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The membrane and actin cortex of a motile cell can autonomously differentiate into two states, one typical of the front, the other of the tail. On the substrate-attached surface of Dictyostelium discoideum cells, dynamic patterns of front-like and tail-like states are generated that are well suited to monitor transitions between these states. To image large-scale pattern dynamics independently of boundary effects, we produced giant cells by electric-pulse-induced cell fusion. In these cells, actin waves are coupled to the front and back of phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-rich bands that have a finite width. These composite waves propagate across the plasma membrane of the giant cells with undiminished velocity. After any disturbance, the bands of PIP3 return to their intrinsic width. Upon collision, the waves locally annihilate each other and change direction; at the cell border they are either extinguished or reflected. Accordingly, expanding areas of progressing PIP3 synthesis become unstable beyond a critical radius, their center switching from a front-like to a tail-like state. Our data suggest that PIP3 patterns in normal-sized cells are segments of the self-organizing patterns that evolve in giant cells.
Recent work has demonstrated that the receptor-mediated signaling system in chemotactic amoeboid cells shows typical properties of an excitable system. Here, we delivered spatially confined stimuli of the chemoattractant cAMP to the membrane of differentiated Dictyostelium discoideum cells to investigate whether localized receptor stimuli can induce the spreading of excitable waves in the G-protein-dependent signal transduction system. By imaging the spatiotemporal dynamics of fluorescent markers for phosphatidylinositol (3,4,5)-trisphosphate (PIP3), PTEN and filamentous actin, we observed that the activity of the signaling pathway remained spatially confined to the stimulated membrane region. Neighboring parts of the membrane were not excited and no receptor-initiated spatial spreading of excitation waves was observed. To generate localized cAMP stimuli, either particles that carried covalently bound cAMP molecules on their surface were brought into contact with the cell or a patch of the cell membrane was aspirated into a glass micropipette to shield this patch against freely diffusing cAMP molecules in the surrounding medium. Additionally, the binding site of the cAMP receptor was probed with different surface-immobilized cAMP molecules, confirming results from earlier ligand-binding studies.