Regensburg 2019 – wissenschaftliches Programm

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BP: Fachverband Biologische Physik

BP 31: Cell adhesion and migration, multicellular systems II

BP 31.8: Vortrag

Freitag, 5. April 2019, 11:30–11:45, H10

Visualization of intracellular calcium levels in Dictyostelium discoideum with a genetically encoded reporter — •Manuel Frey, Sven Flemming, Serena Cucinotta, and Carsten Beta — University of Potsdam, Potsdam, Germany

Calcium is an important second messenger in eukaryotic cells and is crucial for several signaling pathways related to cellular functions such as chemotaxis and cell motility. To visualize calcium in the social amoeba Dictyostelium discoideum, we expressed a genetically encoded GFP based calcium reporter at the plasma membrane. This enabled us to monitor spatiotemporal changes in the intracellular Ca2+ levels.

We could detect global increases in Ca2+ levels after chemotactic stimulation with cAMP. Mechanical stimulation of cells led to a local Ca2+ response. Furthermore, we could detect short, focal increases of Ca2+ at the basal plasma membrane, which coincided with the appearance of F-actin foci at the same location. In cells exposed to continuous shear flow, we observed periodic oscillations of the intracellular Ca2+ levels. Interestingly, once excited these oscillations continued for several minutes even after the shear flow was stopped. In contrast, application of a short pulse of shear flow induced only single responses.

Inhibitor experiments suggested that the observed Ca2+ response at the plasma membrane is caused by an influx of Ca2+ needed to replenish the internal stores for Ca2+. Our results show that localized increases in calcium can be visualized with our new reporter in live cell imaging experiments and revealed interesting oscillatory behavior under shear flow.