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Non-chemotactic Dictyostelium discoideum mutants with altered cGMP signal transduction
H Kuwayama1, S Ishida, P J Van Haastert
1Department of Biochemistry, University of Groningen, The Netherlands.
Abstract:
Folic acid and cAMP are chemoattractants in Dictyostelium discoideum, which bind to different surface receptors. The signal is transduced from the receptors via different G proteins into a common pathway which includes guanylyl cyclase and acto-myosin. To investigate this common pathway, ten mutants which do not react chemotactically to both cAMP and folic acid were isolated with a simple new chemotactic assay. Genetic analysis shows that one of these mutants (KI-10) was dominant; the other nine mutants were recessive, and comprise nine complementation groups. In wild-type cells, the chemoattractants activate adenylyl cyclase, phospholipase C, and guanylyl cyclase in a transient manner. In mutant cells the formation of cAMP and IP3 were generally normal, whereas the cGMP response was altered in most of the ten mutants. Particularly, mutant KI-8 has strongly reduced basal guanylyl cyclase activity; the enzyme is present in mutant KI-10, but can not be activated by cAMP or folic acid. The cGMP response of five other mutants is altered in either magnitude, dose dependency, or kinetics. These observations suggest that the second messenger cGMP plays a key role in chemotaxis in Dictyostelium.
Insights
Dictyostelium discoideum uses folic acid and cyclic AMP (cAMP) for chemotaxis. This study identifies key roles for cyclic GMP (cGMP) in the common signaling pathway, revealing new insights into cellular movement.
Area of Science:
- Cellular Biology
- Biochemistry
- Genetics
Background:
- Folic acid and cyclic AMP (cAMP) are crucial chemoattractants in Dictyostelium discoideum.
- Signal transduction involves G proteins, guanylyl cyclase, and acto-myosin.
- Understanding the common pathway is vital for deciphering chemotaxis.
Purpose of the Study:
- To investigate the common signaling pathway for folic acid and cAMP chemotaxis.
- To identify and characterize mutants defective in chemotaxis to both signals.
- To elucidate the role of cyclic GMP (cGMP) in Dictyostelium chemotaxis.
Main Methods:
- Isolation of ten non-chemotactic mutants using a novel chemotactic assay.
- Genetic analysis including complementation tests to determine mutant types (dominant/recessive).
- Biochemical assays measuring adenylyl cyclase, phospholipase C, and guanylyl cyclase activities in wild-type and mutant cells.
Main Results:
- Nine recessive and one dominant mutant were identified, representing nine complementation groups.
- Mutant analysis revealed alterations in the cyclic GMP (cGMP) response in most strains.
- Specific mutants showed reduced basal guanylyl cyclase activity (KI-8) or impaired enzyme activation (KI-10).
Conclusions:
- The cyclic GMP (cGMP) second messenger plays a critical role in Dictyostelium discoideum chemotaxis.
- Defects in guanylyl cyclase activity or its regulation significantly impair chemotactic responses.
- This research provides a deeper understanding of the molecular mechanisms underlying cellular chemotaxis.