scholarly journals Proteomic and Transcriptomic Profiling Identifies Early Developmentally Regulated Proteins in Dictyostelium Discoideum

Cells ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 1187 ◽  
Author(s):  
González-Velasco ◽  
De Las Rivas ◽  
Lacal
Keyword(s):  
Dictyostelium Discoideum ◽  
Proteome Analysis ◽  
Wild Type ◽  
Cellular Functions ◽  
Developmentally Regulated ◽  
Transcriptomic Profiling ◽  
Novel Proteins ◽  
Proteomic Data ◽  
Secondary Messenger ◽  
Substrate Adhesion

Cyclic AMP acts as a secondary messenger involving different cellular functions in eukaryotes. Here, proteomic and transcriptomic profiling has been combined to identify novel early developmentally regulated proteins in eukaryote cells. These proteomic and transcriptomic experiments were performed in Dictyostelium discoideum given the unique advantages that this organism offers as a eukaryotic model for cell motility and as a nonmammalian model of human disease. By comparing whole-cell proteome analysis of developed (cAMP-pulsed) wild-type AX2 cells and an independent transcriptomic analysis of developed wild-type AX4 cells, our results show that up to 70% of the identified proteins overlap in the two independent studies. Among them, we have found 26 proteins previously related to cAMP signaling and identified 110 novel proteins involved in calcium signaling, adhesion, actin cytoskeleton, the ubiquitin-proteasome pathway, metabolism, and proteins that previously lacked any annotation. Our study validates previous findings, mostly for the canonical cAMP-pathway, and also generates further insight into the complexity of the transcriptomic changes during early development. This article also compares proteomic data between parental and cells lacking glkA, a GSK-3 kinase implicated in substrate adhesion and chemotaxis in Dictyostelium. This analysis reveals a set of proteins that show differences in expression in the two strains as well as overlapping protein level changes independent of GlkA.

Incomplete contact site A glycoprotein in HL220, a modB mutant of Dictyostelium discoideum

1985 ◽  
Vol 73 (1) ◽  
pp. 49-68
Author(s):  
G. Gerisch ◽  
U. Weinhart ◽  
G. Bertholdt ◽  
M. Claviez ◽  
J. Stadler
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Apparent Molecular Weight ◽  
Uniform Structure ◽  
Cell Contact ◽  
Wild Type ◽  
Developmentally Regulated ◽  
Contact Formation

HL220, a modB mutant that lacks a modification of certain membrane proteins of Dictyostelium discoideum, has been shown to aggregate and to form EDTA-stable intercellular contacts typical of aggregating wild-type cells. A developmentally regulated glycoprotein of 80 X 10(3) apparent molecular weight has been identified as a target site of adhesion-blocking Fab and thought to be involved in EDTA-stable cell contact formation (Muller & Gerisch, 1978). In the HL220 mutant this glycoprotein is no longer recognized by a modB-specific antibody. Therefore, it has been suggested that the 80 X 10(3) Mr glycoprotein, or a modification on it, is not required for the EDTA-stable cell contact of aggregating cells. We show that HL220 synthesizes an equivalent of the 80 X 10(3) Mr glycoprotein with an apparent molecular weight of 68 X 10(3). The mutant product reacted with certain monoclonal antibodies highly specific for the 80 X 10(3) Mr glycoprotein in the wild type, and was developmentally regulated like the 80 X 10(3) Mr glycoprotein. These results indicate that the 68 X 10(3) Mr protein of the mutant lacks a modification, most likely an oligosaccharide residue, the absence of which causes the substantial shift of the apparent molecular weight from 80 X 10(3) to 68 X 10(3). Monoclonal antibodies that did not react with proteins of the mutant could be classified according to their reactions with different sub-sets of wild-type proteins. These results indicate that the proteins that reacted with either one or the other antibody were not modified by a uniform structure. The modification rather varies from one sub-set of cross-reacting proteins to another, suggesting differences between the glycosyl residues of the partially cross-reacting proteins. HL220 cells showed strongly reduced EDTA-stable contact formation under our conditions. EDTA-sensitive intercellular adhesion was undetectable in the mutant, whereas adhesion of the cells to the substratum appeared to be strengthened. The rear ends of the cells, in particular, were tightly attached to glass or Teflon surfaces. The mutant cells were capable of responding chemotactically. Propagated excitation waves like those known to be based on periodic cyclic AMP production and relay were clearly seen. Extracellular phosphodiesterase induction by cyclic AMP and phosphodiesterase inhibitor production were normal. These results indicate that the generation of chemotactic signals and the cellular responses to cyclic AMP are not severely affected by the mutation.

Investigation of a potential role for aldose reductase AlrA in tetrahydropteridine synthesis in Dictyostelium discoideum Ax2

Pteridines ◽  
2017 ◽  
Vol 28 (2) ◽  
pp. 97-103
Author(s):  
Hye-Lim Kim ◽  
Hyun-Chul Ryu ◽  
Young Shik Park
Keyword(s):  
Dictyostelium Discoideum ◽  
Aldose Reductase ◽  
Potential Role ◽  
Amino Acid Residues ◽  
Wild Type ◽  
High Sequence Identity ◽  
Physiological Conditions ◽  
Antioxidant Function ◽  
Cellular Extract ◽  
Sr Gene

AbstractDictyostelium discoideum Ax2 is well-known for the synthesis of d-threo-tetrahydrobiopterin (DH4) with a smaller amount of l-erythro-tetrahydrobiopterin (BH4). DH4 synthesis from 6-pyruvoyltetrahydropterin (PPH4) is catalyzed by aldose reductase (AR)-like protein and sepiapterin reductase (SR) via an intermediate 1′-oxo-2′-d-hydroxypropyl tetrahydropterin, which is non-enzymatically oxidized to d-sepiapterin in the absence of SR. However, l-sepiapterin was a dominant product in the reaction of a cellular extract of spr− disrupted in the SR gene. In order to investigate its potential role in tetrahydropteridine synthesis, the enzyme catalyzing l-sepiapterin synthesis from PPH4 was purified from spr−. Via mass spectrometry, the protein was identified to be encoded by alrA. AlrA consists of 297 amino acid residues sharing a high sequence identity with human AR. However, in the co-incubation assay, DH4 synthesis was not detected and, furthermore, the recombinant AlrA was observed to suppress BH4 synthesis by SR, which was known to prefer 1′-oxo-2′-d-hydroxypropyl tetrahydropterin to PPH4. Although intracellular DH4 level in alrA− was decreased to 60% of the wild type, it is presumed to result from the antioxidant function of DH4. Therefore, despite the structural and catalytic identities with human AR, AlrA seems to be involved in neither BH4, nor DH4 synthesis under normal physiological conditions.

scholarly journals A Putative Ariadne-Like Ubiquitin Ligase Is Required for Dictyostelium discoideum Development

2006 ◽  
Vol 5 (10) ◽  
pp. 1820-1825 ◽  
Author(s):  
Nathaniel Whitney ◽  
Lacey J. Pearson ◽  
Ryan Lunsford ◽  
Lisa McGill ◽  
Richard H. Gomer ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Ubiquitin Ligase ◽  
Fruiting Bodies ◽  
Wild Type ◽  
Cell Numbers

ABSTRACT The Dictyostelium rbrA gene encodes a putative Ariadne ubiquitin ligase. rbrA − cells form defective slugs that cannot phototax. Prestalk cell numbers are reduced in rbrA − slugs, and these prestalk cells do not localize to the tip of slugs. Chimeric slugs containing wild-type cells could phototax and form fruiting bodies.

scholarly journals Genetic disruption of calpain correlates with loss of membrane blebbing and differential expression of RhoGDI-1, cofilin and tropomyosin

2008 ◽  
Vol 411 (3) ◽  
pp. 657-666 ◽  
Author(s):  
Anna K. Larsen ◽  
René Lametsch ◽  
John S. Elce ◽  
Jørgen K. Larsen ◽  
Bo Thomsen ◽  
...  
Keyword(s):  
Membrane Surface ◽  
Proteome Analysis ◽  
Time Lapse ◽  
Wild Type ◽  
Genetic Rescue ◽  
Dynamic Regulation ◽  
Membrane Blebbing ◽  
Signalling Molecules ◽  
Membrane Blebs

Dynamic regulation of the actin cytoskeleton is important for cell motility, spreading and the formation of membrane surface extensions such as lamellipodia, ruffles and blebs. The ubiquitous calpains contribute to integrin-mediated cytoskeletal remodelling during cell migration and spreading, by cleavage of focal adhesion components and signalling molecules. In the present study, the live-cell morphology of calpain-knockout and wild-type cells was examined by time-lapse fluorescence microscopy, and a role of calpain in mediating the formation of sporadic membrane blebs was established. Membrane blebbing was significantly reduced in calpain-knockout cells, and genetic rescue fully restored the wild-type phenotype in knockout cells. Proteomic comparison of wild-type and knockout cells identified decreased levels of RhoGDI-1 (Rho GDP-dissociation inhibitor) and cofilin 1, and increased levels of tropomyosin in calpain-knockout cells, suggesting a role of calpain in regulating membrane extensions involving these proteins. RhoGDI, cofilin and tropomyosin are known regulators of actin filament dynamics and membrane extensions. The reduced levels of RhoGDI-1 in calpain-knockout cells observed by proteome analysis were confirmed by immunoblotting. Genetic rescue of the calpain-knockout cells enhanced RhoGDI-1-expression 2-fold above that normally present in wild-type cells. These results suggest a regulatory connection between calpain and RhoGDI-1 in promoting formation of membrane blebs.

scholarly journals Functional Promiscuity of Gene Regulation by Serpentine Receptors in Dictyostelium discoideum

1998 ◽  
Vol 18 (10) ◽  
pp. 5744-5749 ◽  
Author(s):  
Irene Verkerke-Van Wijk ◽  
Ji-Yun Kim ◽  
Raymond Brandt ◽  
Peter N. Devreotes ◽  
Pauline Schaap
Keyword(s):  
Gene Expression ◽  
Cell Fate ◽  
Dictyostelium Discoideum ◽  
Developmental Regulation ◽  
Mutant Cell ◽  
Gene Induction ◽  
Specific Gene ◽  
Wild Type ◽  
Animal Development ◽  
Camp Stimulation

ABSTRACT Serpentine receptors such as smoothened and frizzled play important roles in cell fate determination during animal development. InDictyostelium discoideum, four serpentine cyclic AMP (cAMP) receptors (cARs) regulate expression of multiple classes of developmental genes. To understand their function, it is essential to know whether each cAR is coupled to a specific gene regulatory pathway or whether specificity results from the different developmental regulation of individual cARs. To distinguish between these possibilities, we measured gene induction in car1 car3 double mutant cell lines that express equal levels of either cAR1, cAR2, or cAR3 under a constitutive promoter. We found that all cARs efficiently mediate both aggregative gene induction by cAMP pulses and induction of postaggregative and prespore genes by persistent cAMP stimulation. Two exceptions to this functional promiscuity were observed. (i) Only cAR1 can mediate adenosine inhibition of cAMP-induced prespore gene expression, a phenomenon that was found earlier in wild-type cells. cAR1’s mediation of adenosine inhibition suggests that cAR1 normally mediates prespore gene induction. (ii) Only cAR2 allows entry into the prestalk pathway. Prestalk gene expression is induced by differentiation-inducing factor (DIF) but only after cells have been prestimulated with cAMP. We found that DIF-induced prestalk gene expression is 10 times higher in constitutive cAR2 expressors than in constitutive cAR1 or cAR3 expressors (which still have endogenous cAR2), suggesting that cAR2 mediates induction of DIF competence. Since in wild-type slugs cAR2 is expressed only in anterior cells, this could explain the so far puzzling observations that prestalk cells differentiate at the anterior region but that DIF levels are actually higher at the posterior region. After the initial induction of DIF competence, cAMP becomes a repressor of prestalk gene expression. This function can again be mediated by cAR1, cAR2, and cAR3.

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