scholarly journals Off-Target Effects of the Septin Drug Forchlorfenuron on Nonplant Eukaryotes

2014 ◽  
Vol 13 (11) ◽  
pp. 1411-1420 ◽  
Author(s):  
Lydia R. Heasley ◽  
Galo Garcia ◽  
Michael A. McMurray
Keyword(s):  
Mammalian Cells ◽  
Budding Yeast ◽  
Yeast Cells ◽  
Detectable Effect ◽  
Wild Type ◽  
Content Type ◽  
Cellular Effects ◽  
Wide Range ◽  
W303 Strain ◽  
Target Effects

ABSTRACTThe septins are a family of GTP-binding proteins that form cytoskeletal filaments. Septins are highly conserved and evolutionarily ancient but are absent from land plants. The synthetic plant cytokinin forchlorfenuron (FCF) was shown previously to inhibit budding yeast cell division and induce ectopic septin structures (M. Iwase, S. Okada, T. Oguchi, and A. Toh-e, Genes Genet. Syst. 79:199–206, 2004,http://dx.doi.org/10.1266/ggs.79.199). Subsequent studies in a wide range of eukaryotes have concluded that FCF exclusively inhibits septin function, yet the mechanism of FCF action in nonplant cells remains poorly understood. Here, we report that the cellular effects of FCF are far more complex than previously described. The reported growth arrest of budding yeast cells treated with 1 mM FCF partly reflects sensitization caused by abud4mutation present in the W303 strain background. In wild-type (BUD4+) budding yeast, growth was inhibited at FCF concentrations that had no detectable effect on septin structure or function. Moreover, FCF severely inhibited the proliferation of fission yeast cells, in which septin function is nonessential. FCF induced fragmentation of budding yeast mitochondrial reticula and the loss of mitochondrial membrane potential. Mitochondria also fragmented in cultured mammalian cells treated with concentrations of FCF that previously were assumed to target septins only. Finally, FCF potently inhibited ciliation and motility and induced mitochondrial disorganization inTetrahymena thermophilawithout apparent alterations in septin structure. None of these effects was consistent with the inhibition of septin function. Our findings point to nonseptin targets as major concerns when using FCF.

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4084-4092
Author(s):  
P C McCabe ◽  
H Haubruck ◽  
P Polakis ◽  
F McCormick ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Bound States ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Amino Terminal ◽  
Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

scholarly journals Virulence and Metabolic Characteristics of Salmonella enterica Serovar Enteritidis Strains with DifferentsefDVariants in Hens

2012 ◽  
Vol 78 (18) ◽  
pp. 6405-6412 ◽  
Author(s):  
Cesar A. Morales ◽  
Jean Guard ◽  
Roxana Sanchez-Ingunza ◽  
Devendra H. Shah ◽  
Mark Harrison
Keyword(s):  
Salmonella Enterica ◽  
Egg Production ◽  
The Body ◽  
Wild Type ◽  
Salmonella Enterica Serovar Enteritidis ◽  
Blood Calcium ◽  
Content Type ◽  
Metabolic Characteristics ◽  
Wide Range ◽  
Metabolic Properties

ABSTRACTSalmonella entericaserovar Enteritidis is one of a fewSalmonella entericaserotypes that has SEF14 fimbriae encoded by thesefoperon, which consists of 4 cotranscribed genes,sefABCD, regulated bysefR. A parental strain was used to construct asefDmutant and its complement, and all 3 strains were compared for gene expression, metabolic properties, and virulence characteristics in hens. Transcription ofsefDby wild type was suppressed at 42°C and absent for the mutant under conditions where the complemented mutant had 103times higher transcription. Growth of the complemented mutant was restricted in comparison to that of the mutant and wild type. Hens infected with the wild type and mutant showed decreased blood calcium and egg production, but infection with the complemented mutant did not. Thus, the absence ofsefDcorrelated with increased metabolic capacity and enhanced virulence of the pathogen. These results suggest that any contribution thatsefDmakes to egg contamination is either unknown or would be limited to early transmission from the environment to the host. Absence ofsefD, either through mutation or by suppression of transcription at the body temperature of the host, may contribute to the virulence ofSalmonella entericaby facilitating growth on a wide range of metabolites.

scholarly journals Sml1 Inhibits the DNA Repair Activity of Rev1 in Saccharomyces cerevisiae during Oxidative Stress

2020 ◽  
Vol 86 (7) ◽  
Author(s):  
Rui Yao ◽  
Pei Zhou ◽  
Chengjin Wu ◽  
Liming Liu ◽  
Jing Wu
Keyword(s):  
Oxidative Stress ◽  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Survival Rate ◽  
Type Strain ◽  
Mutation Frequency ◽  
Wild Type Strain ◽  
Yeast Cells ◽  
Wild Type ◽  
Content Type

ABSTRACT In Saccharomyces cerevisiae, Y family DNA polymerase Rev1 is involved in the repair of DNA damage by translesion DNA synthesis (TLS). In the current study, to elucidate the role of Rev1 in oxidative stress-induced DNA damage in S. cerevisiae, REV1 was deleted and overexpressed; transcriptome analysis of these mutants along with the wild-type strain was performed to screen potential genes that could be associated with REV1 during response to DNA damage. When the yeast cells were treated with 2 mM H2O2, the deletion of REV1 resulted in a 1.5- and 2.8-fold decrease in the survival rate and mutation frequency, respectively, whereas overexpression of REV1 increased the survival rate and mutation frequency by 1.1- and 2.9-fold, respectively, compared to the survival rate and mutation frequency of the wild-type strain. Transcriptome and phenotypic analyses identified that Sml1 aggravated oxidative stress in the yeast cells by inhibiting the activity of Rev1. This inhibition was due to the physical interaction between the BRCA1 C terminus (BRCT) domain of Rev1 and amino acid residues 36 to 70 of Sml1; the cell survival rate and mutation frequency increased by 1.8- and 3.1-fold, respectively, when this interaction was blocked. We also found that Sml1 inhibited Rev1 phosphorylation under oxidative stress and that deletion of SML1 increased the phosphorylation of Rev1 by 46%, whereas overexpression of SML1 reduced phosphorylation of Rev1. Overall, these findings demonstrate that Sml1 could be a novel regulator that mediates Rev1 dephosphorylation to inhibit its activity during oxidative stress. IMPORTANCE Rev1 was critical for cell growth in S. cerevisiae, and the deletion of REV1 caused a severe growth defect in cells exposed to oxidative stress (2 mM H2O2). Furthermore, we found that Sml1 physically interacted with Rev1 and inhibited Rev1 phosphorylation, thereby inhibiting Rev1 DNA antioxidant activity. These findings indicate that Sml1 could be a novel regulator for Rev1 in response to DNA damage by oxidative stress.

scholarly journals A Family of Small Intrinsically Disordered Proteins Involved in Flagellum-Dependent Motility inSalmonella enterica

2018 ◽  
Vol 201 (2) ◽  
Author(s):  
Tamiko Oguri ◽  
Youjeong Kwon ◽  
Jerry K. K. Woo ◽  
Gerd Prehna ◽  
Hyun Lee ◽  
...  
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Expression Profiles ◽  
Functional Characterization ◽  
Disordered Proteins ◽  
Wild Type ◽  
Content Type ◽  
Cellular Pathway ◽  
Intrinsically Disordered ◽  
Small Proteins ◽  
Wide Range

ABSTRACTBy screening a collection ofSalmonellamutants deleted for genes encoding small proteins of ≤60 amino acids, we identified three paralogous small genes (ymdF,STM14_1829, andyciG) required for wild-type flagellum-dependent swimming and swarming motility. TheymdF,STM14_1829, andyciGgenes encode small proteins of 55, 60, and 60 amino acid residues, respectively. A bioinformatics analysis predicted that these small proteins are intrinsically disordered proteins, and circular dichroism analysis of purified recombinant proteins confirmed that all three proteins are unstructured in solution. A mutant deleted for STM14_1829 showed the most severe motility defect, indicating that among the three paralogs, STM14_1829 is a key protein required for wild-type motility. We determined that relative to the wild type, the expression of the flagellin protein FliC is lower in the ΔSTM14_1829mutant due to the downregulation of theflhDCoperon encoding the FlhDC master regulator. By comparing the gene expression profiles between the wild-type and ΔSTM14_1829strains via RNA sequencing, we found that the gene encoding the response regulator PhoP is upregulated in the ΔSTM14_1829mutant, suggesting the indirect repression of theflhDCoperon by the activated PhoP. Homologs of STM14_1829 are conserved in a wide range of bacteria, includingEscherichia coliandPseudomonas aeruginosa. We showed that the inactivation of STM14_1829 homologs inE. coliandP. aeruginosaalso alters motility, suggesting that this family of small intrinsically disordered proteins may play a role in the cellular pathway(s) that affects motility.IMPORTANCEThis study reports the identification of a novel family of small intrinsically disordered proteins that are conserved in a wide range of flagellated and nonflagellated bacteria. Although this study identifies the role of these small proteins in the scope of flagellum-dependent motility inSalmonella, they likely play larger roles in a more conserved cellular pathway(s) that indirectly affects flagellum expression in the case of motile bacteria. Small intrinsically disordered proteins have not been well characterized in prokaryotes, and the results of our study provide a basis for their detailed functional characterization.

scholarly journals RSR1, a ras-like gene homologous to Krev-1 (smg21A/rap1A): role in the development of cell polarity and interactions with the Ras pathway in Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (2) ◽  
pp. 758-766 ◽  
Author(s):  
R Ruggieri ◽  
A Bender ◽  
Y Matsui ◽  
S Powers ◽  
Y Takai ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Polarity ◽  
Copy Number ◽  
Mammalian Cells ◽  
Direct Interaction ◽  
Dominant Negative ◽  
Yeast Cells ◽  
Wild Type ◽  
Ras Gene ◽  
Ras Pathway

The Saccharomyces cerevisiae ras-like gene RSR1 is particularly closely related to the mammalian gene Krev-1 (also known as smg21A and rap1A). RSR1 was originally isolated as a multicopy suppressor of a cdc24 mutation, which causes an inability to bud or establish cell polarity. Deletion of RSR1 itself does not affect growth but causes a randomization of bud position. We have now constructed mutant alleles of RSR1 encoding proteins with substitutions of Val for Gly at position 12 (analogous to constitutively activated Ras proteins) or Asn for Lys at position 16 (analogous to a dominant-negative Ras protein). rsr1Val-12 could not restore a normal budding pattern to an rsr1 deletion strain but could suppress a cdc24 mutation when overexpressed. rsr1Asn-16 could randomize the budding pattern of a wild-type strain even in low copy number but was not lethal even in high copy number. These and other results suggest that Rsr1p functions only in bud site selection and not in subsequent events of polarity establishment and bud formation, that this function involves a cycling between GTP-bound and GDP-bound forms of the protein, and that the suppression of cdc24 involves direct interaction between Rsr1p[GTP] and Cdc24p. Functional homology between Rsr1p and Krev-1 p21 was suggested by the observations that expression of the latter protein in yeast cells could both suppress a cdc24 mutation and randomize the budding pattern of wild-type cells. As Krev-1 overexpression can suppress ras-induced transformation of mammalian cells, we looked for effects of RSR1 on the S. cerevisiae Ras pathway. Although no suppression of the activated RAS2Val-19 allele was observed, overexpression of rsr1Val-12 suppressed the lethality of strains lacking RAS gene function, apparently through a direct activation of adenyl cyclase. This interaction of Rsr1p with the effector of Ras in S. cerevisiae suggests that Krev-1 may revert ras-induced transformation of mammalian cells by affecting the interaction of ras p21 with its effector.

scholarly journals Zap1 Control of Cell-Cell Signaling in Candida albicans Biofilms

2011 ◽  
Vol 10 (11) ◽  
pp. 1448-1454 ◽  
Author(s):  
Shantanu Ganguly ◽  
Andrew C. Bishop ◽  
Wenjie Xu ◽  
Suman Ghosh ◽  
Kenneth W. Nickerson ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Yeast Cells ◽  
Gas Chromatography Mass Spectrometry ◽  
Wild Type ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Hypha Formation ◽  
Dependent Signal ◽  
Reporter Strains ◽  
Biosensor Strain

ABSTRACTBiofilms ofCandida albicansinclude both yeast cells and hyphae. Prior studies indicated that azap1Δ/Δ mutant, defective in zinc regulator Zap1, has increased accumulation of yeast cells in biofilms. This altered yeast-hypha balance may arise from internal regulatory alterations or from an effect on the production of diffusible quorum-sensing (QS) molecules. Here, we develop biosensor reporter strains that express yeast-specificYWP1-RFPor hypha-specificHWP1-RFP, along with a constitutiveTDH3-GFPnormalization standard. Seeding these biosensor strains into biofilms allows a biological activity assay of the surrounding biofilm milieu. Azap1Δ/Δ biofilm induces the yeast-specificYWP1-RFPreporter in a wild-type biosensor strain, as determined by both quantitative reverse transcription-PCR (qRT-PCR) gene expression measurements and confocal microscopy. Remediation of thezap1Δ/Δ zinc uptake defect through zinc transporter geneZRT2overexpression reverses induction of the yeast-specificYWP1-RFPreporter. Gas chromatography-mass spectrometry (GC-MS) measurements of known organic QS molecules show that thezap1Δ/Δ mutant accumulates significantly less farnesol than wild-type or complemented strains and thatZRT2overexpression does not affect farnesol accumulation. Farnesol is a well-characterized inhibitor of hypha formation; hence, a reduction in farnesol levels inzap1Δ/Δ biofilms is unexpected. Our findings argue that a Zap1- and zinc-dependent signal affects the yeast-hypha balance and that it is operative in the low-farnesol environment of thezap1Δ/Δ biofilm. In addition, our results indicate that Zap1 is a positive regulator of farnesol accumulation.

scholarly journals Proteolysis by calpains: a possible contribution to degradation of p53.

1997 ◽  
Vol 17 (5) ◽  
pp. 2806-2815 ◽  
Author(s):  
M Pariat ◽  
S Carillo ◽  
M Molinari ◽  
C Salvat ◽  
L Debüssche ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Tertiary Structure ◽  
P53 Protein ◽  
Calcium Ionophore ◽  
Expression Vectors ◽  
Wild Type ◽  
Experimental Conditions ◽  
Null Cell ◽  
Wide Range ◽  
Wild Type P53

p53 is a short-lived transcription factor that is frequently mutated in tumor cells. Work by several laboratories has already shown that the ubiquitin-proteasome pathway can largely account for p53 destruction, at least under specific experimental conditions. We report here that, in vitro, wild-type p53 is a sensitive substrate for milli- and microcalpain, which are abundant and ubiquitous cytoplasmic proteases. Degradation was dependent on p53 protein conformation. Mutants of p53 with altered tertiary structure displayed a wide range of susceptibility to calpains, some of them being largely resistant to degradation and others being more sensitive. This result suggests that the different mutants tested here adopt slightly different conformations to which calpains are sensitive but that cannot be discriminated by using monoclonal antibodies such as PAb1620 and PAb240. Inhibition of calpains by using the physiological inhibitor calpastatin leads to an elevation of p53 steady-state levels in cells expressing wild-type p53. Conversely, activation of calpains by calcium ionophore led to a reduction of p53 in mammalian cells, and the effect was blocked by cell-permeant calpain inhibitors. Cotransfection of p53-null cell lines with p53 and calpastatin expression vectors resulted in an increase in p53-dependent transcription activity. Taken together, these data support the idea that calpains may also contribute to the regulation of wild-type p53 protein levels in vivo.

scholarly journals The number of cytokinesis nodes in mitotic fission yeast scales with cell volume

2021 ◽  
Author(s):  
Wasim A Sayyad ◽  
Thomas D Pollard
Keyword(s):  
Plasma Membrane ◽  
Fission Yeast ◽  
Large Population ◽  
Yeast Cells ◽  
Wild Type ◽  
Contractile Ring ◽  
Node Number ◽  
Nmt1 Promoter ◽  
Wide Range ◽  
Mutant Cells

Cytokinesis nodes are assemblies of stoichiometric ratios of proteins associated with the plasma membrane, which serve as precursors for the contractile ring during cytokinesis by fission yeast. The total number of nodes is uncertain, because of the limitations of the methods used previously. Here we used the ~140 nm resolution of Airyscan confocal microscopy to resolve a large population of dim, unitary cytokinesis nodes in 3D reconstructions of whole fission yeast cells. Wild-type fission yeast cells make about 200 unitary cytokinesis nodes. Most, but not all of these nodes condense into a contractile ring. The number of cytokinesis nodes scales with cell size in four strains tested, although wide rga4Δ mutant cells form somewhat fewer cytokinesis nodes than expected from the overall trend. The surface density of Pom1 kinase on the plasma membrane around the equators of cells is similar with a wide range of node numbers, so Pom1 does not control cytokinesis node number. However, varying protein concentrations with the nmt1 promoter showed that the numbers of nodes increase above a baseline of about 200 with the total cellular concentration of either Pom1 or the kinase Cdr2.

scholarly journals Deletion of Budding Yeast MAD2 Suppresses Clone-to-Clone Differences in Artificial Linear Chromosome Copy Numbers and Gives Rise to Higher Retention Rates

2020 ◽  
Vol 8 (10) ◽  
pp. 1495
Author(s):  
Scott C. Schuyler ◽  
Lin-Ing Wang ◽  
Yi-Shan Ding ◽  
Yi-Chieh Lee ◽  
Hsin-Yu Chen
Keyword(s):  
Budding Yeast ◽  
Retention Rate ◽  
Retention Rates ◽  
Yeast Cells ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Linear Chromosome ◽  
Copy Numbers ◽  
Clone Differences ◽  
Linear Chromosomes

Our goal was to investigate the changes in artificial short-linear chromosome average copy numbers per cell arising from partial or full loss of Mitotic Arrest-Deficient 2 (MAD2) spindle checkpoint function in budding yeast Saccharomyces cerevisiae. Average artificial linear chromosome copy numbers in a population of cells, as measured by quantitative polymerase chain reactions (qPCR), and retention rates, as measured by fluctuation analyses, were performed on a total of 62 individual wild type and mad2∆ mutant haploid and diploid clones. Wild type cells, both haploids and diploids, displayed phenotypically unique clone-to-clone differences: one group of 15 clones displayed low-copy numbers per cell and high retention rates, were 1 clone was found to have undergone a genomic integration event, and the second group of 15 clones displayed high copy numbers per cell and low retention rates, with the latter values being consistent with the previously published results where only a single clone had been measured. These chromosome states were observed to be unstable when propagated for 10 days under selection, where high copy-low retention rate clones evolved into low copy-high retention rate clones, but no evidence for integration events was observed. By contrast, mad2∆ haploid and mad2∆/mad2∆ diploids displayed a suppression of the clone-to-clone differences, where 20 out of 21 clones had mid-level artificial linear chromosome copy numbers per cell, but maintained elevated chromosome retention rates. The elevated levels in retention rates in mad2∆ and mad2∆/mad2∆ cells were also maintained even in the absence of selection during growth over 3 days. MAD2/mad2∆ heterozygous diploids displayed multiple clonal groups: 4 with low copy numbers, 5 with mid-level copy numbers, and 1 with a high copy number of artificial linear chromosomes, but all 10 clones uniformly displayed low retention rates. Our observations reveal that MAD2 function contributes to the ability of yeast cells to maintain a high number of artificial linear chromosomes per cell in some clones, but, counter-intuitively, mad2∆ suppresses clone-to-clone differences and leads to an improvement in artificial linear chromosome retention rates yielding a more uniform and stable clonal population with mid-level chromosome copy numbers per cell.

Global SLAM-Seq for accurate mRNA decay determination and identification of NMD targets

2021 ◽  
Author(s):  
Hanna Alalam ◽  
Jorge Zepeda ◽  
Per Sunnerhagen
Keyword(s):  
Half Life ◽  
Mammalian Cells ◽  
Rna Degradation ◽  
Stability Study ◽  
Yeast Cells ◽  
Invasive Technique ◽  
Cell Physiology ◽  
Wild Type ◽  
Nonsense Mediated Decay ◽  
Degradation Rates

Gene expression analysis requires accurate measurements of global RNA degradation rates, earlier problematic with methods disruptive to cell physiology. Recently, metabolic RNA labeling emerged as an efficient and minimally invasive technique applied in mammalian cells. Here, we have adapted SH-Linked Alkylation for the Metabolic Sequencing of RNA (SLAM-Seq) for a global mRNA stability study in yeast using 4-thiouracil pulse-chase labeling. We assign high-confidence half-life estimates for 67.5 % of expressed ORFs, and measure a median half-life of 9.4 min. For mRNAs where half-life estimates exist in the literature, their ranking order was in good agreement with previous data, indicating that SLAM-Seq efficiently classifies stable and unstable transcripts. We then leveraged our yeast protocol to identify targets of the Nonsense-mediated decay (NMD) pathway. There are currently no global reports of half-lives in both wild type and NMD defective yeast cells; instead steady-state RNA level changes are used as a proxy. With SLAM-Seq, we assign 580 transcripts as putative NMD targets, based on their measured half-lives in wild-type and upf3Δ mutants. We find 230 novel targets, and observe a strong agreement with previous reports of NMD targets, 60 % of our candidates being identified in previous studies. This indicates that SLAM-Seq is a simpler and more economic method for global quantification of mRNA half-lives. Our adaptation for yeast yielded global quantitative measures of the NMD effect on transcript half-lives, high correlation with RNA half-lives measured previously with more technically challenging protocols, and identification of novel NMD regulated transcripts that escaped prior detection.

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