Komponenten und Wege von post-Golgi Vesikeln beim Transport zur apikalen Plasmamembran
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Philipps-Universität Marburg
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Abstract
The plasma membrane of epithelial cells shows an asymmetric organization,
characterized by a functionally and morphologically distinct apical and basolateral
domain. This polarity is a prerequisite for the integrity of an epithelial tissue and for the
directional transport. The establishment and the maintenance of cellular polarity depend
on a highly specialized cellular sorting and transport machinery.
Microtubules are an important component of this transport machinery and they
provide tracks for the vesicular transport. Important features of microtubules are diverse
posttranslational modifications. The detyrosination represents a form of modification that
occurs on the C-terminus of α-tubulin. Within the tyrosination/detyrosination-cycle the
tyrosine is removed by a vasohibin/chaperon complex and subsequently re-added by
tubulin tyrosine ligase (TTL). As part of this work, the influence of tubulin detyrosination
on cellular polarity of MDCK kidney epithelial cells was studied. Knocking out TTL
resulted in a dramatically increase in the level of detyrosinated tubulin as well as
acetylated tubulin. Changing tubulin contents towards detyrosinated or tyrosinated
microtubules by modulating TTL expression affected the epithelial morphology.
Interestingly, TTL knockout cells were more mobile than cells overexpressing TTL.
Furthermore, detyrosinated microtubule possibly influenced the number and size of focal
adhesions. The presented observations indicate that a dynamic adaptation of
detyrosinated and tyrosinated microtubules is essential for the regulation of cellular
polarization, migration and adhesion.
Beside microtubules, the sorting and transport machinery consist of a diverse set of
proteins, which are crucial for cargo delivery to the correct membrane domain. These
include motor proteins, adaptor proteins, lectins, Rab-GTPases and cargo molecules.
Among these components, the large GTPase Myxovirus Resistance Protein 1 (Mx1), a
member of the dynamin superfamily, was identified on apical post-Golgi vesicles. Mx-
proteins are known as interferon induced proteins, which are expressed after viral
infection. In MDCK cells, Mx1 shows no antiviral activity and is constitutively expressed.
Mx-proteins exhibit many characteristic properties that suggest functions in basic cellular
processes. The large GTPase was identified as an important regulatory component of
the apical transport system. Thereby, the large GTPase associates with compartments of
the early and late secretory pathway. Since vesicular structures positive for Mx1 are
highly dynamic, the influence of the microtubule cytoskeleton on this motility was studied
using live cell imaging. Microtubule disruption induced by nocodazole, inhibits long-range
trafficking. Using biochemical and fluorescence microscopic methods, the association of
canine Mx1 with α-tubulin was verified. Furthermore, it was demonstrated for the first
time that Mx1 interacts with the motor protein Kif5B. In agreement with these
observations the knockout of Mx1 or mutation in the membrane interaction motif of the
unstructured L4 loop decreased the efficiency of apical cargo delivery. The L4 loop
mutant still interacts with Kif5B, however the mutation causes elongation of Mx1-positive
compartments. It is thus conceivable that Mx1 functions in the fission of transport
vesicles based on structural similarities to classical dynamins.
For the first time, this work shows a functional role of the large GTPase Mx1 in the
apical protein trafficking. Mx1 not only stabilizes the interaction between Kif5B,
microtubules and apical transport carriers, but also aids in vesicle fission.
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Dates
Created: 2019Issued: 2020-06-25Updated: 2020-06-25
Faculty
Medizin
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
DynmainproteinepitheliatraffickingMx1GTPaseposttranslatiolanle TubulinmodifikationenvesicleZellpolaritätTubulin
DFG-subjects
BiowissenschaftenProteintransportCytologieBiologie
DDC-Numbers
610
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Ringer, Karina: Komponenten und Wege von post-Golgi Vesikeln beim Transport zur apikalen Plasmamembran. : Philipps-Universität Marburg 2020-06-25. DOI: https://doi.org/10.17192/z2020.0269.