Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Burgstaller, S.
Visualization of local intracellular and cell surface cation alterations using fluorescent protein-based probes.
PhD-Studium (Doctor of Philosophy); Humanmedizin; [ Dissertation ] Graz Medical University; 2020. pp. 106
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Graier Wolfgang
- Hallström Seth
- Malli Roland
- Altmetrics:
- Abstract:
- The maintenance and regulation of intracellular pH levels is of utmost importance for every cell, since many essential metabolic processes rely on a tightly controlled pH homeostasis. Eventually, small pH alterations might disturb cell metabolism by affecting important cellular pathways, thereby promoting various diseases. However, not only intracellular, but also extracellular pH alterations play an important role. Hence, the determination and visualization of intra- and extracellular pH values might lead to crucial insight into the development and progression of various diseases. In order to measure pH levels and alterations with high precision, we developed pH-Lemon, a pH indicator based on Förster resonance energy transfer (FRET). pH-Lemon, a probe consisting of a pH stable and a pH sensitive fluorescent protein (FP), is perfectly suitable for the visualization of intracellular acidic pH values. Localization of pH-Lemon within secretory vesicles and autolysosomes revealed a great heterogeneity of vesicular pH values. Reversible alterations of these intravesicular pH levels could be measured with high precision in real-time. Considering the application of pH-Lemon to detect extracellular pH levels, different techniques to obtain cell membrane localization of the probe have been developed. Besides expression of GPI-anchored pH-Lemon, we describe novel techniques comprising the application of recombinant purified genetically-encoded sensors. The immobilization of purified sensors on the plasma membrane of living cells opens new doors for yet unknown applications of genetically-encoded probes. Furthermore, our method proved perfectly suitable for the immobilization of recombinant sensor proteins on glass surfaces, leading to new approaches in the field of cell biology, medicine and biotechnology.