Proteinkomponenten und Transportwege an der apikalen Membran polarer Epithelzellen

Polare Epithelzellen stellen einen essentiellen Baustein für die korrekte Funktion eines Organes dar. Sie bilden ein Monolayer an den Organaußengrenzen aus und stellen sowohl eine Barriere gegen, als auch ein Austauschsystem mit der Umwelt dar. Um diese speziellen Aufgaben wahrzunehmen, bedarf es ei...

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1. Verfasser: Greb, Christoph
Beteiligte: Maier, Uwe G. (Prof. Dr.) (BetreuerIn (Doktorarbeit))
Format: Dissertation
Sprache:Deutsch
Veröffentlicht: Philipps-Universität Marburg 2011
Biologie
Ausgabe:http://dx.doi.org/10.17192/z2011.0103
Schlagworte:
Mx1
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topic apical protein transport
Polare Epithelzellen
Mx1
Polare Epithelzellen
polarized epithelial cells
apikaler Proteintransport
Mx1
Mx1
Galectin-3
Biowissenschaften, Biologie
apikaler Proteintransport
Galectin-3
galectin-3
spellingShingle apical protein transport
Polare Epithelzellen
Mx1
Polare Epithelzellen
polarized epithelial cells
apikaler Proteintransport
Mx1
Mx1
Galectin-3
Biowissenschaften, Biologie
apikaler Proteintransport
Galectin-3
galectin-3
Proteinkomponenten und Transportwege an der apikalen Membran polarer Epithelzellen
Greb, Christoph
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.
title_alt Protein-Components and Transport-Pathways at the Apical Membrane of Polarized Epithelial Cells
format Dissertation
oai_set_str_mv doc-type:doctoralThesis
open_access
ddc:570
xMetaDissPlus
title Proteinkomponenten und Transportwege an der apikalen Membran polarer Epithelzellen
title_short Proteinkomponenten und Transportwege an der apikalen Membran polarer Epithelzellen
title_full Proteinkomponenten und Transportwege an der apikalen Membran polarer Epithelzellen
title_fullStr Proteinkomponenten und Transportwege an der apikalen Membran polarer Epithelzellen
title_full_unstemmed Proteinkomponenten und Transportwege an der apikalen Membran polarer Epithelzellen
title_sort Proteinkomponenten und Transportwege an der apikalen Membran polarer Epithelzellen
ref_str_mv references
publisher Philipps-Universität Marburg
license_str http://archiv.ub.uni-marburg.de/adm/urhg.html
doi_str_mv http://dx.doi.org/10.17192/z2011.0103
edition http://dx.doi.org/10.17192/z2011.0103
publishDate 2011
era_facet 2011
author2 Maier, Uwe G. (Prof. Dr.)
author2_role ths
institution Biologie
url http://archiv.ub.uni-marburg.de/diss/z2011/0103/pdf/dcg.pdf
description Polare Epithelzellen stellen einen essentiellen Baustein für die korrekte Funktion eines Organes dar. Sie bilden ein Monolayer an den Organaußengrenzen aus und stellen sowohl eine Barriere gegen, als auch ein Austauschsystem mit der Umwelt dar. Um diese speziellen Aufgaben wahrzunehmen, bedarf es einer geordneten und spezialisierten Aufteilung der Zytoplasmamembran. Die apikale Zytoplasmamembran ist dabei zur Außenwelt orientiert, während die basolaterale Zytoplasmamembran an benachbarte Zellen oder die Basallamina angrenzt. Für die Aufrechterhaltung dieser Polarität ist ein spezielles Protein- und Lipidsortiersystem nötig. Dabei sind bis dato zwei Grundlegende Proteinsortierplattformen bekannt. Zum einen handelt es sich um die Lipid-Raft-assoziierte Sortierung, bei der sich Proteine mit Vorlieben für solche Detergens-resistenten Membranmikrodomänen in diesen sammeln und als hochmolekulare Cluster zur Oberfläche transportiert werden. Zum anderen existiert ein Sortierweg, der auf der Bindung von Galectin-3 an die zu sortierenden Glykoproteine basiert. Auch in diesem Lipid-Raft-unabhängigen Weg bilden die beteiligten Proteine hochmolekulare Cluster. Zunächst wurde die Rolle der Alphakinase ALPK1 im Transport apikaler Proteine im Mäusedarm untersucht und eine Verwicklung in den Transport der Lipid-Raft-Proteine SI und DPP IV festgestellt. Im Hauptteil dieser Dissertation ging es um die Erweiterung des Verständnisses des Lipid-Raft-unabhängigen Proteinsortierweges. Insbesondere wurde festgestellt, dass für das vesikuläre post-TGN-Trafficking im Galectin-3-Sorting vermutlich keine Mantelproteine nötig sind, wohl aber ein Dynamin-ähnliches Protein involviert ist. Dabei handelt es sich um die große GTPase Mx1. Der knock-down dieses Proteins führte zu einer leicht reduzierten Transporteffizienz des von Galectin-3 sortierten Glykoproteins P75-GFP zur apikalen Zytoplasmamembran. Des Weiteren konnte durch Mx1 eine Verbindung zur unkonventionellen Sekretion von Galectin-3 gefunden werden. Durch siRNA-Experimente gegen Mx1 wurde festgestellt, dass dadurch die Sekretion von Galectin-3 abnahm. Interessanterweise scheint sekretiertes Galectin-3 für seine Wiederaufnahme in die Zelle eine Präferenz für Lipid-Rafts zu entwickeln, da gezeigt werden konnte, dass die Galectin-Endozytose in DRMs abläuft und durch einen Inhibitor der Lipid-Raft-abhängigen Endozytose gehemmt werden kann. Unter Zuhilfenahme der TIRF-Mikroskopie konnte die Oberfläche von MDCK-Zellen in bisher nicht gekannter Detailtreue abgebildet werden. Dabei konnten sowohl Mikrovilli, als auch dicht unter der apikalen Oberfläche befindliche Recycling-Endosomen detektiert werden, die möglicherweise als Sortierstation von Galectin-3 und P75-GFP dienen. Neben den Aufgaben von Galectin-3 bei der Epithelzellpolarisierung scheint dieses Lektin außerdem auch bei der Ausbildung von klarzelligen Nierenzellkarzinomen eine Rolle zu spielen. In einem Teilprojekt konnte gezeigt werden, dass Galectin-3 dort ein erhöhtes Expressionslevel aufweist und verstärkt mit β-Catenin interagiert. Zusammengefasst legen die in dieser Dissertation vorgelegten Studien nahe, dass das vielseitige und in der Entwicklung klarzelliger Nierenzellkarzinome beteiligte Protein Galectin-3 während seines Lebenszyklus, zu dem die Sortierung apikaler nicht mit Lipid-Rafts assoziierter Glykoproteine gehört, eine alternierende Präferenz für diese Detergens-resistenten Membranen aufzeigt und seine unkonventionelle Sekretion offensichtlich mit der Dynamin-ähnlichen GTPase Mx1 in Verbindung steht.
author Greb, Christoph
language German
contents 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.
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dewey-search 570
genre Life sciences
genre_facet Life sciences
topic_facet Biowissenschaften, Biologie
cites_str_mv http://archiv.ub.uni-marburg.de/diss/z2015/0608
last_indexed 2011-08-08T23:59:59Z
first_indexed 2011-06-28T00:00:00Z
building Fachbereich Biologie
thumbnail http://archiv.ub.uni-marburg.de/diss/z2011/0103/cover.png
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Die apikale Zytoplasmamembran ist dabei zur Außenwelt orientiert, während die basolaterale Zytoplasmamembran an benachbarte Zellen oder die Basallamina angrenzt. Für die Aufrechterhaltung dieser Polarität ist ein spezielles Protein- und Lipidsortiersystem nötig. Dabei sind bis dato zwei Grundlegende Proteinsortierplattformen bekannt. Zum einen handelt es sich um die Lipid-Raft-assoziierte Sortierung, bei der sich Proteine mit Vorlieben für solche Detergens-resistenten Membranmikrodomänen in diesen sammeln und als hochmolekulare Cluster zur Oberfläche transportiert werden. Zum anderen existiert ein Sortierweg, der auf der Bindung von Galectin-3 an die zu sortierenden Glykoproteine basiert. Auch in diesem Lipid-Raft-unabhängigen Weg bilden die beteiligten Proteine hochmolekulare Cluster. Zunächst wurde die Rolle der Alphakinase ALPK1 im Transport apikaler Proteine im Mäusedarm untersucht und eine Verwicklung in den Transport der Lipid-Raft-Proteine SI und DPP IV festgestellt. Im Hauptteil dieser Dissertation ging es um die Erweiterung des Verständnisses des Lipid-Raft-unabhängigen Proteinsortierweges. Insbesondere wurde festgestellt, dass für das vesikuläre post-TGN-Trafficking im Galectin-3-Sorting vermutlich keine Mantelproteine nötig sind, wohl aber ein Dynamin-ähnliches Protein involviert ist. Dabei handelt es sich um die große GTPase Mx1. Der knock-down dieses Proteins führte zu einer leicht reduzierten Transporteffizienz des von Galectin-3 sortierten Glykoproteins P75-GFP zur apikalen Zytoplasmamembran. Des Weiteren konnte durch Mx1 eine Verbindung zur unkonventionellen Sekretion von Galectin-3 gefunden werden. Durch siRNA-Experimente gegen Mx1 wurde festgestellt, dass dadurch die Sekretion von Galectin-3 abnahm. Interessanterweise scheint sekretiertes Galectin-3 für seine Wiederaufnahme in die Zelle eine Präferenz für Lipid-Rafts zu entwickeln, da gezeigt werden konnte, dass die Galectin-Endozytose in DRMs abläuft und durch einen Inhibitor der Lipid-Raft-abhängigen Endozytose gehemmt werden kann. Unter Zuhilfenahme der TIRF-Mikroskopie konnte die Oberfläche von MDCK-Zellen in bisher nicht gekannter Detailtreue abgebildet werden. Dabei konnten sowohl Mikrovilli, als auch dicht unter der apikalen Oberfläche befindliche Recycling-Endosomen detektiert werden, die möglicherweise als Sortierstation von Galectin-3 und P75-GFP dienen. Neben den Aufgaben von Galectin-3 bei der Epithelzellpolarisierung scheint dieses Lektin außerdem auch bei der Ausbildung von klarzelligen Nierenzellkarzinomen eine Rolle zu spielen. In einem Teilprojekt konnte gezeigt werden, dass Galectin-3 dort ein erhöhtes Expressionslevel aufweist und verstärkt mit β-Catenin interagiert. Zusammengefasst legen die in dieser Dissertation vorgelegten Studien nahe, dass das vielseitige und in der Entwicklung klarzelliger Nierenzellkarzinome beteiligte Protein Galectin-3 während seines Lebenszyklus, zu dem die Sortierung apikaler nicht mit Lipid-Rafts assoziierter Glykoproteine gehört, eine alternierende Präferenz für diese Detergens-resistenten Membranen aufzeigt und seine unkonventionelle Sekretion offensichtlich mit der Dynamin-ähnlichen GTPase Mx1 in Verbindung steht. 2011-05-05 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. 2011-08-08 2011-06-28 Philipps-Universität Marburg ths Prof. Dr. Maier Uwe G. Maier, Uwe G. (Prof. Dr.) Greb, Christoph Greb Christoph
recordtype opus
id urn:nbn:de:hebis:04-z2011-0103
urn_str urn:nbn:de:hebis:04-z2011-01037
collection Monograph
uri_str http://archiv.ub.uni-marburg.de/diss/z2011/0103
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callnumber-label diss z2011 0103
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