Copine A plays a role in the differentiation of stalk cells and the initiation of culmination in Dictyostelium development (original) (raw)
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Copine A is required for cytokinesis, contractile vacuole function, and development in Dictyostelium
Eukaryotic …, 2007
Copines make up a family of soluble, calcium-dependent, membrane binding proteins found in a variety of eukaryotic organisms. In an earlier study, we identified six copine genes in the Dictyostelium discoideum genome and focused our studies on cpnA. Our previous localization studies of green fluorescent protein-tagged CpnA in Dictyostelium suggested that CpnA may have roles in contractile vacuole function, endolysosomal trafficking, and development. To test these hypotheses, we created a cpnA ؊ knockout strain, and here we report the initial characterization of the mutant phenotype. The cpnA ؊ cells exhibited normal growth rates and a slight cytokinesis defect. When placed in starvation conditions, cpnA ؊ cells appeared to aggregate into mounds and form fingers with normal timing; however, they were delayed or arrested in the finger stage. When placed in water, cpnA ؊ cells formed unusually large contractile vacuoles, indicating a defect in contractile vacuole function, while endocytosis and phagocytosis rates for the cpnA ؊ cells were similar to those seen for wild-type cells. These studies indicate that CpnA plays a role in cytokinesis and contractile vacuole function and is required for normal development, specifically in the later stages prior to culmination. We also used real-time reverse transcription-PCR to determine the expression patterns of all six copine genes during development. The six copine genes were expressed in vegetative cells, with each gene exhibiting a distinct pattern of expression throughout development. All of the copine genes except cpnF showed an upregulation of mRNA expression at one or two developmental transitions, suggesting that copines may be important regulators of Dictyostelium development.
Development, Growth & Differentiation, 2011
Copines are calcium-dependent membrane-binding proteins found in many eukaryotic organisms. We are studying the function of copines using the model organism, Dictyostelium discoideum. When under starvation conditions, Dictyostelium cells aggregate into mounds that become migrating slugs, which can move toward light and heat before culminating into a fruiting body. Previously, we showed that Dictyostelium cells lacking the copine A (cpnA) gene are not able to form fruiting bodies and instead arrest at the slug stage. In this study, we compared the slug behavior of cells lacking the cpnA gene to the slug behavior of wild-type cells. The slugs formed by cpnA-cells were much larger than wild-type slugs and exhibited no phototaxis and negative thermotaxis in the same conditions that wild-type slugs exhibited positive phototaxis and thermotaxis. Mixing as little as 5% wild-type cells with cpnA-cells rescued the phototaxis and thermotaxis defects, suggesting that CpnA plays a specific role in the regulation of the production and ⁄ or release of a signaling molecule. Reducing extracellular levels of ammonia also partially rescued the phototaxis and thermotaxis defects of cpnA-slugs, suggesting that CpnA may have a specific role in regulating ammonia signaling. Expressing the lacZ gene under the cpnA promoter in wild-type cells indicated cpnA is preferentially expressed in the prestalk cells found in the anterior part of the slug, which include the cells at the tip of the slug that regulate phototaxis, thermotaxis, and the initiation of culmination into fruiting bodies. Our results suggest that CpnA plays a role in the regulation of the signaling pathways, including ammonia signaling, necessary for sensing and ⁄ or orienting toward light and heat in the prestalk cells of the Dictyostelium slug.
Regulation of cell differentiation and pattern formation in Dictyostelium development
The International journal of developmental biology, 1994
Free-living cells of Dictyostelium discoideum aggregate to form a slug-shaped cell mass and differentiate into prestalk and prespore cells. The differentiation of prespore cells is characterized by expression of Dp87 gene, the earliest event of prespore differentiation. It encodes a protein which first appears in ER of aggregating cells in a precursor form, is then translocated to prespore vacuoles and modified to a mature form and finally exocytosed to constitute the sorus matrix. The transcription of Dp87 is regulated by the cis-acting region consisting of positive, prespore-specific, negative, non-prespore-specific and positive, cell-type-non-specific elements. Cells expressing Dp87 appear at random in early aggregation streams and centers and then sort out to the posterior part of the slug. Intercellular signals required for prestalk and prespore differentiation were investigated by incubation at a low cell density of disaggregated cells. cAMP is inhibitory at the first and seco...
The International journal of developmental biology, 2000
Spatial gradients of sequestered and free cellular calcium (Ca2+) exist in the slug of Dictyostelium discoideum (Maeda and Maeda, 1973; Tirlapur et al., 1991; Azhar et al., 1995; Cubitt et al., 1995). When we vary intracellular Ca2+ with the help of calcium buffers and the ionophore Br-A23187, there are striking effects on slug morphology, patterning and cell differentiation. In the presence of a calcium ionophore, high external Ca2+ levels lead to an increase of intracellular sequestered and free Ca2+, the formation of long slugs, a decrease in the fraction of genetically defined prespore cells and 'stalky' fruiting bodies. Conversely, a lowering of external Ca2+ levels results in a decrease of intracellular Ca2+, the formation of short slugs, an increase in the prespore fraction and 'spory' fruiting bodies. We infer that Ca2+ plays a significant morphogenetic role in D. discoideum development, by selectively promoting the prestalk pathway relative to the prespore p...
Cell, 1995
We present evidence that Dictyostelium slug tip cells, the pstA cells, may arise by positional differentiation, but at asite remote from that which they will eventually occupy. When first detectable, the pstA cells form a peripheral ring surrounding the other prestalk cell subtype, the pst0 cells, but subsequently move above the pstOcellstoform thetip. BecausepstAcell differentiation requires a lo-fold higher concentration of differention-inducing factor, the stalkcell inducer, the initial patterning seems likely to reflect the existence of a morphogenetic gradient. The subsequent redistribution of the two cell types is explicable by their different rates of chemotaxis to cyclic AMP. These results help reconcile the two apparently opposing views of pattern formation in Dictyostelium, that there is positional differentiation and that pattern formation occurs by cell sorting.
Signal Transduction and Dictyostelium Development
Protist 152 15 20, 2001
Abe K, Yanagisawa K (1983) A new class of rapidly developing mutants in Dictyostelium discoideum: implications for cyclic AMP metabolism and cell differentiation. Dev Biol 95: 200-210 Anjard C, Pinaud S, Kay RR, Reymond CD (1992) Overexpression of Dd PK2 protein kinase causes rapid development and affects the intracellular cAMP pathway of Dictyostelium discoideum. Development 115: 785-790 Anjard C, van Bemmelen M, Veron M, Reymond CD (1997) A new spore differentiation factor (SDF) secreted by Dictyostelium cells is phosphorylated by the cAMP dependent protein kinase. Differentiation 62: 43-49 Araki T, Gamper M, Early A, Fukuzawa M, Abe T, Kawata T, Kim E, Firtel RA, Williams JG (1998) Developmentally and spatially regulated activation of a Dictyostelium STAT protein by a serpentine receptor. EMBO J 17: 4018-4028 Baldauf SL, Roger AJ, Wenk-Siefert I, Doolittle WF (2000) A kingdom-level phylogeny of eukaryotes based on combined protein data. Science 290: 972-977 Brazill DT, Lindsey DF, Bishop JD, Gomer RH (1998) Cell density sensing mediated by a G protein-coupled receptor activating phospholipase C. J Biol Chem 273: 8161-8168 Buczynski G, Grove B, Nomura A, Kleve M, Bush J, Firtel RA, Cardelli J (1997) Inactivation of two Dictyostelium discoideum genes, DdPIK1 and DdPIK2, encoding proteins related to mammalian phosphatidylinositide 3-kinases, results in defects in endocytosis, lysosome to postlysosome transport, and actin cytoskeleton organization. J Cell Biol 136: 1271-1286 Cadigan KM, Nusse R (1997) Wnt signaling: a common theme in animal development. Genes Dev 11: 3286-3305 Chang WT, Thomason PA, Gross, JD, Newell, PC (1998) Evidence that the RdeA protein is a component of a multistep phosphorelay, modulating rate of development in Dictyostelium. EMBO J 17: 2809-2816 Chen MY, Long Y, Devreotes PN (1997) A novel cytosolic regulator, Pianissimo, is required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase in Dictyostelium. Genes Dev 11: 3218-3231 Chung CY, Firtel RA (1999) PAKa, a putative PAK family member, is required for cytokinesis and the regulation of the cytoskeleton in Dictyostelium discoideum cells during chemotaxis. J Cell Biol 147: 559-576 Cotter DA, Mahadeo DC, Cervi DN, Kishi Y, Gale K, Sands T, Sameshima M (2000) Environmental regulation of pathways controlling sporulation, dormancy and germination utillizes bacterial-like signalling complexes in Dictyostelium discoideum. Protist 151:111-126 Cubitt AB, Firtel RA (1992) Characterization of phospholipase activity in Dictyostelium discoideum. Identification of a Ca 2+ -dependent polyphosphoinositide-specific phospholipase C. Biochem J 283: 371-378 Deery WJ, Gomer RH (1999) A putative receptor mediating cell-density sensing in Dictyostelium. J Biol Chem 274: 34476-3482 Dormann D, Vasiev B, Weijer CJ (2000) The control of chemotactic cell movement during Dictyostelium morphogenesis. Phil Trans R Soc Lond B 355: 983-991 Drayer AL, Van der Kaay J, Mayr GW, Van Haastert PJ (1994) Role of phospholipase C in Dictyostelium: formation of inositol (1,4,5) trisphosphate and normal development in cells lacking phospholipase C activity. EMBO J 13: 1601-1609 Firtel RA, Chung CY (2000) The molecular genetics of chemotaxis: sensing and responding to chemoattractant gradients. Bioessays 22: 603-615 Ginger RS, Dalton EC, Ryves WJ, Fukuzawa M, Williams JG, Harwood AJ (2000) Glycogen synthase kinase-3 enhances nuclear export of a Dictyostelium STAT protein. EMBO J 19: 5483-5491 Grimson MJ, Coates JC, Reynolds JP, Shipman M, Blanton RL, Harwood AJ (2000) Adherens junctions and β-catenin-mediated cell signalling in a non-metazoan organism. Nature 408: 727-731 Harwood AJ, Hopper NA, Simon M-N, Driscoll DM, Veron M, Williams JG (1992) Culmination in Dictyostelium is regulated by the cAMP-dependent protein kinase. Cell 69: 615-624 Harwood AJ, Early A, Williams JG (1993) A repressor controls the timing and spatial localisation of stalk cellspecific gene expression in Dictyostelium. Development 118: 1041-1048
Cell-cell adhesion and signal transduction during Dictyostelium development
Journal of cell science, 2001
The development of the non-metazoan eukaryote Dictyostelium discoideum displays many of the features of animal embryogenesis, including regulated cell-cell adhesion. During early development, two proteins, DdCAD-1 and csA, mediate cell-cell adhesion between amoebae as they form a loosely packed multicellular mass. The mechanism governing this process is similar to epithelial sheet sealing in animals. Although cell differentiation can occur in the absence of cell contact, regulated cell-cell adhesion is an important component of Dictyostelium morphogenesis, and a third adhesion molecule, gp150, is required for multicellular development past the aggregation stage. Cell-cell junctions that appear to be adherens junctions form during the late stages of Dictyostelium development. Although they are not essential to establish the basic multicellular body plan, these junctions are required to maintain the structural integrity of the fruiting body. The Dictyostelium beta-catenin homologue Aa...
Developmental Biology, 2001
The protein tyrosine phosphatase PTP1, which mediates reversible phosphorylation on tyrosine, has been shown to play an important regulatory role during Dictyostelium development. Mutants lacking PTP1 develop more rapidly than normal, while strains that overexpress PTP1 display aberrant morphology. However, the signalling pathways involved have not been characterised. In reexamining these strains, we have found that there is an inverse correlation between levels of PTP1 activity, the extent of tyrosine phosphorylation on Dictyostelium STATa after treatment with cAMP, and the proportion of the slug population exhibiting STATa nuclear enrichment in vivo. This suggests that PTP1 acts to attenuate the tyrosine phosphorylation of STATa and downstream STATa-mediated pathways. Consistent with this, we show that when PTP1 is overexpressed, there is increased expression of a prestalk cell marker at the slug posterior, a phenocopy of STATa null slugs. In ptp1 null strains, STATa tyrosine phosphorylation and nuclear enrichment in the slug anterior is increased. There is also a change in the prestalk to prespore cell ratio. Synergy experiments suggest that this is due to a cell-autonomous defect in forming the subset of prespore cells that are located in the anterior prespore region.
Cellular signalling, 2017
Multicellular development in Dictyostelium discoideum involves tightly regulated signaling events controlling the entry into development, initiation of aggregation and chemotaxis, and cellular differentiation. Here we show that PkcA, a Dictyostelium discoideum Protein Kinase C-orthologue, is involved in quorum sensing and the initiation of development, as well as cAMP sensing during chemotaxis. Additionally, by epistasis analysis we provide evidence that PkcA and PldB (a Phospholipase D-orthologue) functionally interact to regulate aggregation, differentiation, and cell-cell adhesion during development. Finally, we show that PkcA acts as a positive regulator of intracellular PLD-activity during development. Taken together, our results suggest that PkcA act through PldB, by regulating PLD-activity, in order to control events during development.
Cell cycle phase, cellular Ca2+ and development in Dictyostelium discoideum
The International journal of developmental biology, 2001
In Dictyostelium discoideum, the initial differentiation of cells is regulated by the phase of the cell cycle at starvation. Cells in S and early G2 (or with a low DNA content) have relatively high levels of cellular Ca2+ and display a prestalk tendency after starvation, whereas cells in mid to late G2 (or with a high DNA content) have relatively low levels of Ca2+ and display a prespore tendency. We found that there is a correlation between cytosolic Ca2+ and cell cycle phase, with high Ca2+ levels being restricted to cells in the S and early G2 phases. As expected on the basis of this correlation, cell cycle inhibitors influence the proportions of amoebae containing high or low Ca2+. However, it has been reported that in the rtoA mutant, which upon differentiation gives rise to many more stalk cells than spores (compared to the wild type), initial cell-type choice is independent of cell cycle phase at starvation. In contrast to the wild type, a disproportionately large fraction of...