monograph Epithelzellen charakterisieren sich durch die polare Organisation ihrer Zellmembran, welche sich in eine apikale und basolaterale Domäne aufteilt. Diese Struktur wird durch einen hoch spezialisierten Transport- und Sortiermechanismus aufrecht erhalten, welcher neu synthetisierte Plasmamembranproteine effizient zu ihrer korrekten Zielmembran befördert. In MDCK Zellen erfolgt der apikale Proteintransport nach Verlassen des TGN Exits über mindestens zwei verschiedene Sortiermechanismen, zum einen über einen lipid raft-abhängigen und zum anderen über einem lipid raft-unabhängigen Transportweg. Gegenstand dieser Arbeit war die Identifizierung von Proteinen, welche für den Transport apikaler Proteine essentiell sind. Im ersten Teil der vorliegenden Arbeit konnte ein Mitglied der Kinesin-1 Gruppe/Familie, KIF5C, als Kinesin Motor für den apikalen Transport der raft-assoziierten Saccharase-Isomaltase, als auch der nicht-raft-assoziierten p75 identifiziert werden. KIF5C konnte mit Hilfe von Massenspektrometrie in immunisolierten post-Golgi Vesikeln, welche spezifisch apikale Proteine transportieren, nachgewiesen werden. Hierbei wurde es sowohl im raft-abhängigen Vesikeln von SI als auch im raft-unabhängigen Vesikeln von p75 und LPH direkt nach Verlassen des TGN-Exits identifiziert. Die spezifische Unterdrückung der KIF5C-Expression zeigte, dass der apikale Transport von sowohl raft-assoziierten als auch nicht-raft-assoziierten Markerproteinen signifikant reduziert wurde (Astanina and Jacob, 2010). Im zweiten Teil der Arbeit konnte ein weiteres Protein, Annexin XIIIb, mit Hilfe der Massenspektrometrie, Western Blot Analysen und konfokaler Mikroskopie im raft-unabhängigen apikalen Transport identifiziert werden. Hier konnte gezeigt werden, dass Annexin XIIIb in endosomalen Kompartimenten lokalisiert ist, welche sowohl von raft-unabhängigen als auch raft-abhängigen apikalen Proteinen nach Verlassen des TGN durchlaufen werden. Darüber hinaus führte die siRNA-vermittelte Reduktion der Annexin XIIIb-Expression zu einer Reduktion des apikalen Proteinanteils / des apikalen Proteintransportes von sowohl der raft-assoziierten SI als auch der nicht.raft-assoziierten Markerproteine p75 und LPH (Astanina et al., 2010). Zusammenfassend zeigt sich, dass beide in dieser Arbeit identifizierten Proteine, KIF5C and Annexin XIIIb, eine Rolle als endosomale Organisatoren beim apikalen Proteintransport in polaren Epithelzellen spielen. Die konfokalmikroskopischen Analysen sowie die TGN-release Experimente lassen den Rückschluss zu, dass beide Proteine direkt nach Verlassen des TGN eine Rolle im apikalen Transport raft und nicht-raft-assoziierte Proteine spielen. 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This architecture is maintained by highly specific cargo sorting machinery that efficiently delivers newly synthesized polypeptides to their correct target membrane. After TGN exit apical cargo is segregated by at least two distinct sorting mechanisms into lipid-raft-dependent or lipid-raft-independent apical pathways in MDCK cells. The aim of this study was the identification of proteins which are essential for the transport of apically sorted proteins. In the first part of the current study, a member of kinesin-1 group, KIF5C, was identified as a kinesin motor for apical trafficking of sucrase-isomaltase, the marker for the raft-associated pathway, and of non-raft-associated p75. KIF5C was found by mass spectrometry in vesicle enriched fractions and on immunoisolated post-Golgi vesicles carrying apical cargo. KIF5C associates with vesicles of both raft-dependent and raft-independent pathways directly after TGN exit. The specific knockdown of KIF5C interfered the apical trafficking of both raft-associated and non-raft associated marker proteins significantly (Astanina and Jacob, 2010). In the second part, annexin XIIIb was identified in raft-independent apical trafficking by mass spectrometry, immunoblotting and confocal microscopy. Annexin XIIIb accumulated in endosomal compartments that are traversed by raft-dependent and raft-independent apical cargo after TGN release. Finally, a specific reduction of annexin XIIIb expression by RNA interference resulted in a significant decrease in the apical delivery of the raft- as well as non-raft apical markers (Astanina et al., 2010). Taken together, both proteins – KIF5C and annexin XIIIb – act as endosomal organizers of apical protein trafficking in polarized epithelial cells. Based on the confocal microscopy studies and TGN release experiments we came to the conclusion that both proteins function on the first transport steps after TGN exit and accomplish trafficking of raft-associated, as well as non-raft-associated apical cargo. Publikationsserver der Universitätsbibliothek Marburg Universitätsbibliothek Marburg Epithelzellen urn:nbn:de:hebis:04-z2010-06537 ths Prof. Dr. Maier Uwe Maier, Uwe (Prof. Dr.) Endosomal Organizers of post-Golgi trafficking in polarized epithelial cells Medizin 2011-08-10 2010-12-29 Klinische Zytobiologie und Zytopathologie Annexin Teile publiziert in Cellular and Molecular Life Sciences, European Journal of Cell Biology 2010-10-25 application/pdf