Figure 1.
Heat-induced membrane changes visualised by Laurdan two-photon microscopy.
K562 cells in RPMI medium were treated at 42°C for 1 h and imaged (A) before and (B) after HS at room temperature. The colour chart in Panel A represents Laurdan GP values as indicated. Bars in A and B, 6 µm. (C) Histogram of GP changes observed in whole cell and in subcellular (perinuclear, internal and plasma membrane) regions. An example set of ROIs used for quantification of subcellular GP distribution is given in panel A. Data are expressed as means ± SD, n = 30, *p<0.05 compared to the control, unpaired t-test.
Figure 2.
High resolution images of the general polarisation distribution at the cell border.
K562 cells in RPMI medium were treated at 42°C for 1 h and imaged (A) before and (B) after HS at room temperature. Bars in A and B, 1 µm. (C) GP profile along blue lines shown in panels A and B. For other details see Figure 1.
Figure 3.
Anisotropy changes as detected by different fluorescent probes.
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 t-test.
Figure 4.
Fluorescence changes during heat treatment are different in cells compared to isolated plasma membranes.
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.
Figure 5.
Changes in anisotropy upon benzyl alcohol addition also differ between cells and isolated plasma membranes.
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 was followed at 37°C (representative traces are shown from n = 4 independent experiments). BA was administered as indicated (blue arrows).
Figure 6.
Heat-induced membrane heterogeneity changes followed by DPH lifetime distribution in K562 cells.
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).
Figure 7.
Different fluorescent probes show distinct patterns of localization in K562 cells.
Cells were incubated with different DPH analogues at 37°C or 42°C for 5 or 30 min as indicated. Fluorescence video microscopic images were recorded within 2 min following the incubation (representative results are shown from n = 3 independent experiments). Bar, 6 µm.
Figure 8.
Colocalization of DPH with lipid droplets in K562 cells.
Cells were double-stained with DPH and LD540 (which stains LDs) and examined with a CytoScout fluorescence microscope. Bar, 6 µm.
Figure 9.
Spin-labelling of K562 cells reveals changes in membrane rigidization following heat stress.
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 (mI = 0). Data are expressed as means ± SD, n = 3 (independent preparations), *p<0.05 compared to 37°C control, unpaired t-test.
Table 1.
Characteristic properties of different probes used in the present study.