Regulation der Autophagie durch Miz1 in Brustdrüsenzellen und embryonalen Fibroblasten

Die Autophagie beschreibt einen katabolischen Prozess, bei dem für den Abbau bestimmtes Zellmaterial in Autophagosomen eingeschlossen und durch deren Verschmelzung mit Lysosomen zersetzt wird. Durch diesen Selbstreinigungs-Vorgang trägt sie zur Aufrechterhaltung der zellulären Homöostase bei und hil...

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Bibliographic Details
Main Author: Paul, Tanja
Contributors: Elsässer, Hans-Peter (Prof.Dr.) (Thesis advisor)
Format: Dissertation
Published: Philipps-Universität Marburg 2016
Klinische Zytobiologie und Zytopathologie
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Table of Contents: Autophagy is a catabolic process in which cellular content determined for degradation is engulfed by autophagosomes and finally degraded by their fusion with lysosomes. With this self-cleaning mechanism it contributes to the maintainence of the cellular homeostasis and helps with the adaptation to nutrient-poor conditions by obtaining energy from intracellular resources. In addition to these main functions, autophagy fulfills important functions in the context of cell maturation, differentiation and for the immune system. Aside from that, an impaired autophagic process is associated with different disorders. The correct functioning of autophagy requires interaction of numerous autophagy-associated proteins which are tightly regulated. In previous works, it was already shown that the transcription factor Miz 1 is involved in the regulation of autophagy. Both morphological and gene expression level differences were observed between cells with wildtype Miz 1 and cells with truncated Miz 1. The aim of this thesis was to reproduce these previously found differences in an improved cell culture model and furthermore to analyze the influence of the interaction between Miz 1 and Myc on the autophagic process. For that purpose, the murine mammary cell line HC11 was used. Firstly, the change of gene expression levels from Miz 1 and other genes were analyzed over time when cells were incubated with nutrient-low medium. After an initial upregulation of CDKN1a expression, Miz 1 and Myc levels increased as CDKN1a went down again. In the further course of this work, HC11 cells were infected with different constructs aiming to overexpress Miz 1, Myc and the Myc-mutant V394D. This mutation makes Myc interaction with Miz1 not possible. These infected cells were treated with EBSS for 6 hours and subsequently immunocytochemically analyzed. Cells overexpressing Miz 1 showed a slightly increased autophagic activity, as quantified by LC 3 immunofluorescence, compared to the corresponding control. While cells overexpressing Myc showed a boosted autophagy, cells with the mutated Myc-sequence did not differ from control cells. The latter results could be reproduced with electron microscopy and are indicative for a role of the interaction between Miz 1 and Myc in the regulation of the autophagic process. Also, differences in gene expression levels between the two cell lines could be assessed after induction of autophagy. Cells that overexpressed MycV394D were characterized by a significantly induced mRNA expression of Atg 5, Atg 12 and Spast compared to cells overexpressing the non mutated Myc gene. Some additional genes showed different expression tendencies between the two constructs. A knockdown of Miz 1 via interference shRNA was unfortunately not successful. For that reason, another cell model was established in which the exons that code for the POZ-domain of Miz 1 are excised by the Cre-ER recombinase after tamoxifen induction. In accordance with previous results, deletion of Miz 1 led to a decreased adaptation ability to a nutrient-low environment in these modified mouse embryonic fibroblasts. In contrast to previous experiments, no Miz 1-dependent expression differences in Atg 4c, Atg 5 and Atg 7 were observed. Furthermore, other cell lines overexpressing GFP-labeled LC 3 and a corresponding control vector, were developed. These can be used for real time investigation of the autophagic process. Summarizing, this work not only confirmed previous results on the influence of Miz 1 on autophagy but additionally demonstrated the relevance of the interaction between Miz 1 and Myc in this essential process.