Chemoattractant and GTP gamma S-mediated stimulation of adenylyl cyclase in Dictyostelium requires translocation of CRAC to membranes.

Lilly, Pamela J.; Devreotes, Peter N.

doi:10.1083/jcb.129.6.1659

Cited by 61 publications

(56 citation statements)

References 34 publications

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“…During stimulation, the pleckstrin homology domain of CRAC binds to the phospholipid phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P 3 ], causing CRAC-GFP to translocate to the leading edge of the cell. The GFP fluorescence along the cell membrane therefore provides a reporter of directional sensing at the single cell level (12)(13)(14)(15)(16). A spatiotemporal cAMP gradient was formed by uncaging a known concentration of cAMP with a circular UV beam (Fig.…”

Section: Resultsmentioning

confidence: 99%

Cellular asymmetry and individuality in directional sensing

Samadani

Mettetal

Oudenaarden

2006

Proc. Natl. Acad. Sci. U.S.A.

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It is generally assumed that single cells in an isogenic population, when exposed to identical environments, exhibit the same behavior. However, it is becoming increasingly clear that, even in a genetically identical population, cellular behavior can vary significantly among cells. Here we explore this variability in the gradientsensing response of Dictyostelium cells when exposed to repeated spatiotemporal pulses of chemoattractant. Our experiments show the response of a single cell to be highly reproducible from pulse to pulse. In contrast, a large variability in the response direction and magnitude is observed from cell to cell, even when different cells are exposed to the same pulse. First, these results indicate that the gradient-sensing network has inherent asymmetries that can significantly impact the ability of cells to faithfully sense the direction of extracellular signals (cellular asymmetry). Second, we find that the magnitude of this asymmetry varies greatly among cells. Some cells are able to accurately follow the direction of an extracellular stimulus, whereas, in other cells, the intracellular asymmetry dominates, resulting in a polarization axis that is independent of the direction of the extracellular cue (cellular individuality). We integrate these experimental findings into a model that treats the effective signal a cell detects as the product of the extracellular signal and the asymmetric intracellular signal. With this model we successfully predict the population response. This cellular individuality and asymmetry might fundamentally limit the fidelity of signal detection; in contrast, however, it might be beneficial by diversifying phenotypes in isogenic populations.T he low number of molecules involved in biological systems can lead to large stochastic effects and population heterogeneity even within a genetically identical population (1-3). For example, the swimming behavior of Escherichia coli cells varies greatly from cell to cell (4), and recent studies start to link this variability in swimming behavior to concentration fluctuations in regulatory proteins (5-7). It is an open question whether a similar variability can be observed in eukaryotic chemotactic cells, such as the slime mold Dictyostelium discoideum, which has the exquisite ability to sense and respond to shallow gradients of chemoattractants. In these spatially sensitive systems, signaling errors might be introduced in two different ways. First, the concentrations of intracellular signaling components might vary from cell to cell; second, spatial inhomogeneities or asymmetries in the cellular distributions of molecules might influence the ability of cells to sense slight spatial differences in the extracellular environment.To explore this question, we employ a quantitative approach to systematically study directional sensing in single Dictyostelium cells. Recent experiments have demonstrated that an extracellular signal induces spatial localization of several signaling proteins along the plasma membrane (8-12). The localiz...

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Section: Resultsmentioning

confidence: 99%

Cellular asymmetry and individuality in directional sensing

Samadani

Mettetal

Oudenaarden

2006

Proc. Natl. Acad. Sci. U.S.A.

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“…One possible function of RIP3 is to control the function of other components of the pathway that are required for G␤␥-stimulated adenylyl cyclase activity. As the function of the pleckstrin homology domain-containing protein CRAC is essential for activation of adenylyl cyclase (Insall et al, 1994a;Lilly and Devreotes, 1995;Parent et al, 1998), one possible role of RIP3 is to potentiate one of these processes. Previous results and the results presented here indicate that aimless and rip3 null cells exhibit similar defects in adenylyl cyclase activation.…”

Section: Discussionmentioning

confidence: 99%

“…The activation is mediated by the G␤␥ subunit and requires cytosolic proteins including CRAC, a pleckstrin homology domain containing protein that translocates to the plasma membrane in response to receptor activation, and Pianissimo (Lilly et al, 1993;Insall et al, 1994b;Lilly and Devreotes, 1995;Chen et al, 1997). In addition, activation of adenylyl cyclase and cAMP accumulation requires the function of the putative Ras exchange factor (GEF) Aimless (AleA) and the MAPK ERK2 (Segall et al, 1995;Insall et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

A Novel Ras-interacting Protein Required for Chemotaxis and Cyclic Adenosine Monophosphate Signal Relay inDictyostelium

Lee

Parent

Insall

et al. 1999

MBoC

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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 ϳ60% 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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“…(20). For CRAC supplementation, cells were lysed into cytosol derived from cells overexpressing CRAC (21). Typically, 540 l of cell lysates with or without GTP␥S and cAMP were mixed with 120 l of CRAC-containing supernatants and incubated on ice for 4 min, and 200 l of this mixture was assayed.…”

Section: Mutagenesis Library Construction and Transformation Into Acamentioning

confidence: 99%

Isolation of Inactive and G Protein-resistant Adenylyl Cyclase Mutants Using Random Mutagenesis

Parent

Devreotes

1995

Journal of Biological Chemistry

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We used random mutagenesis and phenotypic rescue of adenylyl cyclase-null Dictyostelium cells to isolate loss-of-function mutations in the enzyme. Mutants were (i) catalytically inactive or (ii) resistant to chemoattractant receptor and guanosine 5-3-O-(thio)triphosphate stimulation. Both classes of mutants harbored substitutions within the cytoplasmic C1a domain. Mutations that inactivated the enzyme were often at highly conserved positions. Those that blocked activation were grouped in two distinct regions: one close to the plane of the plasma membrane and another halfway within the C1 loop. Missense mutations or deletions within the transmembrane domains resulted in missorting of the protein. Our screen provides a simple and efficient method to separately define the sites of catalysis and regulation of this important class of enzymes.Adenylyl cyclases catalyze the conversion of ATP into the second messenger cAMP. Modulation of adenylyl cyclase activity by hormone and neurotransmitter receptors, through heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins), underlies a wide variety of physiological events (1). The genes for eight types of mammalian, one Drosophila, and one Dictyostelium adenylyl cyclase all encode proteins that are predicted to span the membrane 12 times and contain two large cytoplasmic domains (about 40 kDa each) (2-5). Each cytoplasmic domain contains a region of homology (designated C1a and C2a) with the catalytic domains of several adenylyl and guanylyl cyclases (2, 3). Deletion analysis and site-directed mutagenesis have shown that the interaction between the two halves of the enzyme is necessary for activity and suggested that the two homologous cytoplasmic domains are not equivalent (6, 7). 1The receptor and G protein regulation of the adenylyl cyclase (ACA) 2 in Dictyostelium is analogous to that in mammalian cells (8, 9). The chemoattractant receptor, cAR1, mediates, in addition to the events involved in chemotaxis, the activation of ACA and synthesis of cAMP. The regulation of ACA is similar to mammalian type II and IV adenylyl cyclases, which are synergistically stimulated by G protein ␤␥-subunits and activated by G s ␣ (10). Indeed, genetic and biochemical analyses have established that receptor and GTP␥S activation of ACA requires two components: the ␤␥-subunits of the heterotrimeric G protein G2 and a novel cytosolic protein named CRAC for cytosolic regulator of adenylyl cyclase (11,12). Proper regulation of ACA is essential for the early stages of development during which 10 5 cells spontaneously aggregate and differentiate into fruiting bodies. Secretion of cAMP serves to relay chemotactic signals to distal cells which, in turn, migrate toward the center to form aggregates. Consequently, aca Ϫ , g␤ Ϫ , and crac Ϫ cells cannot carry out aggregation and remain as smooth monolayers when plated on non-nutrient agar (5,11,12). In this study we used complementation of the aca Ϫ phenotype as a convenient and efficient readout to identify loss-of-function mutation...

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Chemoattractant and GTP gamma S-mediated stimulation of adenylyl cyclase in Dictyostelium requires translocation of CRAC to membranes.

Abstract: Abstract. We have previously reported that activation of adenylyl cyclase by chemoattractant receptors in Dictyostelium requires, in addition to a heterotrimeric G-protein, a cytosolic protein, designated CRAC (Lilly, P., and P. N.

Cited by 61 publications

References 34 publications

Cellular asymmetry and individuality in directional sensing

Cellular asymmetry and individuality in directional sensing

A Novel Ras-interacting Protein Required for Chemotaxis and Cyclic Adenosine Monophosphate Signal Relay inDictyostelium

Isolation of Inactive and G Protein-resistant Adenylyl Cyclase Mutants Using Random Mutagenesis

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