Imaging of mobile long-lived nanoplatforms in the live cell plasma membrane

Abstract

The plasma membrane has been hypothesized to contain nanoscopic lipid platforms, which are discussed in the context of "lipid rafts" or "membrane rafts." Based on biochemical and cell biological studies, rafts are believed to play a crucial role in many signaling processes. However, there is currently not much information on their size, shape, stability, surface density, composition, and heterogeneity. We present here a method that allows for the first time the direct imaging of nanoscopic long-lived platforms with raft-like properties diffusing in the live cell plasma membrane. Our method senses these platforms by their property to assemble a characteristic set of fluorescent marker proteins or lipids on a time scale of seconds. A special photobleaching protocol was used to reduce the surface density of labeled mobile platforms down to the level of well isolated diffraction-limited spots without altering the single spot brightness. The statistical distribution of probe molecules per platform was determined by single molecule brightness analysis. For demonstration, we used the consensus raft marker glycosylphosphatidylinositol-anchored monomeric GFP and the fluorescent lipid analog BODIPY-G(M1), which preferentially partitions into liquid-ordered phases. For both markers, we found cholesterol-dependent homo-association in the plasma membrane of living CHO and Jurkat T cells in the resting state, thereby demonstrating the existence of small, mobile, long-lived platforms containing these probes. We further applied the technology to address structural changes in the plasma membrane during fever-type heat shock: at elevated temperatures, the glycosylphosphatidylinositol-anchored monomeric GFP homo-association disappeared, accompanied by an increase in the expression of the small heat shock protein Hsp27.

FIGURE 1.

Observation of mGFP-GPI clusters in the plasma membrane of living CHO cells. The principle of the TOCCSL method is shown in A–D. A, overlay of a white light image and the fluorescent image of a mGFP-GPI-expressing CHO cell. The box indicates the region of interest chosen for the TOCCSL sequence. B–D, different steps of the illumination protocol. The upper panels present the original data; the lower panels present a sketch of the effect on putative membrane platforms. Active and photobleached fluorophores are indicated by green and white dots, respectively. The white hatched areas indicate the position of the field stop (width of 7 μm). After recording the pre-bleach image (B), the selected area was totally photobleached by a laser pulse (450 ms). The efficiency of photobleaching was controlled by an image recorded immediately after the bleach pulse (recovery time of 0.5 ms). After a recovery time of 800 ms, the first fluorescent spots entering the field of view could be observed as diffraction-limited signals. For display, the color axis range was reduced by a factor of 5 in C and D. The brightness distributions of single mGFP-GPI spots in CHO cells without (E) and after cholesterol depletion with MβCD (F) are plotted as probability density functions. G, data obtained after cholesterol depletion and subsequent replenishment with MβCD. Data (black line) were fitted by Equation S1 (red line); monomer (dotted line) and dimer (solid line) contributions are indicated in blue. H, the dimer fraction (α₂) as a function of mGFP-GPI surface density (σ). Each data point resulted from the analysis of a single cell on which multiple TOCCSL runs were performed. Experiments are shown under standard conditions (black circles) or after cholesterol depletion using MβCD (red triangles) or cholesterol oxidase (red squares). Data obtained after cholesterol depletion and subsequent replenishment with MβCD are indicated as black triangles. σ was obtained from the pre-bleach image by dividing the overall signal by the single molecule brightness. All experiments were performed at 37 °C. Scale bars = 5 μm.

FIGURE 2.

Dimer fraction of the externally applied probe BODIPY-G_M1. Cells were stained with BODIPY-G_M1, and the dimer fraction (α₂) was determined for different surface densities (σ). We found a linear increase in α₂ with σ in CHO cells (A, black circles) and Jurkat T cells (B, black circles). Upon cholesterol depletion using MβCD, the dimer fraction was dramatically reduced (red triangles). Cholesterol replenishment recovered the original dimer fraction (black triangle). We included one experiment in which BODIPY-G_M1 labeling was performed at 37 °C (white circle), indicating that the labeling temperature of 4 °C used in all other experiments had no influence on the observed association. Microscopy was performed at 37 °C.

FIGURE 3.

Stoichiometric composition of BODIPY-G_M1 clusters. TOCCSL experiments were performed on Jurkat T cells at 25 °C using BODIPY-G_M1 as the label. Brightness distributions were analyzed for various stainings, with A providing a showcase for σ = 1140 μm⁻²; the display is analogous to Fig. 1E. In this case, higher order cluster sizes up to tetramers were observed and included in the fit function. B, relative amount of cluster size n (α_n) obtained for five different surface densities (σ; black circles). Fit results based on a generalized Poisson model (Equation 1) are shown as white circles. C, the fit parameter (λ) is plotted as a function of the surface density (σ; triangles). Linear fitting yielded a slope (ζ) of 1300 platforms/μm². For comparison, we also included λ calculated for CHO cells at 37 °C (black circles; ζ = 3500 μm⁻²) and Jurkat T cells at 37 °C (white circles; ζ = 3100 μm⁻²). This experiment was performed in non-total internal reflection configuration.

FIGURE 4.

Effect of temperature on the expression of heat shock proteins and the homo-association of mGFP-GPI. A, CHO cells stably expressing mGFP-GPI were subjected to heat at the indicated temperatures or left at 37 °C for 60 min. After a 16-h recovery at 37 °C, the expression levels of Hsp27 and Hsp70 were determined by Western blotting. Whereas Hsp70 expression remained at basal levels at mild heat stress, Hsp27 expression showed already a considerable increase at 39 °C. B, TOCCSL experiments were performed on the same cells at the indicated temperatures. The dimer fraction (α₂) was found to decline substantially when increasing the temperature (top). The mean surface density of mGFP-GPI did not change with temperature (bottom).