Evidence that cyclic gmp regulates myosin interaction with the cytoskeleton during chemotaxis of Dictyostelium

Abstract

Amoebae of Dictyostelium discoideum respond to a chemotactic cyclic AMP stimulus within 10 s by the formation of an intracellular peak of cyclic GMP. In wild-type cells the cyclic GMP is rapidly degraded by a cyclic GMP-specific phosphodiesterase. In ‘streamer F’ mutants this enzyme is defective, due to mutation in the enzyme structural gene, and the cyclic GMP persists several times longer than the normal period, an effect that is correlated with a persistence in cell elongation during the chemotactic movement phase. In this study we have used the streamer mutants NP368 and NP377, and their parental strain XP55, to study changes in cytoskeletal proteins during the chemotactic response. We have studied three proteins that change their association with the cytoskeleton after stimulation of amoebae with the chemoattractant cyclic AMP: (1) actin, (2) a protein with an apparent M_r of 190x10³ and (3) myosin heavy chain. Both actin and the 190x10³M_r protein were found to accumulate rapidly in the cytoskeleton after cyclic AMP stimulation, with a sharp peak at 5 s, and showed similar changes in the parental and streamer mutants. However, the cytoskeletal level of myosin heavy chain showed a different pattern of changes, which also clearly differed in the streamer mutants compared with the parental strain XP55. In XP55 myosin heavy chain showed an initial drop after cyclic AMP stimulation, with a trough at 3–10 s followed by a rapid rise to a sharp peak at 20–25 s. In contrast, the myosin heavy chain in the streamer mutants produced a broad peak that persisted several times longer than the parental strain. We conclude that in the streamer mutants the defect in cyclic GMP phosphodiesterase that produces the broad peak of cyclic GMP is causally correlated with the broad peak of cytoskeletal myosin, and we suggest that this is connected with the observed phenotype of prolonged cell elongation during chemotaxis in these mutants.