The Organizing Potential of Sphingolipids in Intracellular Membrane ...
The Organizing Potential of Sphingolipids in Intracellular Membrane ...
The Organizing Potential of Sphingolipids in Intracellular Membrane ...
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1700 HOLTHUIS, POMORSKI, RAGGERS, SPRONG, AND VAN MEER<br />
4. Microscopy on fixed cells<br />
Independent evidence for cluster<strong>in</strong>g <strong>of</strong> glycosph<strong>in</strong>golipids<br />
on the cell surface has been obta<strong>in</strong>ed by microscopy.<br />
A first approach has utilized glycosph<strong>in</strong>golipid-b<strong>in</strong>d<strong>in</strong>g<br />
prote<strong>in</strong>s, which were visualized by a fluorescent or<br />
electron-dense tag or by a secondary labeled prote<strong>in</strong>. <strong>The</strong><br />
major problem <strong>in</strong> such studies is that lipids cannot be<br />
fixed at their orig<strong>in</strong>al location. Artificial cluster<strong>in</strong>g was<br />
<strong>in</strong>duced when the b<strong>in</strong>d<strong>in</strong>g <strong>of</strong> a primary antibody to globoside<br />
(Gb 4Cer) or Forssman glycosph<strong>in</strong>golipid (IV 3 --<br />
GalNAc-Gb 4Cer) was followed by label<strong>in</strong>g with a dimeric<br />
secondary antibody, tetrameric prote<strong>in</strong> A, or when multimeric<br />
complexes were used <strong>of</strong> the primary or secondary<br />
ligand to ferrit<strong>in</strong> or colloidal gold (47, 96, 377). Cluster<strong>in</strong>g<br />
<strong>of</strong> the ganglioside GM1 was observed when it was labeled<br />
with pentameric cholera tox<strong>in</strong> B subunit by itself (9) or<br />
conjugated with gold <strong>in</strong>to a multimeric complex (276), or<br />
when a biot<strong>in</strong>ylated GM1 was labeled with anti-biot<strong>in</strong>gold<br />
(246). <strong>The</strong> latter studies were performed on freezesubstituted<br />
samples <strong>in</strong> which redistribution seems rather<br />
unlikely. In an <strong>in</strong>dependent approach, label<strong>in</strong>g <strong>of</strong> glycosph<strong>in</strong>golipids<br />
with a primary antibody was followed by<br />
fixation before the addition <strong>of</strong> the secondary antibodygold<br />
complex, a condition that had been shown to prevent<br />
redistribution <strong>of</strong> Forssman glycosph<strong>in</strong>golipid (47, 96).<br />
Clusters <strong>of</strong> GM3 were still observed <strong>in</strong> one study (354),<br />
while cluster<strong>in</strong>g <strong>of</strong> a number <strong>of</strong> sph<strong>in</strong>golipids <strong>in</strong> caveolae<br />
was no longer observed under these str<strong>in</strong>gent conditions<br />
(96). Still, a local enrichment <strong>of</strong> the ganglioside GM1 <strong>in</strong><br />
caveolae was found by a postembedd<strong>in</strong>g label<strong>in</strong>g protocol<br />
us<strong>in</strong>g cholera tox<strong>in</strong> where redistribution could be excluded<br />
(276). 14 In cells transfected with <strong>in</strong>fluenza virus<br />
hemagglut<strong>in</strong><strong>in</strong> or GPI prote<strong>in</strong>s, the distribution <strong>of</strong> these<br />
prote<strong>in</strong>s overlapped with that <strong>of</strong> GM1 (128). Cluster<strong>in</strong>g <strong>of</strong><br />
GPI prote<strong>in</strong>s has been observed by microscopy under a<br />
variety <strong>of</strong> conditions (7). However, <strong>in</strong> many cases cluster<strong>in</strong>g<br />
was <strong>in</strong>duced by the protocol used. A major po<strong>in</strong>t <strong>of</strong><br />
concern <strong>in</strong> most microscopic studies on lipid and lipidl<strong>in</strong>ked<br />
molecules is the use <strong>of</strong> multimeric reporter ligands<br />
without proper controls.<br />
14 When sections <strong>of</strong> freeze-substituted A431 cells were labeled<br />
with cholera tox<strong>in</strong> B-gold, clusters were observed <strong>in</strong> uncoated <strong>in</strong>vag<strong>in</strong>ations<br />
<strong>of</strong> the plasma membrane. It seems unlikely that cluster<strong>in</strong>g was<br />
<strong>in</strong>duced by the multimeric ligand as lipids most likely do not diffuse on<br />
the surface <strong>of</strong> the section which <strong>in</strong> this case consists <strong>of</strong> Lowicryl<br />
polymer. This is different <strong>in</strong> frozen sections, where dur<strong>in</strong>g the label<strong>in</strong>g <strong>of</strong><br />
the thawed sections lipids are free to diffuse <strong>in</strong> the membranes. In the<br />
freeze-substituted cells, GM1 was also found <strong>in</strong> small vesicles <strong>in</strong> the<br />
cytosol <strong>in</strong> close approximation with the patches on the plasma membrane.<br />
Because the vesicle pr<strong>of</strong>iles were not <strong>in</strong> contact with the plasma<br />
membrane dur<strong>in</strong>g the label<strong>in</strong>g <strong>of</strong> the section, a higher concentration <strong>of</strong><br />
GM1 <strong>in</strong> these vesicles must have been present already <strong>in</strong> the liv<strong>in</strong>g cell.<br />
In the cell, the vesicle membranes are thought to be cont<strong>in</strong>uous with the<br />
plasma membrane or to be derived from the plasma membrane by<br />
endocytosis. In both cases, the results demonstrate that GM1 is enriched<br />
<strong>in</strong> subdoma<strong>in</strong>s <strong>of</strong> the plasma membrane <strong>of</strong> these A431 cells.<br />
Physiol Rev • VOL 81 • OCTOBER 2001 • www.prv.org<br />
5. Optical studies on liv<strong>in</strong>g cells<br />
Already <strong>in</strong> the early 1980s, measurements on the<br />
behavior <strong>of</strong> fluorescent probes <strong>in</strong> biomembranes supported<br />
the concept <strong>of</strong> lipid doma<strong>in</strong>s <strong>in</strong> membranes (157),<br />
and microscopy was performed on liv<strong>in</strong>g cells us<strong>in</strong>g fluorescent<br />
lipids (357). In the latter study, the redistribution<br />
<strong>of</strong> GM1 by cholera tox<strong>in</strong> caused cocapp<strong>in</strong>g <strong>of</strong> the unrelated<br />
ganglioside GM3, which suggested that the headgroup-labeled<br />
GM1 and GM3 were associated by lipidlipid<br />
<strong>in</strong>teractions. Much more recently, a major<br />
breakthrough <strong>in</strong> the field has been the application to liv<strong>in</strong>g<br />
cells <strong>of</strong> novel high-resolution optical techniques, like resonance<br />
energy transfer between fluorescent membrane<br />
molecules, s<strong>in</strong>gle particle track<strong>in</strong>g, two-dimension scann<strong>in</strong>g<br />
resistance, and s<strong>in</strong>gle dye trac<strong>in</strong>g. A number <strong>of</strong> these<br />
studies support the existence <strong>of</strong> locations on the cell<br />
surface that are enriched <strong>in</strong> GPI prote<strong>in</strong>s and glycosph<strong>in</strong>golipids<br />
and, <strong>in</strong> addition, <strong>of</strong> areas with enhanced resistance<br />
to lateral diffusion that are preferred by some but<br />
not by other probes. One controversial issue is the size <strong>of</strong><br />
the doma<strong>in</strong>s. What is their diameter? Whereas the detergent-extraction<br />
studies yielded DRM vesicles with a diameter<br />
<strong>of</strong> 0.1–1 m (39), suggest<strong>in</strong>g a diameter size <strong>of</strong> 200–<br />
2,000 nm, this was a few hundred nanometers for the<br />
small regions to which a GPI prote<strong>in</strong> and GM1 were found<br />
to be conf<strong>in</strong>ed <strong>in</strong> particle track<strong>in</strong>g studies (290, 331). 15<br />
<strong>The</strong> newest s<strong>in</strong>gle particle track<strong>in</strong>g measurements on<br />
these doma<strong>in</strong>s suggest that they are even smaller with a<br />
diameter <strong>of</strong> 50 nm (roughly 3,500 lipids), exist for more<br />
than 1 m<strong>in</strong>, and comprise 50 prote<strong>in</strong>s (289). Chemical<br />
cross-l<strong>in</strong>k<strong>in</strong>g and fluorescence resonance energy transfer<br />
to measure GPI-prote<strong>in</strong> <strong>in</strong>teractions led to estimates <strong>of</strong> 70<br />
nm (94, 401). In contrast to these data, no cluster<strong>in</strong>g <strong>of</strong> a<br />
GPI prote<strong>in</strong> was observed on the apical surface <strong>of</strong> MDCK<br />
cells (162). In addition, a comparison between various<br />
GPI prote<strong>in</strong>s on various cell surfaces did not provide<br />
evidence for the occurrence <strong>of</strong> a sizeable fraction <strong>of</strong> the<br />
GPI prote<strong>in</strong>s as stable clusters (163). <strong>The</strong> authors expla<strong>in</strong>ed<br />
the discrepancies with the earlier work, by conclud<strong>in</strong>g<br />
that lipid rafts either exist only as transiently<br />
stabilized structures or, if stable, comprise at most a<br />
m<strong>in</strong>or fraction <strong>of</strong> the cell surface.<br />
Here, it becomes relevant to discuss the area <strong>of</strong> the<br />
membrane covered by rafts. SM constitutes 20% <strong>of</strong> the<br />
plasma membrane phospholipids. If, as generally believed,<br />
SM is located <strong>in</strong> the outer leaflet, it covers 40% <strong>of</strong><br />
the surface. When saturated phospholipids and choles-<br />
15 It should be noted that each gold particle possessed many<br />
b<strong>in</strong>d<strong>in</strong>g sites aga<strong>in</strong>st the GPI prote<strong>in</strong> (antibodies conjugated to gold) and<br />
to GM1 (cholera tox<strong>in</strong> B subunits conjugated to gold). In the case <strong>of</strong> the<br />
GPI prote<strong>in</strong>, the number <strong>of</strong> b<strong>in</strong>d<strong>in</strong>g sites did not affect the results.<br />
Concern<strong>in</strong>g GM1, cholera tox<strong>in</strong> has been shown to reduce its solubility<br />
<strong>in</strong> detergent, which implies that b<strong>in</strong>d<strong>in</strong>g <strong>of</strong> the pentavalent tox<strong>in</strong> changes<br />
the phase behavior <strong>of</strong> GM1 (117).