A Frizzled like protein in Dictyostelium discoideum

Summary Frizzled genes encode integral membrane proteins that function in multiple signal transduction pathways. They have been identified in diverse animals, from sponges to humans. Frizzled genes are well characterized for their functions in development and diseases. In this study we report the identification and characterisation of a Frizzled like protein in Dictyostelium. We identified a novel Frizzled like protein (FrzA) in the Dictyostelium database, which has a seven transmembrane region at the N-terminus but unlike other Frizzleds it lacks the CRD domain and instead has a Phosphatidylinositol-4-phosphate 5-kinase (PIP5K) domain at the C-terminus. The FrzA gene is present on the chromosome 4 of the Dictyostelium genome. The expression pattern of FrzA revealed a single transcript of ~2.5kb in size in the northern blot analysis. To characterize the function of FrzA protein in vivo, cells carrying an inactivated FrzA gene were generated by homologous recombination. The FrzA- cells grew normal on a bacterial lawn or in axenic medium indicating normal pinocytosis as well as intact cytokinesis. However the cell size was slightly smaller when compared to the Ax2 wild type cells. Notable was the defect in development on bacterial plates. When developed on phosphate agar plates the FrzA- cells did not form tight aggregates during early development. However the loose aggregates gave rise to very few and small fruiting bodies, which were formed with a delay of 24hrs. Also, the FrzA- mutant exhibited a pronounced developmental defect when developed at low cell density, where the mutant cells did not aggregate or develop fruiting bodies. Northern blot analysis to characterise the developmental defect in the FrzA- mutant showed expression of genes expressed early in aggregation such as csA; cAR1, ACA, PDE and PDI. However, the pattern was strongly altered and showed prolonged expression in case of CAR1 nad PDE and delayed expression for ACA and csA. The mutant cells did not exhibit a directed migration towards cAMP and thus showing a defect in chemotaxis. However, application of the exogenous cAMP pulses restored the expression of contact sites A and adenylyl cyclase (ACA) transcripts necessary for aggregation and also rescued the defect in chemotaxis. Calcium oscillation, which is important for initiating aggregation and further development, was disturbed in the mutant. We could show that the FrzA- mutant did not sense the cell density factor (CMF) and produce IP3 in response to the CMF stimulation. We therefore presume that FrzA is involved in the early steps of aggregation during Dictyostelium development. Our results indicate that FrzA is a potential G protein coupled receptor for the ligand CMF to induce the CMF signaling during the early development of Dictyostelium

A Dictyostelium chalone uses G proteins to regulate proliferation

<p>Abstract</p> <p>Background</p> <p>Several studies have shown that organ size, and the proliferation of tumor metastases, may be regulated by negative feedback loops in which autocrine secreted factors called chalones inhibit proliferation. However, very little is known about chalones, and how cells sense them. We previously identified two secreted proteins, AprA and CfaD, which act as chalones in <it>Dictyostelium</it>. Cells lacking AprA or CfaD proliferate faster than wild-type cells, and adding recombinant AprA or CfaD to cells slows their proliferation.</p> <p>Results</p> <p>We show here that cells lacking the G protein components Galpha8, Galpha9, and Gbeta proliferate faster than wild-type cells despite secreting normal or high levels of AprA and CfaD. Compared with wild-type cells, the proliferation of <it>galpha8^-</it>, <it>galpha9^-</it>and <it>gbeta^-</it>cells are only weakly inhibited by recombinant AprA (rAprA). Like AprA and CfaD, Galpha8 and Gbeta inhibit cell proliferation but not cell growth (the rate of increase in mass and protein per nucleus), whereas Galpha9 inhibits both proliferation and growth. <it>galpha8^-</it>cells show normal cell-surface binding of rAprA, whereas <it>galpha9^-</it>and <it>gbeta^-</it>cells have fewer cell-surface rAprA binding sites, suggesting that Galpha9 and Gbeta regulate the synthesis or processing of the AprA receptor. Like other ligands that activate G proteins, rAprA induces the binding of [³H]GTP to membranes, and GTPgammaS inhibits the binding of rAprA to membranes. Both AprA-induced [³H]GTP binding and the GTPgammaS inhibition of rAprA binding require Galpha8 and Gbeta but not Galpha9. Like <it>aprA^-</it>cells, <it>galpha8^-</it>cells have reduced spore viability.</p> <p>Conclusion</p> <p>This study shows that Galpha8 and Gbeta are part of the signal transduction pathway used by AprA to inhibit proliferation but not growth in <it>Dictyostelium</it>, whereas Galpha9 is part of a differealnt pathway that regulates both proliferation and growth, and that a chalone signal transduction pathway uses G proteins.</p

Dictyostelium cells bind a secreted autocrine factor that represses cell proliferation

<p>Abstract</p> <p>Background</p> <p><it>Dictyostelium </it>cells secrete the proteins AprA and CfaD. Cells lacking either AprA or CfaD proliferate faster than wild type, while AprA or CfaD overexpressor cells proliferate slowly, indicating that AprA and CfaD are autocrine factors that repress proliferation. CfaD interacts with AprA and requires the presence of AprA to slow proliferation. To determine if CfaD is necessary for the ability of AprA to slow proliferation, whether AprA binds to cells, and if so whether the binding requires the presence of CfaD, we examined the binding and effect on proliferation of recombinant AprA.</p> <p>Results</p> <p>We find that the extracellular accumulation of AprA increases with cell density and reaches a concentration of 0.3 μg/ml near a stationary cell density. When added to wild-type or <it>aprA</it>^-cells, recombinant AprA (rAprA) significantly slows proliferation at 0.1 μg/ml and higher concentrations. From 4 to 64 μg/ml, the effect of rAprA is at a plateau, slowing but not stopping proliferation. The proliferation-inhibiting activity of rAprA is roughly the same as that of native AprA in conditioned growth medium. Proliferating <it>aprA</it>^-cells show saturable binding of rAprA to 92,000 ± 11,000 cell-surface receptors with a <it>K</it>_{<it>D </it>}of 0.03 ± 0.02 μg/ml. There appears to be one class of binding site, and no apparent cooperativity. Native AprA inhibits the binding of rAprA to <it>aprA</it>^-cells with a <it>K</it>_{<it>i </it>}of 0.03 μg/ml, suggesting that the binding kinetics of rAprA are similar to those of native AprA. The proliferation of cells lacking CrlA, a cAMP receptor-like protein, or cells lacking CfaD are not affected by rAprA. Surprisingly, both cell types still bind rAprA.</p> <p>Conclusion</p> <p>Together, the data suggest that AprA functions as an autocrine proliferation-inhibiting factor by binding to cell surface receptors. Although AprA requires CfaD for activity, it does not require CfaD to bind to cells, suggesting the possibility that cells have an AprA receptor and a CfaD receptor, and activation of both receptors is required to slow proliferation. We previously found that <it>crlA</it>^-cells are sensitive to CfaD. Combined with the results presented here, this suggests that CrlA is not the AprA or CfaD receptor, and may be the receptor for an unknown third factor that is required for AprA and CfaD activity.</p

Linking Ras to myosin function: RasGEF Q, a Dictyostelium exchange factor for RasB, affects myosin II functions

Ras guanine nucleotide exchange factor (GEF) Q, a nucleotide exchange factor from Dictyostelium discoideum, is a 143-kD protein containing RasGEF domains and a DEP domain. We show that RasGEF Q can bind to F-actin, has the potential to form complexes with myosin heavy chain kinase (MHCK) A that contain active RasB, and is the predominant exchange factor for RasB. Overexpression of the RasGEF Q GEF domain activates RasB, causes enhanced recruitment of MHCK A to the cortex, and leads to cytokinesis defects in suspension, phenocopying cells expressing constitutively active RasB, and myosin-null mutants. RasGEF Q− mutants have defects in cell sorting and slug migration during later stages of development, in addition to cell polarity defects. Furthermore, RasGEF Q− mutants have increased levels of unphosphorylated myosin II, resulting in myosin II overassembly. Collectively, our results suggest that starvation signals through RasGEF Q to activate RasB, which then regulates processes requiring myosin II

The Dictyostelium genome encodes numerous RasGEFs with multiple biological roles

BACKGROUND: Dictyostelium discoideum is a eukaryote with a simple lifestyle and a relatively small genome whose sequence has been fully determined. It is widely used for studies on cell signaling, movement and multicellular development. Ras guanine-nucleotide exchange factors (RasGEFs) are the proteins that activate Ras and thus lie near the top of many signaling pathways. They are particularly important for signaling in development and chemotaxis in many organisms, including Dictyostelium. RESULTS: We have searched the genome for sequences encoding RasGEFs. Despite its relative simplicity, we find that the Dictyostelium genome encodes at least 25 RasGEFs, with a few other genes encoding only parts of the RasGEF consensus domains. All appear to be expressed at some point in development. The 25 genes include a wide variety of domain structures, most of which have not been seen in other organisms. The LisH domain, which is associated with microtubule binding, is seen particularly frequently; other domains that confer interactions with the cytoskeleton are also common. Disruption of a sample of the novel genes reveals that many have clear phenotypes, including altered morphology and defects in chemotaxis, slug phototaxis and thermotaxis. CONCLUSION: These results suggest that the unexpectedly large number of RasGEF genes reflects an evolutionary expansion of the range of Ras signaling rather than functional redundancy or the presence of multiple pseudogenes

A Retinoblastoma Orthologue Is Required for the Sensing of a Chalone in Dictyostelium discoideum

Retinoblastoma-like proteins regulate cell differentiation and inhibit cell proliferation. The Dictyostelium discoideum retinoblastoma orthologue RblA affects the differentiation of cells during multicellular development, but it is unclear whether RblA has a significant effect on Dictyostelium cell proliferation, which is inhibited by the secreted proteins AprA and CfaD. We found that rblA(−) cells in shaking culture proliferate to a higher density, die faster after reaching stationary density, and, after starvation, have a lower spore viability than wild-type cells, possibly because in shaking culture, rblA(−) cells have both increased cytokinesis and lower extracellular accumulation of CfaD. However, rblA(−) cells have abnormally slow proliferation on bacterial lawns. Recombinant AprA inhibits the proliferation of wild-type cells but not that of rblA(−) cells, whereas CfaD inhibits the proliferation of both wild-type cells and rblA(−) cells. Similar to aprA(−) cells, rblA(−) cells have a normal mass and protein accumulation rate on a per-nucleus basis, indicating that RblA affects cell proliferation but not cell growth. AprA also functions as a chemorepellent, and RblA is required for proper AprA chemorepellent activity despite the fact that RblA does not affect cell speed. Together, our data indicate that an autocrine proliferation-inhibiting factor acts through RblA to regulate cell density in Dictyostelium, suggesting that such factors may signal through retinoblastoma-like proteins to control the sizes of structures such as developing organs or tumors

(A) Computer-generated cell tracks of and AX2 cells during chemotactic migration in a spatial cAMP gradient using DIAS

Arrowheads indicate turns initiated by formation of lateral pseudopods in cells. (B) Shape changes of aggregation-competent and AX2 cells during chemotactic migration were analyzed by DIAS. Images were taken every 6 s but only every third frame is shown. The green areas indicate new membrane protrusions, the red areas indicate retractions, and the arrows indicate the direction of migration.<p><b>Copyright information:</b></p><p>Taken from "Linking Ras to myosin function: RasGEF Q, a exchange factor for RasB, affects myosin II functions"</p><p></p><p>The Journal of Cell Biology 2008;181(5):747-760.</p><p>Published online 2 Jun 2008</p><p>PMCID:PMC2396803.</p><p></p

(A) Aggregation-competent AX2 and cells were fixed and stained for myosin II

Images were taken with a confocal microscope. Every confocal section is accompanied by a pseudo-3D projection, in which the z axis represents the intensity of myosin II staining over the scanned area. Bar, 5 μm. (B) Myosin II levels in cytoskeletal ghosts from cells in vegetative and aggregation-competent stages. The graph (below) represents a mean of four independent experiments. The error bars indicate SD. (C) Lysates from vegetative wild-type AX2 and cells were subjected to 2D SDS-PAGE analysis for determining the phosphorylation status of myosin II.<p><b>Copyright information:</b></p><p>Taken from "Linking Ras to myosin function: RasGEF Q, a exchange factor for RasB, affects myosin II functions"</p><p></p><p>The Journal of Cell Biology 2008;181(5):747-760.</p><p>Published online 2 Jun 2008</p><p>PMCID:PMC2396803.</p><p></p

(A) Cytokinesis is normal in wild-type AX2 and cells but is defective in GFP-GEF cells

Images were taken using a fluorescent microscope (DMR). Images were acquired with a camera (DC 350 FX). (B) The increase in the number of nuclei in GFP-GEF cells. GFP-GEF cells were grown in suspension, fixed at the indicated times, stained with DAPI, and the nuclei were counted. The data are the mean of three independent determinations ±SD. (C) Cytokinesis defect in cells overexpressing constitutively activated RasB (GFP-RasB; green). Cells were fixed with methanol and nuclei are visualized with DAPI (blue). Arrowheads indicate multinucleated cells. Images were taken using a fluorescent microscope (DMR). Images were acquired with a camera (DC 350 FX). Bars, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Linking Ras to myosin function: RasGEF Q, a exchange factor for RasB, affects myosin II functions"</p><p></p><p>The Journal of Cell Biology 2008;181(5):747-760.</p><p>Published online 2 Jun 2008</p><p>PMCID:PMC2396803.</p><p></p