scholarly journals The ROCO Kinase QkgA Is Necessary for Proliferation Inhibition by Autocrine Signals in Dictyostelium discoideum

2010 ◽  
Vol 9 (10) ◽  
pp. 1557-1565 ◽  
Author(s):  
Jonathan E. Phillips ◽  
Richard H. Gomer
Keyword(s):  
Cell Proliferation ◽  
Dictyostelium Discoideum ◽  
Fluorescent Protein ◽  
Signal Transduction Pathway ◽  
Protein Accumulation ◽  
Proliferation Inhibition ◽  
Wild Type ◽  
Content Type ◽  
Multicellular Development ◽  
Green Fluorescent

ABSTRACT AprA and CfaD are secreted proteins that function as autocrine signals to inhibit cell proliferation in Dictyostelium discoideum. Cells lacking AprA or CfaD proliferate rapidly, and adding AprA or CfaD to cells slows proliferation. Cells lacking the ROCO kinase QkgA proliferate rapidly, with a doubling time 83% of that of the wild type, and overexpression of a QkgA-green fluorescent protein (GFP) fusion protein slows cell proliferation. We found that qkgA − cells accumulate normal levels of extracellular AprA and CfaD. Exogenous AprA or CfaD does not slow the proliferation of cells lacking qkgA, and expression of QkgA-GFP in qkgA − cells rescues this insensitivity. Like cells lacking AprA or CfaD, cells lacking QkgA tend to be multinucleate, accumulate nuclei rapidly, and show a mass and protein accumulation per nucleus like those of the wild type, suggesting that QkgA negatively regulates proliferation but not growth. Despite their rapid proliferation, cells lacking AprA, CfaD, or QkgA expand as a colony on bacteria less rapidly than the wild type. Unlike AprA and CfaD, QkgA does not affect spore viability following multicellular development. Together, these results indicate that QkgA is necessary for proliferation inhibition by AprA and CfaD, that QkgA mediates some but not all of the effects of AprA and CfaD, and that QkgA may function downstream of these proteins in a signal transduction pathway regulating proliferation.

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

2014 ◽  
Vol 13 (3) ◽  
pp. 376-382 ◽  
Author(s):  
Deenadayalan Bakthavatsalam ◽  
Michael J. V. White ◽  
Sarah E. Herlihy ◽  
Jonathan E. Phillips ◽  
Richard H. Gomer
Keyword(s):  
Cell Proliferation ◽  
Dictyostelium Discoideum ◽  
Accumulation Rate ◽  
Protein Accumulation ◽  
Wild Type ◽  
Spore Viability ◽  
Content Type ◽  
Multicellular Development ◽  
Normal Mass ◽  
Inhibit Cell Proliferation

ABSTRACT 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.

scholarly journals mhpTEncodes an Active Transporter Involved in 3-(3-Hydroxyphenyl)Propionate Catabolism by Escherichia coli K-12

2013 ◽  
Vol 79 (20) ◽  
pp. 6362-6368 ◽  
Author(s):  
Ying Xu ◽  
Bing Chen ◽  
Hongjun Chao ◽  
Ning-Yi Zhou
Keyword(s):  
Escherichia Coli ◽  
Wild Type Strain ◽  
Fluorescent Protein ◽  
Gene Knockout ◽  
Transport Activity ◽  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
Green Fluorescent ◽  
K 12

ABSTRACTEscherichia coliK-12 utilizes 3-(3-hydroxyphenyl)propionate (3HPP) as a sole carbon and energy source. Among the genes in its catabolic cluster in the genome,mhpTwas proposed to encode a hypothetical transporter. Since no transporter for 3HPP uptake has been identified, we investigated whether MhpT is responsible for 3HPP uptake. MhpT fused with green fluorescent protein was found to be located at the periphery of cells by confocal microscopy, consistent with localization to the cytoplasmic membrane. Gene knockout and complementation studies clearly indicated thatmhpTis essential for 3HPP catabolism inE. coliK-12 W3110 at pH 8.2. Uptake assays with14C-labeled substrates demonstrated that strain W3110 and strain W3110ΔmhpTcontaining recombinant MhpT specifically transported 3HPP but not benzoate, 3-hydroxybenzoate, or gentisate into cells. Energy dependence assays suggested that MhpT-mediated 3HPP transport was driven by the proton motive force. The change of Ala-272 of MhpT to a histidine, surprisingly, resulted in enhanced transport activity, and strain W3110ΔmhpTcontaining the MhpT A272H mutation had a slightly higher growth rate than the wild-type strain at pH 8.2. Hence, we demonstrated that MhpT is a specific 3HPP transporter and vital forE. coliK-12 W3110 growth on this substrate under basic conditions.

scholarly journals Corrected and Republished from: Activation Status-Coupled Transient S-Acylation Determines Membrane Partitioning of a Plant Rho-Related GTPase

2017 ◽  
Vol 37 (23) ◽  
Author(s):  
Nadav Sorek ◽  
Limor Poraty ◽  
Hasana Sternberg ◽  
Ella Buriakovsky ◽  
Einat Bar ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Wild Type ◽  
Content Type ◽  
Lipid Modifications ◽  
Membrane Partitioning ◽  
Acyl Lipids ◽  
Detergent Resistant Membranes ◽  
Green Fluorescent ◽  
Triton X ◽  
Activation Status

ABSTRACT ROPs or RACs are plant Rho-related GTPases implicated in the regulation of a multitude of signaling pathways that function at the plasma membrane via posttranslational lipid modifications. The relationships between ROP activation status and membrane localization has not been established. Here, we show that endogenous ROPs, as well as a transgenic His6-green fluorescent protein (GFP)-Arabidopsis thaliana ROP6 (AtROP6) fusion protein, were partitioned between Triton X-100-soluble and -insoluble membranes. In contrast, the His6-GFP-Atrop6CA activated mutant accumulated exclusively in detergent-resistant membranes. GDP induced accumulation of ROPs in Triton-soluble membranes, whereas GTPγS induced accumulation of ROPs in detergent-resistant membranes. Recombinant wild-type and constitutively active AtROP6 proteins were purified from Arabidopsis plants, and in turn, their lipids were cleaved and analyzed by gas chromatography-coupled mass spectrometry. In Triton-soluble membranes, the wild-type AtROP6 was only prenylated, primarily by geranylgeranyl. The activated AtROP6 that accumulated in detergent-resistant membranes was modified by prenyl and acyl lipids, identified as palmitic and stearic acids. Consistently, activated His6-GFP-Atrop6CAmS156, in which C156 was mutated into serine, accumulated in Triton-soluble membranes. These findings show that upon GTP binding and activation, AtROP6, and possibly other ROPs, are transiently S-acylated, inducing their partitioning into detergent-resistant membranes.

scholarly journals Phosphodiesterase EdpX1 PromotesXanthomonas oryzaepv. oryzae Virulence, Exopolysaccharide Production, and Biofilm Formation

2018 ◽  
Vol 84 (22) ◽  
Author(s):  
Dingrong Xue ◽  
Fang Tian ◽  
Fenghuan Yang ◽  
Huamin Chen ◽  
Xiaochen Yuan ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Second Messenger ◽  
Fold Increase ◽  
Regulatory Function ◽  
Wild Type ◽  
Content Type ◽  
Genes Encoding ◽  
Bacterial Blight Pathogen ◽  
Domain Protein ◽  
Green Fluorescent

ABSTRACTInXanthomonas oryzaepv. oryzae, the bacterial blight pathogen of rice, there are over 20 genes encoding GGDEF, EAL, and HD-GYP domains, which are potentially involved in the metabolism of second messenger c-di-GMP. In this study, we focused on the characterization of an EAL domain protein, EdpX1. Deletion of theedpX1gene resulted in a 2-fold increase in the intracellular c-di-GMP levels, which were restored to the wild-type levels in the complemented ΔedpX1(pB-edpX1) strain, demonstrating that EdpX1 is an active phosphodiesterase (PDE) inX. oryzaepv. oryzae. In addition, colorimetric assays further confirmed the PDE activity of EdpX1 by showing that the E153A mutation at the EAL motif strongly reduced its activity. Virulence assays on the leaves of susceptible rice showed that the ΔedpX1mutant was severely impaired in causing disease symptoms. Intransexpression of wild-typeedpX1, but notedpX1E153A, was able to complement the weakened virulence phenotype. These results indicated that an active EAL domain is required for EdpX1 to regulate the virulence ofX. oryzaepv. oryzae. We then demonstrated that the ΔedpX1mutant was defective in secreting exopolysaccharide (EPS) and forming biofilms. The expression ofedpX1in the ΔedpX1mutant, but notedpX1E153A, restored the defective phenotypes to near-wild-type levels. In addition, we observed that EdpX1-green fluorescent protein (EdpX1-GFP) exhibited multiple subcellular localization foci, and this pattern was dependent on its transmembrane (TM) region, which did not seem to directly contribute to the regulatory function of EdpX1. Thus, we concluded that EdpX1 exhibits PDE activity to control c-di-GMP levels, and its EAL domain is necessary and sufficient for its regulation of virulence inX. oryzaepv. oryzae.IMPORTANCEBacteria utilize c-di-GMP as a second messenger to regulate various biological functions. The synthesis and degradation of c-di-GMP are catalyzed by GGDEF domains and an EAL or HD-GYP domain, respectively. Multiple genes encoding these domains are often found in one bacterial strain. For example, in the genome ofX. oryzaepv. oryzae PXO99A, 26 genes encoding proteins containing these domains were identified. Therefore, to fully appreciate the complexity and specificity of c-di-GMP signaling inX. oryzaepv. oryzae, the enzymatic activities and regulatory functions of each GGDEF, EAL, and HD-GYP domain protein need to be elucidated. In this study, we showed that the EAL domain protein EdpX1 is a major PDE to regulate diverse virulence phenotypes through the c-di-GMP signaling pathway.

scholarly journals The Phosducin-Like Protein PhLP1 Is Essential for Gβγ Dimer Formation in Dictyostelium discoideum

2005 ◽  
Vol 25 (18) ◽  
pp. 8393-8400 ◽  
Author(s):  
Jaco C. Knol ◽  
Ruchira Engel ◽  
Mieke Blaauw ◽  
Antonie J. W. G. Visser ◽  
Peter J. M. van Haastert
Keyword(s):  
G Protein ◽  
Dictyostelium Discoideum ◽  
Fluorescent Protein ◽  
Correlation Spectroscopy ◽  
Protein Signaling ◽  
Wild Type ◽  
Dimer Formation ◽  
Green Fluorescent

ABSTRACT Phosducin proteins are known to inhibit G protein-mediated signaling by sequestering Gβγ subunits. However, Dictyostelium discoideum cells lacking the phosducin-like protein PhLP1 display defective rather than enhanced G protein signaling. Here we show that green fluorescent protein (GFP)-tagged Gβ (GFP-Gβ) and GFP-Gγ subunits exhibit drastically reduced steady-state levels and are absent from the plasma membrane in phlp1 − cells. Triton X-114 partitioning suggests that lipid attachment to GFP-Gγ occurs in wild-type cells but not in phlp1 − and gβ − cells. Moreover, Gβγ dimers could not be detected in vitro in coimmunoprecipitation assays with phlp1 − cell lysates. Accordingly, in vivo diffusion measurements using fluorescence correlation spectroscopy showed that while GFP-Gγ proteins are present in a complex in wild-type cells, they are free in phlp1 − and gβ − cells. Collectively, our data strongly suggest the absence of Gβγ dimer formation in Dictyostelium cells lacking PhLP1. We propose that PhLP1 serves as a cochaperone assisting the assembly of Gβ and Gγ into a functional Gβγ complex. Thus, phosducin family proteins may fulfill hitherto unsuspected biosynthetic functions.

scholarly journals Increased Apoptosis Arising from Increased Expression of the Alzheimer's Disease–associated Presenilin-2 Mutation (N141I)

1997 ◽  
Vol 139 (2) ◽  
pp. 485-495 ◽  
Author(s):  
Susan Janicki ◽  
Mervyn J. Monteiro
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Cell Death ◽  
Hela Cells ◽  
Fluorescent Protein ◽  
Normal Function ◽  
Protein Accumulation ◽  
Protein Cleavage ◽  
Wild Type ◽  
Green Fluorescent

Mutations in the genes for presenilin 1 and 2 (PS-1 and PS-2) have been linked to development of early-onset Alzheimer's disease (AD). As neither the normal function of either presenilin is known nor why mutations cause disease, we examined the properties of wild-type, truncated, and mutant PS-2 upon expression in HeLa cells. Although HeLa cells are strongly predisposed to continued mitosis, expression of PS-2 induced programmed cell death (apoptosis). Direct evidence for apoptosis was obtained by double staining for terminal deoxynucleotide transferase nick end labeling (TUNEL) and PS-2 expression and by following green fluorescent protein–tagged PS-2 over time. Deletion analysis indicates that as little as 166 NH2-terminal residues of PS-2 are sufficient for endoplasmic reticulum (ER) localization and apoptosis. Moreover, the AD- associated PS-2 missense mutation (N141I) more efficiently induced cell death compared to wild-type PS-2 despite lower mutant protein accumulation. Expression of the presenilins in several other cell lines and transgenic mice has been accompanied by rapid protein cleavage without the induction of cell death. In contrast, PS-2 expressed in HeLa cells was not cleaved, and cell death occurred. We hypothesize that full-length but not cleaved PS-2 may be important in the regulation or induction of apoptosis.

scholarly journals PakD, a Putative p21-Activated Protein Kinase in Dictyostelium discoideum, Regulates Actin

2013 ◽  
Vol 13 (1) ◽  
pp. 119-126 ◽  
Author(s):  
Miguel Garcia ◽  
Sibnath Ray ◽  
Isaiah Brown ◽  
Jon Irom ◽  
Derrick Brazill
Keyword(s):  
Protein Kinase ◽  
Actin Cytoskeleton ◽  
Dictyostelium Discoideum ◽  
Cell Function ◽  
Actin Dynamics ◽  
Wild Type ◽  
Content Type ◽  
Multicellular Development ◽  
Actin Organization ◽  
Cellular Processes

ABSTRACT Proper regulation of the actin cytoskeleton is essential for cell function and ultimately for survival. Tight control of actin dynamics is required for many cellular processes, including differentiation, proliferation, adhesion, chemotaxis, endocytosis, exocytosis, and multicellular development. Here we describe a putative p21-activated protein kinase, PakD, that regulates the actin cytoskeleton in Dictyostelium discoideum . We found that cells lacking pakD are unable to aggregate and thus unable to develop. Compared to the wild type, cells lacking PakD have decreased membrane extensions, suggesting defective regulation of the actin cytoskeleton. pakD − cells show poor chemotaxis toward cyclic AMP (cAMP) but normal chemotaxis toward folate, suggesting that PakD mediates some but not all chemotaxis responses. pakD − cells have decreased polarity when placed in a cAMP gradient, indicating that the chemotactic defects of the pakD − cells may be due to an impaired cytoskeletal response to cAMP. In addition, while wild-type cells polymerize actin in response to global stimulation by cAMP, pakD − cells exhibit F-actin depolymerization under the same conditions. Taken together, the results suggest that PakD is part of a pathway coordinating F-actin organization during development.

scholarly journals Imaging and Analysis of Pseudomonas aeruginosa Swarming and Rhamnolipid Production

2011 ◽  
Vol 77 (23) ◽  
pp. 8310-8317 ◽  
Author(s):  
Joshua D. Morris ◽  
Jessica L. Hewitt ◽  
Lawrence G. Wolfe ◽  
Nachiket G. Kamatkar ◽  
Sarah M. Chapman ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fluorescent Protein ◽  
Nile Red ◽  
Temporal Distribution ◽  
Time Lapse ◽  
Content Type ◽  
Rhamnolipid Production ◽  
Serovar Typhimurium ◽  
Surface Motility ◽  
Green Fluorescent

ABSTRACTMany bacteria spread over surfaces by “swarming” in groups. A problem for scientists who study swarming is the acquisition of statistically significant data that distinguish two observations or detail the temporal patterns and two-dimensional heterogeneities that occur. It is currently difficult to quantify differences between observed swarm phenotypes. Here, we present a method for acquisition of temporal surface motility data using time-lapse fluorescence and bioluminescence imaging. We specifically demonstrate three applications of our technique with the bacteriumPseudomonas aeruginosa. First, we quantify the temporal distribution ofP. aeruginosacells tagged with green fluorescent protein (GFP) and the surfactant rhamnolipid stained with the lipid dye Nile red. Second, we distinguish swarming ofP. aeruginosaandSalmonella entericaserovar Typhimurium in a coswarming experiment. Lastly, we quantify differences in swarming and rhamnolipid production of severalP. aeruginosastrains. While the best swarming strains produced the most rhamnolipid on surfaces, planktonic culture rhamnolipid production did not correlate with surface growth rhamnolipid production.

scholarly journals Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance

2011 ◽  
Vol 55 (5) ◽  
pp. 2438-2441 ◽  
Author(s):  
Zeynep Baharoglu ◽  
Didier Mazel
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Vibrio Cholerae ◽  
Fluorescent Protein ◽  
Resistance Development ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Green Fluorescent ◽  
Regulated Promoters

ABSTRACTAntibiotic resistance development has been linked to the bacterial SOS stress response. InEscherichia coli, fluoroquinolones are known to induce SOS, whereas other antibiotics, such as aminoglycosides, tetracycline, and chloramphenicol, do not. Here we address whether various antibiotics induce SOS inVibrio cholerae. Reporter green fluorescent protein (GFP) fusions were used to measure the response of SOS-regulated promoters to subinhibitory concentrations of antibiotics. We show that unlike the situation withE. coli, all these antibiotics induce SOS inV. cholerae.

scholarly journals Mechanism of Aurora-B Degradation and Its Dependency on Intact KEN and A-Boxes: Identification of an Aneuploidy-Promoting Property

2005 ◽  
Vol 25 (12) ◽  
pp. 4977-4992 ◽  
Author(s):  
Hao G. Nguyen ◽  
Dharmaraj Chinnappan ◽  
Takeshi Urano ◽  
Katya Ravid
Keyword(s):  
Chromosome Segregation ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Degradation Pathway ◽  
Aurora B ◽  
Stable Form ◽  
Wild Type ◽  
Green Fluorescent ◽  
Critical Regions

ABSTRACT The kinase Aurora-B, a regulator of chromosome segregation and cytokinesis, is highly expressed in a variety of tumors. During the cell cycle, the level of this protein is tightly controlled, and its deregulated abundance is suspected to contribute to aneuploidy. Here, we provide evidence that Aurora-B is a short-lived protein degraded by the proteasome via the anaphase-promoting cyclosome complex (APC/c) pathway. Aurora-B interacts with the APC/c through the Cdc27 subunit, Aurora-B is ubiquitinated, and its level is increased upon treatment with inhibitors of the proteasome. Aurora-B binds in vivo to the degradation-targeting proteins Cdh1 and Cdc20, the overexpression of which accelerates Aurora-B degradation. Using deletions or point mutations of the five putative degradation signals in Aurora-B, we show that degradation of this protein does not depend on its D-boxes (RXXL), but it does require intact KEN boxes and A-boxes (QRVL) located within the first 65 amino acids. Cells transfected with wild-type or A-box-mutated or KEN box-mutated Aurora-B fused to green fluorescent protein display the protein localized to the chromosomes and then to the midzone during mitosis, but the mutated forms are detected at greater intensities. Hence, we identified the degradation pathway for Aurora-B as well as critical regions for its clearance. Intriguingly, overexpression of a stable form of Aurora-B alone induces aneuploidy and anchorage-independent growth.

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