Intracellular photoactivation of caged cGMP induces myosin II and actin responses in motile cells

Abstract

Cyclic GMP (cGMP) is a ubiquitous second messenger in eukaryotic cells. It is assumed to regulate the association of myosin II with the cytoskeleton of motile cells. When cells of the social amoeba Dictyostelium discoideum are exposed to chemoattractants or to increased osmotic stress, intracellular cGMP levels rise, preceding the accumulation of myosin II in the cell cortex. To directly investigate the impact of intracellular cGMP on cytoskeletal dynamics in a living cell, we released cGMP inside the cell by laser-induced photo-cleavage of a caged precursor. With this approach, we could directly show in a live cell experiment that an increase in intracellular cGMP indeed induces myosin II to accumulate in the cortex. Unexpectedly, we observed for the first time that also the amount of filamentous actin in the cell cortex increases upon a rise in the cGMP concentration, independently of cAMP receptor activation and signaling. We discuss our results in the light of recent work on the cGMP signaling pathway and suggest possible links between cGMP signaling and the actin system. Insight, innovation, integrationSecond messengers like cGMP play a central role in the regulatory pathways of living cells. Here, we demonstrate that an increase in intracellular cGMP can trigger not only myosin II but also actin responses in motile amoeboid cells. In previous studies, an increase in intracellular cGMP was induced indirectly via membrane receptor stimulation. In the present work, we employ, for the first time, the direct light-induced release of cGMP from a caged precursor to raise the cytosolic cGMP level in Dictyostelium cells that carry fluorescent markers for filamentous actin and myosin II. Based on this advanced combination of live cell photo-uncaging and multi-color confocal microscopy, we could identify a link between cGMP and actin dynamics in motile amoeboid cells.

Moving towards a paradigm: common mechanisms of chemotactic signaling in Dictyostelium and mammalian leukocytes

“…Although this regulation appears to be dispensable for myosin II function in growth and development, it is involved in cell depolarization during chemotaxis as mentioned above [268,247]. A recent report suggests that in addition to its role in myosin II regulation, cGMP can also stimulate actin polymerization [269]. …”

Phosphodiesterase PdeD, dynacortin, and a Kelch repeat‐containing protein are direct GSK3 substrates in Dictyostelium that contribute to chemotaxis towards cAMP

“…We demonstrate that the amount of F-actin polymerization in response to the chemoattractant cAMP is prolonged in pdeDcells compared to wild type cells, presumably because of the prolonged intracellular concentration of cGMP, which suggests a link between PdeD and the actin system. Indeed, the intracellular release of cGMP elicits a response in both actin polymerization and cortical localization of MyoII filaments in wild type cells (Pfannes et al, 2013). Cortical actin polymerization rises in a sharp transient peak within the first 10 s after intracellular release of cGMP, independently of cAMP receptor activation and signalling, while cortical MyoII localization becomes maximal after approximately 30 s (Pfannes et al, 2013).…”

“…Indeed, the intracellular release of cGMP elicits a response in both actin polymerization and cortical localization of MyoII filaments in wild type cells (Pfannes et al, 2013). Cortical actin polymerization rises in a sharp transient peak within the first 10 s after intracellular release of cGMP, independently of cAMP receptor activation and signalling, while cortical MyoII localization becomes maximal after approximately 30 s (Pfannes et al, 2013). Our observations indicate that when we express PdeD or any of its mutant derivatives (PdeD 2Ala or PdeD 2Asp ) in pdeDcells, F-actin levels are reduced compared to wild type cells.…”

Interplay between myosin II and actin dynamics in chemotactic amoeba

“…Dynamical properties of actin and myosin II and their interactions were extensively studied in vitro [2,26,27] and in vivo in motile cells [28][29][30] or in cells responding to the external stimulation with cAMP [31][32][33] or by mechanical confinement [34]. For example, a coupling between Rho and actin was studied in [35], where the actin cell cortex was described as an excitable medium, where pattern formation in the form of cortical waves of Rho activity is coupled to actin in a reaction-diffusion based manner.…”