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Ca(2+) signalling is not required for chemotaxis in Dictyostelium.

ABSTRACT: Dictyostelium cells can move rapidly towards a source of cyclic-AMP (cAMP). This chemoattractant is detected by G-protein-linked receptors, which trigger a signalling cascade including a rapid influx of Ca(2+). We have disrupted an inositol 1,4,5-trisphosphate (InsP(3)) receptor-like gene, iplA, to produce null cells in which Ca(2+) entry in response to chemoattractants is abolished, as is the normal increase in free cytosolic Ca(2+) ([Ca(2+)](c)) that follows chemotactic stimulation. However, the resting [Ca(2+)](c) is similar to wild type. This mutant provides a test for the role of Ca(2+) influx in both chemotaxis and the signalling cascade that controls it. The production of cyclic-GMP and cAMP, and the activation of the MAP kinase, DdERK2, triggered from the cAMP receptor, are little perturbed in the mutant; mobilization of actin into the cytoskeleton also follows similar kinetics to wild type. Mutant cells chemotax efficiently towards cAMP or folic acid and their sensitivity to cAMP is similar to wild type. Finally, they move at similar speeds to wild-type cells, with or without chemoattractant. We conclude that Ca(2+) signalling is not necessary for chemotaxis to cAMP.

SUBMITTER: Traynor D

PROVIDER: S-EPMC302083 | biostudies-literature | 2000 Sep

REPOSITORIES: biostudies-literature

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Ca(2+) signalling is not required for chemotaxis in Dictyostelium.

Traynor D D Milne J L JL Insall R H RH Kay R R RR

The EMBO journal 20000901 17

Dictyostelium cells can move rapidly towards a source of cyclic-AMP (cAMP). This chemoattractant is detected by G-protein-linked receptors, which trigger a signalling cascade including a rapid influx of Ca(2+). We have disrupted an inositol 1,4,5-trisphosphate (InsP(3)) receptor-like gene, iplA, to produce null cells in which Ca(2+) entry in response to chemoattractants is abolished, as is the normal increase in free cytosolic Ca(2+) ([Ca(2+)](c)) that follows chemotactic stimulation. However, t ...[more]

PMID: 10970875

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Project description:BackgroundRas proteins are guanine-nucleotide-binding enzymes that couple cell surface receptors to intracellular signaling pathways controlling cell proliferation and differentiation, both in lower and higher eukaryotes. They act as molecular switches by cycling between active GTP and inactive GDP-bound states, through the action of two classes of regulatory proteins: a) guanine nucleotide exchange factor (GEFs) and b) GTP-ase activating proteins (GAPs). Genome wide analysis of the lower eukaryote Dictyostelium discoideum revealed a surprisingly large number of Ras Guanine Nucleotide Exchange Factors (RasGEFs). RasGEFs promote the activation of Ras proteins by catalyzing the exchange of GDP for GTP, thus conferring to RasGEFs the role of main activator of Ras proteins. Up to date only four RasGEFs, which are all non-redundant either for growth or development, have been characterized in Dictyostelium. We report here the identification and characterization of a fifth non-redundant GEF, RasGEFM.ResultsRasGEFM is a multi-domain protein containing six poly-proline stretches, a DEP, RasGEFN and RasGEF catalytic domain. The rasGEFM gene is differentially expressed during growth and development. Inactivation of the gene results in cells that form small, flat aggregates and fail to develop further. Expression of genes required for aggregation is delayed. Chemotaxis towards cAMP is impaired in the mutant, due to inability to inhibit lateral pseudopods. Endogenous cAMP accumulates during early development to a much lower extent than in wild type cells. Adenylyl cyclase activation in response to cAMP pulses is strongly reduced, by contrast guanylyl cyclase is stimulated to higher levels than in the wild type. The actin polymerization response to cAMP is also altered in the mutant. Cyclic AMP pulsing for several hours partially rescues the mutant. In vitro experiments suggest that RasGEFM acts downstream of the cAMP receptor but upstream of the G protein.ConclusionThe data indicate that RasGEFM is involved in the establishment of the cAMP relay system. We propose that RasGEFM is a component of a Ras regulated pathway, which integrate signals acting as positive regulator for adenylyl cyclase and negative regulator for guanylyl cyclase. Altered guanylyl cyclase, combined with defective regulation of actin polymerization, results in altered chemotaxis.

Project description:We have identified a novel Ras-interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cyclic AMP (cAMP) chemoattractant signal. rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. In addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced approximately 60% in rip3 null cells. This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage. Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenotypes of the rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells. A double-knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras-regulated pathway involved in integrating chemotaxis and signal relay pathways that are essential for aggregation.

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Ca(2+) signalling is not required for chemotaxis in Dictyostelium.

Publications

Ca(2+) signalling is not required for chemotaxis in Dictyostelium.

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