Columnar epithelia lining the kidney, intestine or pancreas are composed of a single layer of polarized cells. They have evolved to create stable apical and baso-lateral membrane domains, which are sealed off from each other by a tight junction barrier. While the basolateral domain of columnar epithelia faces the underlying extracellular matrix and the blood supply, the apical membrane is the one facing the lumen of the renal tubules, of the intestine, or of the pancreas. It has long been known that apical and basolateral membrane domains have a distinct protein composition [68,69]. However, lipids are also distributed differently between the apical and the basolateral membrane. The lipids found in the basolateral membrane resemble those found in the plasma membrane of an unpolarized cell, whereas the apical membrane contains much more glycosphingolipids . In the brush border membrane of the intestine, glycosphingolipids account for more than 30% of the total lipid amount . Considering that they reside exclusively in the exoplasmic leaflet, more than 50% of the lipids in the exoplasmic leaflet should be glycosphingolipids, and together with cholesterol they should leave very little space for glycerophospholipids. Glycosphingolipids mainly contain two long, saturated hydrocarbon chains, as opposed to glycerophospholipids which usually contain unsaturated acyl chains , and have been proposed to form a liquid-ordered phase together with cholesterol. It was this segregation of raft lipids in the outer leaflet of the apical membrane from the more phosphatidylcholine-enriched basolateral membrane that prompted Simons and van Meer to postulate the existence of lipid platforms involved in the biogenesis of the apical membrane  and has led to the formulation of the raft hypothesis .
Recently, we have experimentally explored the domain organization of the apical membrane of epithelial cells in comparison to that of a fibroblast plasma membrane by measuring long-range diffusion of several fluorescent membrane pro teins using fluorescence recovery after photobleaching (FRAP) . By using this technique, the diffusion of millions of proteins can be examined at the same time in a noninvasive manner. As previously reported , all proteins display free diffusion with 100% recovery in the fibroblast plasma membrane. In the apical membrane of epithelial cells, however, we could distinguish two populations of proteins on the basis of their distinct diffusion characteristics at 25 °C. One group displayed free diffusion with recoveries close to 100%, whereas the other group displayed anomalous diffusion [75, 76] with limited recovery. This is indicative of a phase-separated system, in which there are (at least) two coexisting phases - one which has a mass fraction just high enough to be continuous (percolating) over the entire membrane surface, and the other being present in isolated domains . Within the percolating phase, long-range diffusion is unconstrained, results in complete recovery, and can be described with a single apparent diffusion coefficient [78, 79]
- as observed for the first group of proteins. In the non-percolating phase, proteins will be obstructed in their long-range diffusion, resulting in either incomplete or extremely slow recovery [78, 79] - as observed for the second group of proteins. Strikingly, all proteins falling into the first group have been proposed to reside in rafts, while all members of the second group have been proposed to reside outside of rafts. This may suggest that at 25 °C the apical membrane of epithelial cells is a percolating raft phase with isolated non-raft domains.
Phase separation likely exists also in fibroblasts, with the domain organization of the two membranes being inverted. The fact that in the fibroblast plasma membrane the raft and non-raft proteins diffuse with the same kinetics does, however, not contradict the existence of phase separation. Rather, the results can be explained on the basis of partition coefficients. From all we know, a limited set of proteins has the features required to be accommodated in the ordered lipid environment of a raft domain. While non-raft proteins that lack these features are largely excluded from rafts - that is, non-raft proteins have a low propensity to partition into the surrounding raft phase in the apical membrane of epithelial cells
- raft proteins might have a preference for raft domains, but can easily partition into a less-ordered, non-raft environment - that is, raft proteins are not limited to raft domains in the plasma membrane of fibroblasts [28, 80, 81]. With the additional notion that raft domains in fibroblasts are believed to be small and highly dynamic, the differences between the long-range diffusion paths of raft and non-raft proteins in the fibroblasts plasma membrane become too small to be accessible to FRAP measurements.
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