|Titel:||Use of fluorescent sensors to visualize P2X7-mediated changes in local ATP concentrations in the cytosol and at the cell surface||Sprache:||Englisch||Autor*in:||Kaschubowski, Klaus Eric||Schlagwörter:||ATP-release; P2X7; FRET; Live-cell imaging||Erscheinungsdatum:||2020||Tag der mündlichen Prüfung:||2020-12-16||Zusammenfassung:||
Adenosine triphosphate (ATP) is the primary energy source in all living organisms. In addition, ATP is an important extracellular signaling molecule as a ligand for P2X and P2Y receptors. P2X7 has been thoroughly studied in the context of immunology since it is expressed on the majority of cells of the immune system. Activation of P2X7 leads to various downstream effects, such as enhancement of T cell receptor (TCR) signals, inflammasome assembly, pore formation, and cell death. Most cells can release ATP in a regulated fashion, but the mechanisms and kinetics of these processes are not well understood yet.
ATP assays are mostly based on the luciferase-luciferin interaction and report only global changes of ATP. By contrast, fluorescent sensors based on ATP-binding proteins, expressed in different cell compartments or small molecules attached to the cell surface enable the visualization of local ATP concentrations by live-cell imaging to help to better understand the mechanisms underlying ATP release.
This study aimed to find and develop appropriate ATP sensors and methods to visualize ATP concentrations at a sub-cellular level. To achieve this, the model of P2X7-dependent ATP release was used. First, genetically encoded, Förster resonance energy transfer (FRET)-based sensors were expressed in the cytosol or the plasma membrane of 3T3 or Yac-1 cells. These sensors did detect changes in cytosolic and cell-surface ATP concentrations after gating of P2X7 via ATP or NAD+-dependent ADP-ribosylation. Local ATP concentrations in the cytosol and at the cell surface were visualized and measured by live-cell imaging and flow cytometry. Gating of P2X7 resulted in an increase of ATP at the cell surface accompanied by a slow continuous decrease in cytosolic ATP. By contrast, the loss of cytosolic ATP induced by complement-dependent cytotoxicity (CDC) showed an initial delay of 2 min, followed by a steep decline.
Furthermore, small-molecule sensors based on a Zn(II)-dipicolylamine (DPA) backbone were evaluated. These were synthesized with reactive groups to attach them to cells or solid-phase surfaces by click chemistry. By attaching lipophilic cell anchors, these sensors were also integrated directly into cell membranes. However, in some sensors, modification with reactive groups influenced their sensitivity and selectivity towards nucleotides. Finally, genetically encoded single-wavelength sensors were produced and validated in vitro. Compared to their FRET-based counterparts, they could be produced in high quantity in a bacterial expression system. These sensors were targeted to cells of interest by binding them to streptavidincoated beads, which were additionally loaded with an antibody directed against a cell surface antigen.
The results of this study will contribute to the further development of ATP sensors and lead to a better understanding of ATP release mechanisms and their significant role in immunological processes such as T cell activation.
|Enthalten in den Sammlungen:||Elektronische Dissertationen und Habilitationen|
geprüft am 31.07.2021
geprüft am 31.07.2021