondrial lipids are affected and lipid species less susceptible to peroxidation are increased.