Fluorescence-topographic NSOM directly visualizes peak-valley polarities of GM1/GM3 rafts in cell membrane fluctuations

Abstract

Simultaneous fluorescence-topographic nanoscale imaging of cell-surface molecules in the context of membrane ultra-structures has not been reported. Here, near-field scanning optical microscopy (NSOM)-based direct fluorescence-topographic imaging indicated that GM3 rafts/nanodomains (190.0 +/- 49.8 nm ranging 84.5-365.0 nm) were localized predominantly on the peaks of microvillus-like protrusions in the apical membrane of GM3 + Madin-Darby canine kidney cells, whereas GM1 rafts/nanodomains (159.5 +/- 63.8 nm ranging 42-360 nm) were distributed mainly on the slops of protrusions or the valleys between protrusions in the plasma membranes of GM1 + MDCK cells. The data demonstrated that gangliosides polarized not only in a well-known apical-basolateral manner but also in the more microscopic peak-valley manner, implicating unique distribution of GM1 or GM3 in cell-surface fluctuations on the apical membrane of polarized cells. The peak-valley polarities of gangliosides also implicated their different functions relevant to lipid rafts, microvilli, or cellular processes. Importantly, our study demonstrated for the first time that the NSOM-based direct fluorescence-topographic imaging is unique and powerful for elucidating nanoscale distribution of specific cell-surface molecules in membrane fluctuations.

Fig. 1.

Static confocal microscopic images of fluorescent quantum dot (QD) particles on substrate and QD-stained GM1 or GM3 microdomains on apical membrane of prefixed Madin-Darby canine kidney cells (MDCK) cells. A: Confocal microscopy shows that QD aggregates or large QD particles were removed for commercial QD solution by centrifugation and filtration. Treated or untreated preparations of QD solution were deposited on poly-L-lysine-coated cover slips, and imaged under static confocal microscopy. Shown are confocal images of untreated QD stock solution (left), QD supernatant after the first centrifugation at 5,000 g for 5 min (middle) and final QD supernatant treated by all the procedures: the first centrifugation, dilution in PBS, filtration (∼80–100 nm-pore filter) and the second centrifugation at 12,000 g for 5 min. The near-field scanning optical microscopy (NSOM) (or transmission electron microscopy) images of the single-QD particles from the final supernatant were shown in our previous study (4). B and C show the confocal images of GM1 (B stained first with biotinylated CTB and then streptavidin-conjugated QD655) or GM3 (C stained sequentially with anti-GM3 IgM, biotinylated anti-IgM IgG, and streptavidin-conjugated QD655) microdomains on cell membrane of MDCK cells. Square images in the right panels were the top cell-membrane views of GM1 or GM3 microdomains; the rectangle images flanking the square ones were side views of membrane GM1 or GM3 as sectioned across the lines. Note that GM1 or GM3 domains were distributed most on apical membrane (square images) and some on basolateral membrane (rectangle images). D shows two-color confocal images of GM1 [green; stained with CTB- fluorescein isothiocyanate (FITC)] and GM3 (red; stained as described in C) microdomains on GM1 + GM3 + MDCK cells (inset: the DIC image).

Fig. 2.

Real-time confocal microscopic imaging of dynamic QD staining of GM1 or GM3 microdomains in the apical plasma membrane of MDCK cells. A: Schematic diagram for adding QD solution to MDCK cells for staining under confocal microscope. B and C were confocal images showing formation and dynamics of QD-stained GM1 and GM3 microdomains on cell surface, respectively. Left panels: snapshots of the dynamics displaying many regions of interest (ROIs) marked by circles/numbers, from which the mean fluorescence intensity (MFI) was measured. Middle panels: corresponding DIC images. Right panels: MFI vs. time graphs showing changes in mean MFI (mean ± SD) of randomly-chosen 10 ROIs corresponding to the circles as numbered in the images of the left panels. Four types of ROIs were measured: i) lipid raft ROI (circles 1–10 in the left images): GM1 or GM3 microdomains on cells; ii) NC1 (negative control 1; circles 11–20), ROI: the areas nearby the microdomains (but obviously not in the microdomains); iii) PC (positive control; circles 21–30), ROI: the QD solution areas outside cells, which gave rise to fluorescence due to the QD particles suspending in solution; iv) NC2 (negative control 2; circle 31–40), ROI: the areas inside cells, which were dark (no fluorescence) since QDs could not enter the prefixed cells. Note that MFI curves for GM1 or GM3 raft ROI were increased over time, indicating that accumulative QD staining of GM1 or GM3 microdomains instead of preformed QD aggregates binding to the GM1 or GM3 molcules. The MFI curves for PC ROI started early and relatively stable, which could be explained by the presence of QD particles in solution. No apparent increases in MFI were detected for the NC1 and NC2. Of note, the decrease in fluorescence intensity at the late stage as seen in the right graph of B was probably due to photobleaching and/or diffusion of QD particles from one to other areas of the investigated cell. The distance, concentration, and diffusion rate of QD solution together with other factors appeared to affect the speed at which the QD particles reached the cell surface for staining and therefore impacted on the MFI of QD solution around the cells, the increase in MFI for staining GM1 (B) or GM3 (C) microdomains, and the time of starting the increase. This appeared to explain why the increase in MFI for QD staining of GM1 microdomains in B started earlier than that of GM3 microdomains in C.

Fig. 3.

Direct in situ fluorescence-topographic NSOM imaging and quantification of GM1 rafts localized predominantly in membrane valleys. A: NSOM topographic (left), fluorescence (middle), and merged (right) images of a representative GM1+ MDCK cell. B: Higher-magnification topographic (left) and fluorescence-topographic merged (middle and right) two-dimensional (2D) images of an area on the cell in Fig. 3A. The topographic information was pseudo-colored in yellow for the peak of membrane protrusions, and in blue for the planar membrane or the valley between membrane protrusions. In the middle fluorescence-topographic image, the fluorescence information (in red) was above but not merged with the topographic information (in yellow/blue), highlighting all GM1 rafts in red. In the right fluorescence-topographic image, the topographic and fluorescence information were merged, highlighting the valley-localizing GM1 rafts in pink. Scale bar = 1 μm. Note that GM1 rafts were mainly localized in planar or valley membrane. C: The height (upper curves) and corresponding fluorescence (lower curves) profiles of the cross-sections extracted from the left panel of Fig. 3B (dashed lines). Note that GM1 fluorescence corresponded to the low height areas (planar or valley membrane). D: The NSOM topographic (gray)-fluorescence (red) merged three-dimensional (3D) image of Fig. 3B shows GM1 rafts were localized in planar or valley membrane. E: The topographic 3D image of the first panel of Fig. 3B. F: left panel: the fluorescence profile extracted from a cross-section through two GM1 rafts in the middle image of Fig. 3B, with diameters (FWHM) of these nanodomains being ∼100 nm and ∼168 nm, respectively. Right panel: the histogram for the diameters of GM1 rafts (159.5 ± 63.8 nm; ∼42 nm–360 nm). Shown is the representative of three GM1+ MDCK cells from three independent experiments.

Fig. 4.

Direct in situ fluorescence-topographic NSOM imaging and quantification of GM3 rafts predominantly localized in membrane peaks. The legends are the same as described in Fig. 3 except GM3 replacement for GM1. Note that in contrast to GM1, GM3 were predominantly localized in the membrane peaks as seen in B–E. In the left panel of F, one of the five GM3 rafts has a diameter of ∼270 nm. In the histogram for the diameters of GM3 rafts (right panel of F), the mean diameter is 190.0 ± 49.8 nm (∼84 nm–365 nm). Shown is the representative of three GM3 + MDCK cells from three independent experiments.