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Polar epithelial cells are the prerequisite for a functional organ. They build a monolayer on the outer surface of the organ, providing both a barrier and an exchange system to the surrounding environment. To fulfill these requirements they have developed a special partitioning of their plasma membrane, where the apical part faces the exterior and the basolateral part faces the neighboring cells or the basal lamina. To keep this polarity, epithelial cells possess both a lipid and protein sorting system, which follow two known sorting platforms. There is a lipid raft associated pathway, where lipid raft proteins gather in these detergent resistant membranes to build high molecular weight clusters, which are then transported to the apical cell surface. In addition there is a lectin based pathway, where galectin-3 binds to non-raft glycoproteins to also form high molecular weight clusters.
Initially the role of ALPK1, an alphakinase, was analyzed in the mouse small intestinal tract, where an involvement in the transport of the lipid-raft proteins SI and DPP IV was discovered.
The main project dealt with the characterization of the non-raft apical sorting pathway. It was revealed that there were no coat proteins involved in this galectin-3 dependent post golgi trafficking, however a GTPase named Mx1 was discovered to be present. When Mx1 was knocked down the transport efficiency of P75-GFP, which is sorted by galectin-3, was reduced. Furthermore it seems like Mx1 is implicated in unconventional galectin-3 secretion. In Mx1 knock down cells the lectin was secreted to a much lesser extent and interestingly galectin-3 reuptake is dependent on the integrity of lipid-rafts as shown by the biochemical and microscopic data.
With the help of TIRF microscopy it was possible to reveal previously unknown details of the apical MDCK cell surface. Microvilli were observed, recycling endosomes close to the apical plasma membrane, which are thought to be sorting stations for galectin-3 and P75-GFP were also seen.
Besides its role in cell polarization galectin-3 seems to be involved in the development of clear cell renal cell carcinoma. Here the lectin is expressed at a higher level and shown to interact with β-catenin.
In summary the data shown in this dissertation suggests that galectin-3, which is involved in the development of clear cell renal cell carcinoma and the apical sorting of non-raft proteins, has an alternating preference for detergent resistant membranes during its life cycle and that its unconventional secretion is dependent on the GTPase Mx1.