The structure and functions of lipid rafts and the mechanisms of intracellular membrane trafficking are major topics in current cell biological research. Rafts have been proposed to act as sorting platforms during biosynthetic transport, especially along pathways that deliver proteins to the apical membrane of polarised cells. Based on this, the aim of this work was to contribute to the understanding of apical sorting in epithelial cells. The study of how lipid rafts are structured has been hampered by the scarcity of techniques for their purification. Rafts are thought to be partially resistant to solubilisation by mild detergents, which has made the isolation of detergent-resistant membranes (DRMs) the primary method to characterise them biochemically. While a growing number of detergents is being used to prepare DRMs, it is not clear what can be inferred about the native structure of cell membranes from the composition of different DRMs. This issue was addressed by an analysis of DRMs prepared with a variety of mild detergents. The protein and lipid content of different DRMs from two cell lines, Madin-Darby canine kidney (MDCK) and Jurkat cells, was compared. It was shown that the detergents differed considerably in their ability to selectively solubilise membrane proteins and lipids. These results make it unlikely that different DRMs reflect the same underlying principle of membrane organisation. Another obstacle for understanding apical sorting is that the evidence implicating certain proteins in this process has come from various disparate approaches. It would be helpful to re-examine the putative components of the apical sorting machinery in a single experimental system. To this end, a retroviral system for RNA interference (RNAi) in MDCK cells was established. Efficient suppression of thirteen genes was achieved by retroviral co-expression of short hairpin RNAs and a selectable marker. In addition, the system was extended to simultaneously target two genes, giving rise to double knockdowns.Retroviral RNAi was applied to deplete proteins implicated in apical sorting. Surprisingly, none of the knockdowns analysed caused defects in surface delivery of influenza virus hemagglutinin, a common marker protein for apical transport. Therefore, none of the proteins examined is absolutely required for transport to the apical membrane of MDCK cells. Cells may adapt to the depletion of proteins involved in membrane trafficking by activating alternative pathways. To avoid such adaptation, a visual transport assay was established. It is based on the adenoviral expression of fluorescent marker proteins whose surface transport can be followed microscopically as soon as RNAi has become effective. With this assay, it should now be possible to screen the knockdowns for defects in surface transport. Taken together, this work has provided a number of experimental tools for the study of membrane trafficking in epithelial cells. First, the biochemical analysis of DRMs highlighted that DRMs obtained with different detergents are unlikely to correspond to distinct types of membrane microdomains in cell membranes. Second, the retroviral RNAi system should be valuable for defining the function of proteins, not only in membrane transport, but also in processes like epithelial polarisation. Third, the visual assay for monitoring the surface transport of adenovirally expressed marker proteins should be suitable to detect defects in polarised sorting.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa.de:swb:14-1105952715531-53222 |
| Date | 18 December 2004 |
| Creators | Schuck, Sebastian |
| Contributors | Technische Universität Dresden, Mathematik und Naturwissenschaften, Biologie, Max-Planck-Institut für Molekulare Zellbiologie und Genetik, Prof. Kai Simons, Prof. Lukas Huber, Prof. Kai Simons, Prof. Bernard Hoflack |
| Publisher | Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis |
| Format | application/pdf |
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