Characterization of dictyostelium discoideum coronin A and its role in starvation induced development

The work presented here relates the observations and results of a detailed investigation of the protein coronin A in the slime mould Dictyostelium discoideum.
Since the discovery of coronin A in D. discoideum, homologous proteins have been identified in a great number of eukaryotic species.
These proteins are now grouped together into the coronin family of proteins. Coronins have been implied to participate in diverse processes, such as cell migration, calcium signaling, phagocytosis, and brain morphogenesis.
The exact mechanisms with which coronin proteins influence these processes remain elusive, but are often explained by citing coronin’s role in governing actin dynamics with reference to the original observations made in D. discoideum.
Coronin A was indeed first co-purified from an actin-myosin complex isolated from Dictyostelium discoideum.
The subsequently generated coronin A deletion mutant exhibited pleiotropic defects, including defects in processes considered to be dependent on F-actin regulation. Hence, the family of coronin proteins are generally described as being F-actin interacting and modulatory proteins.
However, more recent observations on the function of mammalian coronins suggest that coronin proteins are also involved in F-actin independent processes.
So, do coronin proteins act by modulating F-actin dynamics or not?
To investigate this apparent discrepancy, we decided to revisit the coronin A deletion phenotypes in Dictyostelium discoideum. To this end, we generated new coronin A deletion mutants in a distinct wild type background.
We report here that the phenotypes of the novel corA-deletion mutant follow the same trend as previously reported, albeit to a lesser degree of severity with respect to phagocytosis and cell motility.
Furthermore, in the new deletion strains, the cytokinesis defects are more pronounced in cells grown in suspension culture, rather than in adherent cells as had been previously reported.
In a next step we aimed at characterizing the purified coronin A protein. For easy purification, we generated a coronin A deletion mutant that expresses a FLAG-tagged version of coronin A.
The isolated coronin A proteins were of high purity and were used for in vitro interaction studies and biophysical characterization. We could not confirm association of coronin A with F-actin in vivo, but did observe co-precipitation of coronin A with F-actin in vitro at low concentrations of NaCl.
Furthermore, we present evidence that purified D.discoideum coronin A forms dimers, and not trimers as has been reported for several other coronin proteins.
In mammalian leukocytes, coronin 1, the closest mammalian homologue to D. discoideum coronin A, was found to influence cell signaling pathways in a Ca2+-dependent manner.
For this reason we decided to investigate, if coronin A also has an impact on signaling pathways of the D.discoideum cell. Many biological systems respond to environmental changes by activating intracellular signaling cascades that elicit an appropriate response.
One such system, a prominent example of an environmental sensing mechanism, is presented by the social amoeba Dictyostelium discoideum.
While normally living as unicellular organisms, when food sources become scarce, individual cells spontaneously release cyclic AMP.
Amplification of the cAMP production by a positive feedback loop triggers a developmental program that results in the formation of a multicellular slug that can develop into a spore-bearing fruiting body, allowing long term survival.
How the initial starvation signal is transduced into the first pulses of cAMP release has remained unclear.
We show here that cells lacking the evolutionary conserved WD repeat protein coronin A, were unable to initiate cAMP release following starvation, and as a result failed to initiate the developmental program required for fruiting body formation.
External addition of cAMP to a coronin A-deficient population fully restored the developmental program.
These results suggest that coronin A is part of a signal transduction cascade essential for system initiation leading to multicellular development in D.discoideum.
In all metazoans, cAMP is an important second messenger involved in a variety of processes.
That the evolutionarily conserved family of coronin proteins might be involved in generating an appropriate cellular response to environmental stimuli via cAMP-signaling, presents an intriguing possibility.
Of course, the work related here cannot rule out that coronin proteins mediate all their effects via manipulation of the cytoskeleton.
The exact mechanism of coronin activity still remains to be elucidated. But we provide evidence that the deficiencies in D.dictyostelium cells lacking coronin A are not simply a product of impaired cytoskeletal dynamics.