Cellular response to heat shock studied by multiconfocal fluorescence correlation spectroscopy

Abstract

Heat shock triggers a transient and ubiquitous response, the function of which is to protect cells against stress-induced damage. The heat-shock response is controlled by a key transcription factor known as heat shock factor 1 (HSF1). We have developed a multiconfocal fluorescence correlation spectroscopy setup to measure the dynamics of HSF1 during the course of the heat-shock response. The system combines a spatial light modulator, to address several points of interest, and an electron-multiplying charge-coupled camera for fast multiconfocal recording of the photon streams. Autocorrelation curves with a temporal resolution of 14 μs were analyzed before and after heat shock on eGFP and HSF1-eGFP-expressing cells. Evaluation of the dynamic parameters of a diffusion-and-binding model showed a slower HSF1 diffusion after heat shock. It is also observed that the dissociation rate decreases after heat shock, whereas the association rate is not affected. In addition, thanks to the multiconfocal fluorescence correlation spectroscopy system, up to five spots could be simultaneously located in each cell nucleus. This made it possible to quantify the intracellular variability of the diffusion constant of HSF1, which is higher than that of inert eGFP molecules and increases after heat shock. This finding is consistent with the fact that heat-shock response is associated with an increase of HSF1 interactions with DNA and cannot be explained even partially by heat-induced modifications of nuclear organization.

Figure 1

ACFs obtained with eGFP cells. (A) Example of ACF curves acquired from four spots located in the nucleus (spots 1–4) and one in the cytoplasm (spot 5) of a single eGFP cell at 37°C. (Inset) Locations of the spots, marked by crosses, on a wide-field fluorescence image of the cell. (B) Averaged ACF curves corresponding to 38 spots at 37°C (blue circles) and 38 spots at 43°C (red triangles) located in the nuclei of five eGFP cells. Superimposed black solid lines correspond to the fits. (Inset) Estimated parameters.

Figure 2

Averaged ACF curves corresponding to 32 spots at 37°C (blue circles) and 13 spots at 43°C (red triangles) located in the nuclei of five HSF1-eGFP cells. (A) Fits without any prior normalization of the individual curves, to reveal the difference of amplitude due to the change in the number of molecules. Superimposed black solid lines correspond to the fits. (Inset) Estimated parameters. (B) Fits with normalization of the amplitude (G(0) = 2) of each individual ACF curve, to emphasize the difference in temporal behavior.

Figure 3

Relative variation of intensities during the time course of acquisitions before and after heat shock. The successive series of five acquisitions are separated by dotted vertical lines, whereas data for the two temperatures are separated by a solid line. Open symbols correspond to eGFP cells (blue circles, 37°C; red diamonds, 43°C) and solid symbols to HSF1-eGFP cells (blue squares, 37°C; red triangles, 43°C).

Figure 4

Mean values and SDs (vertical bars) of the diffusion time, τ_D (A), residence time, τ_off (B), and fraction of free molecules, F_eq (C), before (left) and after heat shock (right), obtained by fitting 330 ACF curves at 37°C and 344 ACF curves at 43°C. The symbol ^∗∗∗ corresponds to a significance level P < 10⁻³ between the bracketed groups.