scholarly journals Exposure of Cells to a Cell Number-Counting Factor Decreases the Activity of Glucose-6-Phosphatase To Decrease Intracellular Glucose Levels in Dictyostelium discoideum

2005 ◽  
Vol 4 (1) ◽  
pp. 72-81 ◽  
Author(s):  
Wonhee Jang ◽  
Richard H. Gomer
Keyword(s):  
Signal Transduction ◽  
Dictyostelium Discoideum ◽  
Group Size ◽  
Signal Transduction Pathway ◽  
Glucose Production ◽  
Cell Number ◽  
Glucose Levels ◽  
A Cell ◽  
Induced Changes ◽  
Intracellular Glucose

ABSTRACT The development of Dictyostelium discoideum is a model for tissue size regulation, as these cells form groups of ≈2 × 104 cells. The group size is regulated in part by a negative feedback pathway mediated by a secreted multipolypeptide complex called counting factor (CF). CF signal transduction involves decreasing intracellular CF glucose levels. A component of CF, countin, has the bioactivity of the entire CF complex, and an 8-min exposure of cells to recombinant countin decreases intracellular glucose levels. To understand how CF regulates intracellular glucose, we examined the effect of CF on enzymes involved in glucose metabolism. Exposure of cells to CF has little effect on amylase or glycogen phosphorylase, enzymes involved in glucose production from glycogen. Glucokinase activity (the first specific step of glycolysis) is inhibited by high levels of CF but is not affected by an 8-min exposure to countin. The second enzyme specific for glycolysis, phosphofructokinase, is not regulated by CF. There are two corresponding enzymes in the gluconeogenesis pathway, fructose-1,6-bisphosphatase and glucose-6-phosphatase. The first is not regulated by CF or countin, whereas glucose-6-phosphatase is regulated by both CF and an 8-min exposure to countin. The countin-induced changes in the Km and V max of glucose-6-phosphatase cause a decrease in glucose production that can account for the countin-induced decrease in intracellular glucose levels. It thus appears that part of the CF signal transduction pathway involves inhibiting the activity of glucose-6-phosphatase, decreasing intracellular glucose levels and affecting the levels of other metabolites, to regulate group size.

scholarly journals A Cell Number-Counting Factor Regulates Levels of a Novel Protein, SslA, as Part of a Group Size Regulation Mechanism in Dictyostelium

2007 ◽  
Vol 6 (9) ◽  
pp. 1538-1551 ◽  
Author(s):  
Tong Gao ◽  
Celine Roisin-Bouffay ◽  
R. Diane Hatton ◽  
Lei Tang ◽  
Debra A. Brock ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Group Size ◽  
Signal Transduction Pathway ◽  
Cell Number ◽  
Regulation Mechanism ◽  
Wild Type ◽  
Coding Region ◽  
Glucose Levels ◽  
Type Size ◽  
Novel Protein

ABSTRACT Developing Dictyostelium cells form aggregation streams that break into groups of ∼2 × 104 cells. The breakup and subsequent group size are regulated by a secreted multisubunit counting factor (CF). To elucidate how CF regulates group size, we isolated second-site suppressors of smlA −, a transformant that forms small groups due to oversecretion of CF. smlA − sslA1(CR11) cells form roughly wild-type-size groups due to an insertion in the beginning of the coding region of sslA1, one of two highly similar genes encoding a novel protein. The insertion increases levels of SslA. In wild-type cells, the sslA1(CR11) mutation forms abnormally large groups. Reducing SslA levels by antisense causes the formation of smaller groups. The sslA(CR11) mutation does not affect the extracellular accumulation of CF activity or the CF components countin and CF50, suggesting that SslA does not regulate CF secretion. However, CF represses levels of SslA. Wild-type cells starved in the presence of smlA − cells, recombinant countin, or recombinant CF50 form smaller groups, whereas sslA1(CR11) cells appear to be insensitive to the presence of smlA − cells, countin, or CF50, suggesting that the sslA1(CR11) insertion affects CF signal transduction. We previously found that CF reduces intracellular glucose levels. sslA(CR11) does not significantly affect glucose levels, while glucose increases SslA levels. Together, the data suggest that SslA is a novel protein involved in part of a signal transduction pathway regulating group size.

scholarly journals A Cell Number Counting Factor Regulates Akt/Protein Kinase B To Regulate Dictyostelium discoideum Group Size

2004 ◽  
Vol 3 (5) ◽  
pp. 1176-1184 ◽  
Author(s):  
Tong Gao ◽  
David Knecht ◽  
Lei Tang ◽  
R. Diane Hatton ◽  
Richard H. Gomer
Keyword(s):  
Protein Kinase ◽  
Dictyostelium Discoideum ◽  
Group Size ◽  
Kinase Activity ◽  
Fluorescent Protein ◽  
Protein Kinase B ◽  
Cell Number ◽  
Pleckstrin Homology Domain ◽  
Wild Type ◽  
High Concentration

ABSTRACT Little is known about how individual cells can organize themselves to form structures of a given size. During development, Dictyostelium discoideum aggregates in dendritic streams and forms groups of ∼20,000 cells. D. discoideum regulates group size by secreting and simultaneously sensing a multiprotein complex called counting factor (CF). If there are too many cells in a stream, the associated high concentration of CF will decrease cell-cell adhesion and increase cell motility, causing aggregation streams to break up. The pulses of cyclic AMP (cAMP) that mediate aggregation cause a transient translocation of Akt/protein kinase B (Akt/PKB) to the leading edge of the plasma membrane and a concomitant activation of the kinase activity, which in turn stimulates motility. We found that countin− cells (which lack bioactive CF) and wild-type cells starved in the presence of anticountin antibodies (which block CF activity) showed a decreased level of cAMP-stimulated Akt/PKB membrane translocation and kinase activity compared to parental wild-type cells. Recombinant countin has the bioactivity of CF, and a 1-min treatment of cells with recombinant countin potentiated Akt/PKB translocation to membranes and Akt/PKB activity. Western blotting of total cell lysates indicated that countin does not affect the total level of Akt/PKB. Fluorescence microscopy of cells expressing an Akt/PKB pleckstrin homology domain-green fluorescent protein (PH-GFP) fusion protein indicated that recombinant countin and anti-countin antibodies do not obviously alter the distribution of Akt/PKB PH-GFP when it translocates to the membrane. Our data indicate that CF increases motility by potentiating the cAMP-stimulated activation and translocation of Akt/PKB.

scholarly journals An improved shotgun antisense method for mutagenesis and gene identification

BioTechniques ◽  
2020 ◽  
Vol 68 (3) ◽  
pp. 163-165
Author(s):  
Yu Tang ◽  
Richard H Gomer
Keyword(s):  
Signal Transduction ◽  
Cdna Library ◽  
Dictyostelium Discoideum ◽  
Signal Transduction Pathway ◽  
Gene Identification ◽  
Cdna Clones ◽  
Transduction Pathway ◽  
Improved Method ◽  
Transcription Level ◽  
Transformed Cell

Shotgun expression of antisense cDNA, where each transformed cell expresses a different antisense cDNA, has been used for mutagenesis and gene identification in Dictyostelium discoideum. However, the method has two limitations. First, there were too few clones in the shotgun antisense cDNA library to have an antisense cDNA for every gene in the genome. Second, the unequal transcription level of genes resulted in many antisense cDNAs in the library for some genes but relatively few antisense cDNAs for other genes. Here we report an improved method for generating a larger antisense cDNA library with a reduced percentage of cDNA clones from highly prevalent mRNAs and demonstrate its utility by screening for signal transduction pathway components in D. discoideum.

Expression of a cell surface antigen in Dictyostelium discoideum in relation to the cell cycle

1989 ◽  
Vol 93 (1) ◽  
pp. 199-204
Author(s):  
M. Krefft ◽  
C.J. Weijer
Keyword(s):  
Cell Cycle ◽  
Cell Surface ◽  
Dictyostelium Discoideum ◽  
Cell Number ◽  
S Phase ◽  
Volume Distribution ◽  
Quantitative Microscopy ◽  
Great Heterogeneity ◽  
A Cell ◽  
The Moment

We have previously shown binding of a monoclonal antibody MUD 9 to the cell surface of Dictyostelium discoideum amoebae and slug cells. In the slug stage the prestalk region was predominantly labelled, while vegetative amoebae showed a great heterogeneity in binding. In the present paper it is shown that the heterogeneous label of vegetative amoebae is due to differences in MUD 9 binding by cells in different cell cycle phases. Cells were synchronized by dilution from stationary phase and the level of MUD 9 binding was determined. Synchrony was determined by investigating increase in cell number and changes in the volume distribution of the cells, and by estimating the number of cells in S phase by monitoring bromodeoxyuridine (BUdR) incorporation. Simultaneously the amount of MUD 9 binding was determined by quantitative microscopy and flow cytometry. The amount of MUD 9 label varies during the cell cycle. The highest amount of label is found on cells early in the cell cycle, i.e. S-phase. These results support the finding that the developmental fate of Dictyostelium discoideum cells depends among other things on the cell cycle position of the cells at the moment of starvation.

CD40/CD154 ligation induces mononuclear cell adhesion to human renal proximal tubule cells via increased ICAM-1 expression

2005 ◽  
Vol 289 (1) ◽  
pp. F145-F153 ◽  
Author(s):  
Hongye Li ◽  
Edward P. Nord
Keyword(s):  
Signal Transduction ◽  
Cell Adhesion ◽  
Proximal Tubule ◽  
Mononuclear Cell ◽  
Mononuclear Cells ◽  
Gene Array ◽  
Signal Transduction Pathway ◽  
Renal Proximal Tubule ◽  
Transduction Pathway ◽  
A Cell

The role of CD40/CD154 ligation in the upregulation of genes of the proinflammatory nuclear factor-κB (NF-κB) signal transduction pathway was explored in primary cultures of human renal proximal tubule epithelial cells. Using a cDNA gene array specific for human NF-κB signal pathway genes, 38 genes were upregulated at 1 h, and 7 of these genes remained upregulated at 3 h. Of these genes, intercellular adhesion molecule-1 (ICAM-1) was explored in further detail. Quantitative real-time PCR for ICAM-1 mRNA expression confirmed the gene array findings. Western blot analysis and quantitative sandwich-enzyme ELISA confirmed this observation at the protein level. A cell-surface ELISA assay showed that ICAM-1 expression doubled by 48 h of CD154 exposure, and fluorescence-activated cell sorter analysis suggested that both the number of cells expressing ICAM-1 and the expression of ICAM-1 on these cells had increased. A cell adhesion assay using fluorescein-labeled human peripheral mononuclear cells showed that ICAM-1 upregulation resulted in increased mononuclear cell adhesion to the monolayer, which was abrogated by pretreatment of the monolayer with a neutralizing ICAM-1 antibody. The p38 mitogen-activated protein kinase (MAPK) inhibitor SB-203580 but not the extracellular signal-regulated kinase 1/2 inhibitor (PD-98059) nor the protein kinase C inhibitor (calphostin) blunted ICAM-1 expression and mononuclear cell adhesion to the monolayer. We conclude that, in human renal proximal tubule epithelial cells, CD40 activation upregulates ICAM-1 (and other NF-κB pathway genes) expression with concomitant enhanced adhesion of mononuclear cells, which is mediated via the p38 MAPK signal transduction pathway.

Modification of β-adrenoceptors and adenylyl cyclase in hearts perfused with hypochlorous acid

1998 ◽  
Vol 76 (10-11) ◽  
pp. 961-966 ◽  
Author(s):  
Sujata Persad ◽  
Naranjan S Dhalla
Keyword(s):  
Signal Transduction ◽  
Adenylyl Cyclase ◽  
Hypochlorous Acid ◽  
Signal Transduction Pathway ◽  
Ischemic Heart ◽  
Beta Adrenoceptors ◽  
Beta Adrenoceptor ◽  
Adrenoceptor Agonist ◽  
Rat Hearts ◽  
Induced Changes

It is now well known that the signal transduction pathway involving beta-adrenoceptors and adenylyl cyclase is altered in ischemic heart disease. Since leukocytes accumulate in the ischemic heart and produce hypochlorous acid (HOCl), we investigated the effects of HOCl upon beta-adrenoceptors and adenylyl cyclase activities by perfusing rat hearts with 0.1 mM HOCl for 10 min and isolating cardiac membranes. Marked depressions in both the density and affinity of beta1-adrenoceptors were observed, whereas no significant change in the affinity or density of beta2-adrenoceptors was seen in hearts perfused with HOCl. After treatment of hearts with HOCl, competition curves using isoproterenol, a beta-adrenoceptor agonist, revealed a decrease in the proportion of high affinity binding sites. The adenylyl cyclase activities in the absence and presence of forskolin, NaF, Gpp(NH)p, or isoproterenol were depressed in hearts perfused with HOCl; however, the stimulatory effects of these agents on adenylyl cyclase were either unaltered or augmented. The presence of methionine in the perfusion medium prevented the HOCl-induced changes in beta1-adrenoceptors and adenylyl cyclase activity. These results suggest that HOCl may produce a defect in the beta-adrenoceptor linked signal transduction mechanism by affecting both beta1-adrenoceptors and adenylyl cyclase enzyme in the myocardium.Key words: signal transduction, adrenergic mechanisms, beta-adrenoceptors, adenylyl cyclase, heart membranes.

scholarly journals The importance of preexisting drug resistance due to overexpression of P-glycoprotein for the formation of resistance to bortezomib

2020 ◽  
Vol 7 (1) ◽  
pp. 23-31
Author(s):  
L. A. Laletina ◽  
N. I. Moiseeva ◽  
A. F. Karamysheva
Keyword(s):  
Signal Transduction ◽  
Drug Resistance ◽  
Signaling Pathways ◽  
Signal Transduction Pathway ◽  
Pten Protein ◽  
Development Of Resistance ◽  
P Glycoprotein ◽  
A Cell ◽  
Kinase Expression ◽  
Human Signal

Objective of the study. In our work we investigated the effect of pre-existing drug resistance by the mechanism of activation of ABC transporters – P-glycoprotein (Pgp) overexpression – on the development of resistance to the proteasome inhibitor bortezomib.Materials and methods. Cultures RPMI8226 and K562 / i-S9 (with Pgp overexpression) and their bortezomib-resistant sublines RPMI8226 / btz-6 and K562 / i-S9vlc were used as models. The methods used were MTT test, flow cytometry, Western blot and real-time polymerase chain reaction using the Human Signal Transduction Pathway Finder system.Results. The expression of the main PI3K-AKT and NF-κB signaling pathways did not change in RPMI8226 / btz-6 subline cells. However, AKT kinase expression was significantly increased and PTEN protein expression was reduced in K562 / i-S9vlc cells with Pgp-overexpression. Significant changes in gene expression (42 %) were found in RPMI8226 / btz-6 cells related to a number of main signaling pathways in the tumor cell, namely: activation of 3–4 genes in signaling pathways related to hypoxia, oxidative stress, PPAR and p53. The highest activation in these cells was found in the TGFβ signaling pathway. In resistant K562 / i-S9vlc cells, expression of only 5 genes (10 %) increased: Fas, HMOX1, CPT2, ICAM, and SOCS3. Three genes were also identified that changed in both resistant sublines: Fas, HMOX1 and CPT2. Further, we showed that in the RPMI8226 / btz-6 subline, along with changes in the expression of signal transduction genes, there is a large pool of CD138-negative cells, and in the K562 / i-S9vlc subline, the number of cells expressing CD34 increases and the number of CD13 decreases.Conclusion. We found that different signaling pathways are involved in the formation of resistance to bortezomib in the absence of Pgp expression and its overexpression. In addition, a cell line without activated resistance pathways requires more extensive rearrangements in the signal system to acquire resistance to bortezomib. However, in both cases, bortezomib leads to a change in the immunophenotype of the cells – to the appearance of dedifferentiated subpopulations.

scholarly journals The negative regulator of Gli, Suppressor of fused (Sufu), interacts with SAP18, Galectin3 and other nuclear proteins

2004 ◽  
Vol 378 (2) ◽  
pp. 353-362 ◽  
Author(s):  
Mélanie PACES-FESSY ◽  
Dominique BOUCHER ◽  
Emile PETIT ◽  
Sandrine PAUTE-BRIAND ◽  
Marie-Françoise BLANCHET-TOURNIER
Keyword(s):  
Signal Transduction ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Signal Transduction Pathway ◽  
Biochemical Properties ◽  
Negative Regulator ◽  
Nuclear Proteins ◽  
Suppressor Of Fused ◽  
Activation Of Transcription ◽  
A Cell

Sufu (Suppressor of fused) is a negative regulator of the Hedgehog signal-transduction pathway, interacting directly with the Gli family of transcription factors. However, its function remains poorly understood. In the present study, we determined the expression, tissue distribution and biochemical properties of mSufu (mouse Sufu) protein. We identified several mSufu variants of which some were phosphorylated. A yeast two-hybrid screen with mSufu as bait allowed us to identify several nuclear proteins as potential partners of mSufu. Most of these partners, such as SAP18 (Sin3-associated polypeptide 18), pCIP (p300/CBP-cointegrator protein) and PIAS1 (protein inhibitor of activated signal transduction and activators of transcription 1), are involved in either repression or activation of transcription and two of them, Galectin3 and hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1), have a nuclear function in pre-mRNA splicing. We confirmed the mSufu–SAP18 and mSufu–Galectin3 interactions by independent biochemical assays. Using a cell transfection assay, we also demonstrated that mSufu protein (484 amino acids) is predominantly cytoplasmic but becomes mostly nuclear when a putative nuclear export signal is mutated or after treatment of the cells with leptomycin B. Moreover, mSufu is translocated to the nucleus when co-expressed with SAP18, which is normally found in this compartment. In contrast, Galectin3 is translocated to the cytoplasm when it is co-expressed with mSufu. Our findings indicate that mSufu is a shuttle protein that appears to be extremely versatile in its ability to bind different proteins in both the cytoplasm and nucleus.

scholarly journals An endogenous chemorepellent directs cell movement by inhibiting pseudopods at one side of cells

2019 ◽  
Vol 30 (2) ◽  
pp. 242-255 ◽  
Author(s):  
Ramesh Rijal ◽  
Kristen M. Consalvo ◽  
Christopher K. Lindsey ◽  
Richard H. Gomer
Keyword(s):  
Signal Transduction ◽  
Actin Polymerization ◽  
Formation Rate ◽  
Cell Movement ◽  
Signal Transduction Pathway ◽  
Pi3 Kinase ◽  
Signal Transduction Pathways ◽  
Ras Protein ◽  
A Cell ◽  
G Protein Coupled

Eukaryotic chemoattraction signal transduction pathways, such as those used by Dictyostelium discoideum to move toward cAMP, use a G protein–coupled receptor to activate multiple conserved pathways such as PI3 kinase/Akt/PKB to induce actin polymerization and pseudopod formation at the front of a cell, and PTEN to localize myosin II to the rear of a cell. Relatively little is known about chemorepulsion. We previously found that AprA is a chemorepellent protein secreted by Dictyostelium cells. Here we used 29 cell lines with disruptions of cAMP and/or AprA signal transduction pathway components, and delineated the AprA chemorepulsion pathway. We find that AprA uses a subset of chemoattraction signal transduction pathways including Ras, protein kinase A, target of rapamycin (TOR), phospholipase A, and ERK1, but does not require the PI3 kinase/Akt/PKB and guanylyl cyclase pathways to induce chemorepulsion. Possibly as a result of not using the PI3 kinase/Akt/PKB pathway and guanylyl cyclases, AprA does not induce actin polymerization or increase the pseudopod formation rate, but rather appears to inhibit pseudopod formation at the side of cells closest to the source of AprA.

Sign in / Sign up

Export Citation Format

Share Document