Hypoxia stimulates retrograde membrane trafficking to the trans-Golgi network via recruitment of T-plastin
by Stephanie Naas
Date of Examination:2018-10-25
Date of issue:2018-10-24
Advisor:Prof. Dr. Dörthe Katschinski
Referee:Prof. Dr. Dörthe Katschinski
Referee:Prof. Dr. Peter Schu
Persistent Address:
http://dx.doi.org/10.53846/goediss-7114
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Abstract
English
Hypoxia is essential for many physiological functions but also plays a pivotal role in the pathogenesis of a variety of epidemiologically highly relevant diseases. Adequate adaptation mechanisms to oxygen deprivation are a crucial prerequisite for cellular survival. The master regulators of oxygen-dependent gene expression of basically all animal cells are the hypoxia-inducible factors (HIFs). Membrane trafficking is a fundamental feature of cells to adapt to their environment. Traffic of membrane-enclosed vesicles comprises the anterograde transport route of cargo from biosynthetic compartments to the plasma membrane but also cargo internalization and the retrieval of plasma membrane components via the retrograde pathway. The modulation of endosomal transport resulting in altered processing of cargo, signaling and membrane composition as a reaction to extracellular stimuli contributes to the preservation of cellular homeostasis. The aim of this study was therefore to analyze the impact of hypoxia on membrane trafficking. To this end, we determined total membrane turnover by using the membrane marker FM® 1-43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino) styryl) pyridinium dibromide) and confocal microscopy. Hypoxia caused an increase in membrane uptake upon hypoxic incubation in MDA-MB 231 and mouse embryonal fibroblast cells. Reoxygenation quickly reconstituted the normoxic phenotype. The influence of oxygen deprivation on different endocytic pathways was investigated by analyzing the uptake of specific marker proteins. The intensified uptake of cholera toxin B subunit suggested an intensified retrograde transport to the trans-Golgi network. This finding was confirmed by performing co-staining experiments with the novel membrane probe mCLING (membrane-binding fluorophore-cysteine-lysine-palmitoyl group) and fluorescently labelled antibodies that were directed against compartment-specific proteins. Analysis of different HIF-1α gain and loss of function cell lines demonstrated that the manifestation of the described phenotype was independent of the HIF pathway. In order to elucidate the mechanism mediating this effect, proteome analysis taking advantage of the SILAC technology and western blotting were used to detect alterations in the abundance and spatiotemporal distribution of proteins owing to restricted oxygen availability. The actin-bundling protein T-plastin was found to be recruited to the plasma membrane due to low oxygen levels. T-plastin knockdown cells demonstrated blunted membrane uptake in hypoxia showing that this plastin isoform facilitates the hypoxia-induced membrane trafficking. In summary, hypoxia causes profound changes in the membrane trafficking activity and the endomembrane system of cells by favoring retrograde transport to the trans-Golgi network. Since this effect is not cell type- or species-specific, hypoxia-induced membrane trafficking constitutes a fundamental biological function. The results obtained in this research project contribute to the understanding of hypoxia-induced changes at the subcellular level that occur in physiological and pathophysiological conditions.
Keywords: hypoxia; retrograde transport; membrane trafficking; T-plastin
