Higher organisms such as mammals are composed of a number of organs that all fulfill specific tasks for which they need to communicate. To enable targeted, controlled communication, these functional units have to be separated, both spatially and biochemically. All animal organs are therefore surrounded by epithelia which, besides protecting them from harmful environmental influences, ensure a controlled exchange of substances with their surroundings. The smallest unit is the polarized epithelial cell. These cells form a barrier exactly the thickness of a cell layer consisting of an uninterrupted row of cells. The tight junctions between the cells create a hermetically sealed surface through which controlled transport processes take place.
Epithelial cells typically have a polar structure. The most striking feature is the difference in the cytoplasmic membrane. There are two different domains: the apical membrane facing the exterior of the organ and the basolateral membrane that faces the interior of the organ or neighboring cells inside the epithelium. The two membrane domains exhibit clearly different protein and lipid compositions. For example, various digestion enzymes such as LPH (lactase-phlorizin-hydrolase) or SI (sucrose-isomaltase) are only transported into the apical cytoplasm membrane of the intestinal epithelium, where they reach their site of action – the intestinal lumen. These enzymes are not found in the basolateral membrane. There would be no point in transporting them there – it would be a waste of energy.
So what are the basic mechanisms of this specific protein sorting? The answer is provided by the proteins themselves, which all carry specific topogenous signals. These can be short amino acid sequences (YXXφ, NPXY, LL, L) like those found in basolaterally transported proteins. The signal effect may also be based on the glycosylation of the proteins as is the case with various apical proteins. GPI anchors (Glycosyl-Phosphatidyl-Inositol) also serve as apical sorting signals. All topogenous signals are an identifying code for other proteins or lipid structures, which then act as a sorting receptor or sorting platform. For example, some basolateral proteins are sorted by adapter proteins (AP1A) of the clathrin-dependent transport in the trans-Golgi network and transported to the target membrane  (Gonzalez and Rodriguez-Boulan, 2009). Proteins with GPI anchors, on the other hand, show a high affinity with specific cholesterin-rich lipid structures – lipid rafts – which are sorting platforms for the apical membrane. There are yet other apical proteins like P75-GFP, that are identified and bound due to their special glycosylation of the sugar-binding protein Galectin-3, which ultimately leads to transport into the correct membrane domain (Figure 1) .
In order to study the processes by which proteins are sorted and transported into the apical membrane of polarized epithelial cells in more detail, the obvious step is to take a closer look at this cell pole under the microscope. TIRF microscopy (Total Internal Reflection Fluorescence) is particularly useful for such studies, as it delivers highly detailed images of structures on, in and immediately under the apical cytoplasm membrane. This applies to both fixed and living cells.