Intracellular Role of Adenylyl Cyclase in Regulation of Lateral Pseudopod Formation during Dictyostelium Chemotaxis (original) (raw)
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Adenylyl cyclase mRNA localizes to the posterior of polarized DICTYOSTELIUM cells during chemotaxis
BMC cell biology, 2017
In Dictyostelium discoideum, vesicular transport of the adenylyl cyclase A (ACA) to the posterior of polarized cells is essential to relay exogenous 3',5'-cyclic adenosine monophosphate (cAMP) signals during chemotaxis and for the collective migration of cells in head-to-tail arrangements called streams. Using fluorescence in situ hybridization (FISH), we discovered that the ACA mRNA is asymmetrically distributed at the posterior of polarized cells. Using both standard estimators and Monte Carlo simulation methods, we found that the ACA mRNA enrichment depends on the position of the cell within a stream, with the posterior localization of ACA mRNA being strongest for cells at the end of a stream. By monitoring the recovery of ACA-YFP after cycloheximide (CHX) treatment, we observed that ACA mRNA and newly synthesized ACA-YFP first emerge as fluorescent punctae that later accumulate to the posterior of cells. We also found that the ACA mRNA localization requires 3' ACA ci...
Requirements for the adenylyl cyclases in the development of Dictyostelium
Development, 2001
It has been suggested that all intracellular signaling by cAMP during development of Dictyostelium is mediated by the cAMP-dependent protein kinase, PKA, since cells carrying null mutations in the acaA gene that encodes adenylyl cyclase can develop so as to form fruiting bodies under some conditions if PKA is made constitutive by overexpressing the catalytic subunit. However, a second adenylyl cyclase encoded by acrA has recently been found that functions in a cell autonomous fashion during late development. We have found that expression of a modified acaA gene rescues acrA− mutant cells indicating that the only role played by ACR is to produce cAMP. To determine whether cells lacking both adenylyl cyclase genes can develop when PKA is constitutive we disrupted acrA in a acaA− PKA-Cover strain. When developed at high cell densities, acrA−acaA− PKA-Cover cells form mounds, express cell type-specific genes at reduced levels and secrete cellulose coats but do not form fruiting bodies o...
cAMP production by adenylyl cyclase G induces prespore differentiation in Dictyostelium slugs
Development, 2007
Encystation and sporulation are crucial developmental transitions for solitary and social amoebae, respectively. Whereas little is known of encystation, sporulation requires both extra- and intracellular cAMP. After aggregation of social amoebae, extracellular cAMP binding to surface receptors and intracellular cAMP binding to cAMP-dependent protein kinase (PKA) act together to induce prespore differentiation. Later, a second episode of PKA activation triggers spore maturation. Adenylyl cyclase B (ACB) produces cAMP for maturation, but the cAMP source for prespore induction is unknown. We show that adenylyl cyclase G (ACG) protein is upregulated in prespore tissue after aggregation. acg null mutants show reduced prespore differentiation,which becomes very severe when ACB is also deleted. ACB is normally expressed in prestalk cells, but is upregulated in the prespore region of acgnull structures. These data show that ACG induces prespore differentiation in wild-type cells, with ACB c...
A temperature-sensitive adenylyl cyclase mutant of Dictyostelium
The EMBO Journal, 2000
Dictyostelium development starts with the chemotactic aggregation of up to 10 6 amoebae in response to propagating cAMP waves. cAMP is produced by the aggregation stage adenylyl cyclase (ACA) and cells lacking ACA (aca null) cannot aggregate. Temperature-sensitive mutants of ACA were selected from a population of aca null cells transformed with a library of ACA genes, a major segment of which had been ampli®ed by error-prone PCR. One mutant (tsaca2) that can complement the aggregation null phenotype of aca null cells at 22°C but not at 28°C was characterized in detail. The basal catalytic activity of the enzyme in this mutant was rapidly and reversibly inactivated at 28°C. Using this mutant strain we show that cell movement in aggregates and mounds is organized by propagating waves of cAMP. Synergy experiments between wild-type and tsaca2 cells, shifted to the restrictive temperature at various stages of development, showed that ACA plays an important role in the control of cell sorting and tip formation.
The Cyclase-associated Protein CAP as Regulator of Cell Polarity and cAMP Signaling in Dictyostelium
Molecular Biology of the Cell, 2004
involved in regulating the adenylyl cyclase activity. We show that cell polarization, F-actin organization, and phototaxis are altered in a Dictyostelium CAP knockout mutant. Furthermore, in complementation assays we determined the roles of the individual domains in signaling and regulation of the actin cytoskeleton. We studied in detail the adenylyl cyclase activity and found that the mutant cells have normal levels of the aggregation phase-specific adenylyl cyclase and that receptor-mediated activation is intact. However, cAMP relay that is responsible for the generation of propagating cAMP waves that control the chemotactic aggregation of starving Dictyostelium cells was altered, and the cAMP-induced cGMP production was significantly reduced. The data suggest an interaction of CAP with adenylyl cyclase in Dictyostelium and an influence on signaling pathways directly as well as through its function as a regulatory component of the cytoskeleton.
Adenylyl cyclase localization to the uropod of aggregating Dictyostelium cells requires RacC
Biochemical and Biophysical Research Communications, 2015
The localization of adenylyl cyclase A (ACA) to uropod of cells is required for the stream formation during Dictyostelium development. RacC is a Dictyostelium orthologue of Cdc42. We identified a streaming defect of racC − cells as they are clearly less polarized and form smaller and fragmented streams. ACA-YFP is mainly associated with intracellular vesicular structures, but not with the plasma membrane in racC − cells. racC − cells have a slightly higher number of vesicles than Ax3 cells, suggesting that the defect of ACA trafficking is not simply due to the lack of vesicle formation. While the ACA-YFP vesicles traveled with an average velocity of 9.1 µm/min in Ax3 cells, a slow and diffusional movement without direction with an average velocity of 4 µm/min was maintained in racC − cells. Images acquired by using total internal reflection fluorescence (TIRF) microscopy and fluorescence recovery after photobleaching (FRAP) analysis revealed that a significantly decreased number of ACA-YFP vesicles appeared near the cell membrane, indicating a defect in ACA-YFP vesicle trafficking. These results suggest an important role of RacC in the rapid and directional movements of ACA vesicles on microtubules to the plasma membrane, especially to the back of polarized cell.
Journal of muscle research and …, 2002
In the natural aggregation process, Dictyostelium amoebae relay the cAMP signal outwardly through the cell population as symmetric, nondissipating waves. Each cell in turn responds in a specific manner to the different phases of the wave. In the front of each wave, each cell experiences an increasing temporal gradient and positive spatial gradient of cAMP; at the peak of each wave, each cell experiences a cAMP concentration inhibitory to locomotion; and in the back of each wave, each cell experiences a decreasing temporal and negative spatial gradient of cAMP. Protocols are described to analyze the basic motile behavior of mutant cells in the absence of a chemotactic signal, and to test the responsiveness of mutant cells to the individual temporal, spatial and concentration components of a natural wave. The results of such an analysis can then be used to develop realistic models of cell motility and chemotaxis. Examples are described in which this contextual framework has been applied to mutant cell lines. The results of these mutant studies result in a model in which independent parallel regulatory pathways emanating from different phases of the wave effect different phase-specific behaviors.
The Journal of Cell Biology, 1985
Using a perfusion chamber, we examined the behavior of individual amebae in increasing and decreasing temporal gradients of cAMP. We demonstrated that amebae respond to increasing temporal gradients of cAMP with stimulated motility and to corresponding decreasing temporal gradients with depressed motility. Depressed motility observed in decreasing temporal gradients corresponded to the inhibited levels observed when cAMP was applied at constant concentrations. These results were consistent with a simple model for the motile behavior of amebae in an early aggregation territory in which nondissipating waves of cAMP originate at the aggregation center and travel outward periodically. We conclude that chemotactically responsive amebae can assess whether a temporal gradient of chemoattractant is increasing or decreasing in the absence of a spatial gradient, and can adjust their motility accordingly.
Journal of cell science, 1991
Dictyostelium cells use extracellular cyclic AMP both as a chemoattractant and as a morphogen inducing cell-type-specific gene expression. Cyclic AMP binds to surface receptors, activates one or more G-proteins, and stimulates adenylate cyclase, guanylate cyclase and phosphoinositidase C. Mutant fgdC showed aberrant chemotaxis, and was devoid of cyclic AMP-induced gene expression and differentiation. Both the receptor- and G-protein-mediated stimulation of adenylate cyclase and guanylate cyclase were unaltered in mutant fgdC as compared to wild-type cells. In wild-type cells phosphoinositidase C was activated about twofold by the cyclic AMP receptor. In mutant fgdC cells, however, the enzyme was inhibited by about 60%. These results suggest that phosphoinositidase C is regulated by a receptor-operated activation/inhibition switch that is defective in mutant fgdC. We conclude that activation of phosphoinositidase C is essential for Dictyostelium development.