Schematic representation of TG synthesis in <i>S</i>. <i>pombe</i>.

<p>Dga1p uses FA-CoA and diacylglycerol (DG) to produce TG, while Plh1p catalyses the direct FA transfer between a glycerophospholipid (GPL) and DG to generate TG and a lysophospholipid (LPL).</p

Changes in membrane lipid compositions.

<p>(A) Double bond index/saturated FA ratios (DBI/sat) for PC, PE and PI. (B) Lipid species compositional alterations for major membrane lipids PC, PE, and PI in the WT strain. (C) Changes in PC/PE ratios. (D) Changes in the contribution of medium chain FA-containing species to membrane lipid composition. (E) Alterations in lyso-lipid species. Cells were untreated (30°C) or stressed at 40°C for 1 h. Values are expressed as mol% of MLs (mean ± SD), n = 3 for <i>plh1Δ</i> and <i>dga1Δ</i>, n = 4 for DKO, and n = 7 for WT; * p<0.05 (30°C vs 40°C), # p<0.05 (WT vs mutants at 30°C), $ p<0.05 (WT vs mutants at 40°C). DBI/sat, double bond index/saturated FA ratio; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol.</p

Growth arrest of heat-stressed DKO cells correlates with enhanced signalling lipid generation.

<p>Changes in the amounts of Cer and DG by lipid class and species levels are shown. Cells were untreated (30°C) or stressed at 40°C for 1 h. Values are expressed as mean ± SD of lipid/protein values (nmol/mg), n = 3 for <i>plh1Δ</i> and <i>dga1Δ</i>, n = 4 for DKO, and n = 7 for WT; * p<0.05 (30°C vs 40°C), # p<0.05 (WT vs mutants at 30°C), $ p<0.05 (WT vs mutants at 40°C).</p

Changes in FA fluxes corroborate crosstalk between membrane and storage lipid metabolism.

<p>(A) Net changes expressed as FA/prot_{(after HS–before HS)} (nmol/mg/h) values for all FAs (upper panels) and for FA groups (medium– C10-C14, long SAT– 16:0 and 18:0, long MUFA– 16:1 and 18:1, very long C20-C32; lower panels). Average values are shown from n = 3 (for <i>plh1Δ</i> and <i>dga1Δ</i>), n = 4 (for DKO), and n = 7 (for WT) independent experiments. Data were reconstituted based on ESI-MS/MS fragmentation results with the exception of FFA, which was determined by GC-MS. (B) Graphical illustration of metabolic crosstalk between membrane and storage lipids.</p

Unsupervised statistics reveal separation of lipidomes due to deletion of TG synthesis genes or HS.

<p>Heatmap representation of hierarchical clustering (A) with or (B) without storage lipids; top 50 most significant species were selected based on ANOVA, Euclidean distance, clustering algorithm Ward; heat color code represents normalized values (z-scores) that range between [−2] (darkest shade of blue) and [+3] (darkest shade of red). Three to seven independent experiments are shown for control and HS-treated (40°C, 1 h) cells.</p

Proposed mechanism of membrane rigidization in response to HS for the <i>dga1Δ</i> strain.

<p>Net changes expressed as lipid/prot_{(after HS–before HS)} (nmol/mg/h) values (left; average data from n = 3 independent experiments are shown), and schematic representation of TG-supported membrane rigidization (right) in the <i>dga1Δ</i> strain. DG, diacylglycerol; GPC/GPE/GPI, phosphorylated headgroups; GPL, glycerophospholipid; LPL, lysophospholipid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; TG, triacylglycerol.</p

Anisotropy changes as detected by different fluorescent probes.

<p>K562 cells in RPMI medium were heat-treated at 42°C for 1 h or left at 37°C, harvested and labelled with DPH-PA, TMA-DPH or DPH. The fluorescence steady-state anisotropy measurement was performed at 37°C and 5 min of trace was averaged. The anisotropy differences were calculated relative to the 37°C control values. Data are represented as means ± SD, n = 4, *p<0.05, paired <i>t</i>-test.</p

Fluorescence changes during heat treatment are different in cells compared to isolated plasma membranes.

<p>K562 cells or plasma membrane fractions (PM) isolated from untreated cells were labelled with DPH-PA, TMA-DPH or DPH and the fluorescence steady-state anisotropy (blue) was followed (representative traces are shown from n = 4 independent experiments). Cyclic temperature shift was applied (red). The arrows indicate the anisotropy difference at 37°C before and after 42°C HS.</p

Spin-labelling of K562 cells reveals changes in membrane rigidization following heat stress.

<p>EPR spectra of K562 cells labelled with 5- (top) and 16-SASL (bottom) are shown. Spectra were recorded at 37°C (blue lines, control), during 1 h HS at 42°C (black lines, HS) and after returning to 37°C (red lines, after HS). The spectra are normalised so that they represent the same number of spins. Total scan range is 10 mT. The corresponding bar graphs show the normalized amplitudes of the center-field ¹⁴N hyperfine EPR lines (m_I = 0). Data are expressed as means ± SD, n = 3 (independent preparations), *p<0.05 compared to 37°C control, unpaired <i>t</i>-test.</p

Heat-induced membrane heterogeneity changes followed by DPH lifetime distribution in K562 cells.

<p>Cells were labelled with DPH. Phase and modulation data were collected using 9 modulation frequencies ranging from 2 to 180 MHz. Measurements were performed at 37°C, during 1 h HS at 42°C and in the post-heat phase after returning to 37°C (representative results are shown from two independent experiments).</p