Laurdan generalized polarization fluctuations measures membrane packing micro-heterogeneity in vivo

Abstract

Cellular membranes are heterogeneous in composition, and the prevailing theory holds that the structures responsible for this heterogeneity in vivo are small structures (10-200 nm), sterol- and sphingolipid-enriched, of different sizes, highly dynamic denominated rafts. Rafts are postulated to be platforms, which by sequestering different membrane components can compartmentalize cellular processes and regulate signaling pathways. Despite an enormous effort in this area, the existence of these domains is still under debate due to the characteristics of the structures itself: small in size and highly mobile, which from the technical point of view implies using techniques with high spatial and temporal resolution. In this report we measured rapid fluctuations of the normalized ratio of the emission intensity at two wavelengths of Laurdan, a membrane fluorescent dye sensitive to local membrane packing. We observed generalized polarization fluctuations in the plasma membrane of intact rabbit erythrocytes and Chinese hamster ovary cells that can be explained by the existence of tightly packed micro-domains moving in a more fluid background phase. These structures, which display different lipid packing, have different sizes; they are found in the same cell and in the entire cell population. The small size and characteristic high lipid packing indicate that these micro-domains have properties that have been proposed for lipid rafts.

Fig. 1.

Schematic of the Laurdan GP fluctuation measurements. (A) Emission spectra of Laurdan solubilized in DPPC small unilamellar vesicles at 50 °C). (B) Emission spectra of Laurdan solubilized in DPPC at 50 °C (red) and at 35 °C (blue) using excitation light at 340 nm. (C) Diagram showing the Laurdan molecules (small black dots) diffusing in a fluid phase (molecule 1) and crossing domains of a more tightly packed phase (molecule 2).

Fig. 2.

Representative results for Laurdan GP fluctuations for the control POPC at 25 °C (A–C) and RRBC at 37 °C (a–c). (A, a) Intensity images taken at the equatorial section show the location of the laser orbit (red circle) used to perform sFCS measurements. The orbit crosses twice the bilayer (see Methods), starts and finishes at the white point and moves clockwise. (B, b) Autocorrelation (AC) analysis applied to the independent channels (AC-CH1 in red and AC-CH2 in blue) together with the cross-correlation of the two channels (in green). (C, c) AC function applied to the pixels corresponding to the membrane on the GP carpet (AC-GP). Solid lines correspond to the fitting of the experimental data. For detailed explanations see Methods.

Fig. 3.

Laurdan GP fluctuation in RRBC (A and B) and CHO cells (C and D). Distribution of D_GP (A and C). D_GP versus G_GP plots for the GP fluctuations obtained on RRBC (B and D, open circles) and CHO cells (D, closed squares). The black dot at (0,0) represents samples not showing autocorrelation. CHO cells incubated with rHDL for 2 h at 37 °C (D, open squares).

Fig. 4.

Model for GP fluctuations in biological membrane. Nano-domains of different sizes will be present in the RRBC membrane: (A) Several small domains with radii in the range of approximately 25 nm, (B) big domains moving slowly with an average estimated radius of approximately 78 nm, and (C) even larger domains with estimated sizes between less than 300 nm and greater than 78 nm. Domains larger than the PSF will not show detectable fluctuations. Dotted circle represents the cross section of the PSF with a diameter of approximately 300 nm, and the black circled inside show the nano-domains responsible for the detected fluctuations.

Fig. 5.

Measurement and analysis of Laurdan GP fluctuations. (A) Intensity image of a rabbit erythrocyte showing the scanning orbit (red circle) of 1.52 um radius used in the circular scanning FCS data acquisition. Sixty-four points were acquired per ms (duration of the orbit). The white point on the orbit shows positions 0 and 64. The laser moves clockwise starting at point zero. (B) The Laurdan emission is recorded in two channels with interference filter center at 440 and 490 nm (blue and black traces). GP trace (red line) is generated using the traces and the GP formula (see Methods). (C) XY transformation of the raw scanning FCS intensity traces in intensity carpet (C1 and C2) and GP trace into GP carpet (C3). The x-position columns represent points along one circular scan (64), and the y-position rows represent successive scans every 1 ms.