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 6 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 1 Madin-Darby canine kidney cells, whereas GM1 rafts/nanodomains (159.5 6 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 1 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 cellsurface molecules in membrane fluctuations.-Chen, Y., J. Qin, and Z. W. Chen. Fluorescence-topographic NSOM directly visualizes peak-valley polarities of GM1/GM3 rafts in cell membrane fluctuations. J. Lipid Res. 2008Res. . 49: 2268Res. -2275 Supplementary key words near-field scanning optical microscopyMorphological, chemical, and functional polarities of membrane lipids (1) are well described and are relevant to lipid raft formation and cellular signaling. While different lipid components, especially glycolipids, are distributed in the apical-basolateral manner in the plasma membranes of epithelial cells (2), the ganglioside-enriched rafts on T lymphocytes can redistribute asymmetrically in a leadingedge-uropod manner upon cell activation (3). Despite identification of apical-basolateral and leading-edge-uropod distributions of membrane lipids, little is known about nanostructures and precise topographic localization of gangliosides or lipid rafts in the context of biological membrane ultrastructures, such as microvilli or protrusions/valleys.Widely used conventional imaging techniques have a limited capability to elucidate fluorescence-topographic distribution of cell-surface molecules at nanoscale. The traditional fluorescence instruments including confocal microscopy do not have enough optical resolution for detection of nanscale ganglioside clusters. Fluorescence resonance energy transfer measurement or single particle tracking technique is unable to reveal the nanoscale topographic localization of molecules on membrane surface. Rapidly developed atomic force microscopy fails to detect fluorescence information. The high-resolution electron microscopy (EM) is often limited by uncertain fidelity or reliability due to the complicated sample-preparing ...

Nanoscale Imaging of Epidermal Growth Factor Receptor Clustering

“…The average diameter for the raft domains visualized using GM1-cholera toxin B was 110 nm, within the predicted range for raft sizes (4) and in reasonable agreement with recent NSOM studies showing nanoscale domains for GM1 and GM3 (diameters of 159 and 190 nm, respectively) in fixed, immunostained Madin-Darby canine kidney cells and for asialo-GM1 in fixed HeLa cells (ϳ90 nm) (30,49). These estimates are larger than those from measurements of hindered diffusion of lipids or raft-anchored proteins in the plasma membrane of live cells.…”

Direct chemical evidence for sphingolipid domains in the plasma membranes of fibroblasts

“…Because the cholesterol-sphingolipid interactions seem to play a minor role in sphingolipid domain formation, we conclude that the sphingolipid domains are not lipid rafts and are instead a distinctly different sphingolipid-enriched plasma membrane domain. On the basis of reports of a size difference between GM1 and GM3 domains and their segregation from one another (4,36,37), we expect that the plasma membrane contains multiple types of microdomains that differ in sphingolipid subspecies composition and size. Accordingly, the micrometer-scale sphingolipid patches we observed in the Clone 15 cells likely consist of microdomains that are each enriched with a different sphingolipid subspecies.…”

“…Visualization of sphingolipid distribution via use of fluorescently labeled sphingolipid analogs or sphingolipid-specific affinity labels has revealed plasma membrane domains that differ in sphingolipid subspecies composition and size (4,36,37,(45)(46)(47). As mentioned above, GM1 and GM3 are located in separate domains that have diameters <300 nm (4,36,37), whereas sphingomyelin is enriched in plasma membrane domains with micrometer-scale dimensions (45)(46)(47). Because micrometer-scale sphingomyelin domains are inconsistent with the expected size range for lipid rafts, these findings have been largely dismissed as artifacts caused by fluorophoreinduced clustering or antibody cross-linking.…”

Imaging the invisible: resolving cellular microcompartments by superresolution microscopy techniques

“…By combination with quantum dot labeling, organization of the T-cell receptor on the surface of T-cells was observed with a resolution < 50 nm (Chen et al , 2008b ). Dual-color NSOM imaging for the first time clearly resolved the localization of proteins (de Bakker et al , 2008 ;van Zanten et al , 2009 ;Zhong et al , 2009 ) and lipids (Chen et al , 2008a ) in different types of domains (Figure 3 A,B). These results, however, were obtained with fixed samples and thus the spatio-temporal dynamics of these domains could not be resolved.…”