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Gewählte Publikation:
SHR Neuro Krebs Kardio Lipid Stoffw Microb
Burgstaller, S; Bischof, H; Gensch, T; Stryeck, S; Gottschalk, B; Ramadani-Muja, J; Eroglu, E; Rost, R; Balfanz, S; Baumann, A; Waldeck-Weiermair, M; Hay, JC; Madl, T; Graier, WF; Malli, R.
pH-Lemon, a Fluorescent Protein-Based pH Reporter for Acidic Compartments.
ACS Sens. 2019; 4(4): 883-891.
Doi: 10.1021/acssensors.8b01599
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- Führende Autor*innen der Med Uni Graz
- Burgstaller Sandra
- Co-Autor*innen der Med Uni Graz
- Bischof Helmut
- EROGLU Emrah
- Gottschalk Benjamin
- Graier Wolfgang
- Madl Tobias
- Malli Roland
- Ramadani-Muja Jeta
- Rost René
- Stryeck Sarah
- Waldeck-Weiermair Markus
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- Abstract:
- Distinct subcellular pH levels, especially in lysosomes and endosomes, are essential for the degradation, modification, sorting, accumulation, and secretion of macromolecules. Here, we engineered a novel genetically encoded pH probe by fusing the pH-stable cyan fluorescent protein (FP) variant, mTurquoise2, to the highly pH-sensitive enhanced yellow fluorescent protein, EYFP. This approach yielded a ratiometric biosensor-referred to as pH-Lemon-optimized for live imaging of distinct pH conditions within acidic cellular compartments. Protonation of pH-Lemon under acidic conditions significantly decreases the yellow fluorescence while the cyan fluorescence increases due to reduced Förster resonance energy transfer (FRET) efficiency. Because of its freely reversible and ratiometric responses, pH-Lemon represents a fluorescent biosensor for pH dynamics. pH-Lemon also shows a sizable pH-dependent fluorescence lifetime change that can be used in fluorescence lifetime imaging microscopy as an alternative observation method for the study of pH in acidic cellular compartments. Fusion of pH-Lemon to the protein microtubule-associated protein 1A/1B-light chain 3B (LC3B), a specific marker of autophagic membranes, resulted in its targeting within autolysosomes of HeLa cells. Moreover, fusion of pH-Lemon to a glycophosphatidylinositol (GPI) anchor allowed us to monitor the entire luminal space of the secretory pathway and the exoplasmic leaflet of the plasma membrane. Utilizing this new pH probe, we revealed neutral and acidic vesicles and substructures inside cells, highlighting compartments of distinct pH throughout the endomembrane system. These data demonstrate, that this novel pH sensor, pH-Lemon, is very suitable for the study of local pH dynamics of subcellular microstructures in living cells.
- Find related publications in this database (Keywords)
- array confocal laser scanning microscopy
- FLIM
- fluorescence microscopy
- FRET
- genetically encoded probes
- Golgi apparatus
- GPI-anchor
- pH