scholarly journals Loss of the F-Actin Binding and Vesicle-Associated Protein Comitin Leads to a Phagocytosis Defect

2002 ◽  
Vol 1 (6) ◽  
pp. 906-914 ◽  
Author(s):  
Thomas Schreiner ◽  
Martina R. Mohrs ◽  
Rosemarie Blau-Wasser ◽  
Alfred von Krempelhuber ◽  
Michael Steinert ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Gene Replacement ◽  
Actin Binding ◽  
Wild Type ◽  
Hyperosmotic Shock ◽  
Latex Beads ◽  
Knockout Mutants ◽  
Mutant Cells

ABSTRACT Comitin is an F-actin binding and membrane-associated protein from Dictyostelium discoideum, which is present on Golgi and vesicle membranes and changes its localization in response to agents affecting the cytoskeleton. To investigate its in vivo functions we have generated knockout mutants by gene replacement. Based on comitin's in vitro functions we examined properties related to vesicular transport and microfilament function. Whereas cell growth, pinocytosis, secretion, chemotaxis, motility, and development were unaltered, comitin-lacking cells were impaired in the early steps of phagocytosis of Saccharomyces cerevisiae particles and of Escherichia coli, whereas uptake of latex beads was unaffected. Furthermore, the lack of comitin positively affected survival of pathogenic bacteria. Mutant cells also showed an altered response to hyperosmotic shock in comparison to the wild type. The redistribution of comitin during hyperosmotic shock in wild-type cells and its presence on early phagosomes suggest a direct involvement of comitin in these processes.

scholarly journals Dictyostelium mutants lacking the cytoskeletal protein coronin are defective in cytokinesis and cell motility.

1993 ◽  
Vol 120 (1) ◽  
pp. 163-173 ◽  
Author(s):  
E L de Hostos ◽  
C Rehfuess ◽  
B Bradtke ◽  
D R Waddell ◽  
R Albrecht ◽  
...  
Keyword(s):  
Leading Edge ◽  
Gene Replacement ◽  
Actin Binding ◽  
Beta Subunits ◽  
Wild Type ◽  
Daughter Cells ◽  
Immunofluorescence Labeling ◽  
Mutant Cells

Coronin is an actin-binding protein in Dictyostelium discoideum that is enriched at the leading edge of the cells and in projections of the cell surface called crowns. The polypeptide sequence of coronin is distinguished by its similarities to the beta-subunits of trimeric G proteins (E. L. de Hostos, B. Bradtke, F. Lottspeich, R. Guggenheim, and G. Gerisch, 1991. EMBO (Eur. Mol. Biol. Organ.) J. 10:4097-4104). To elucidate the in vivo function of coronin, null mutants have been generated by gene replacement. The mutant cells lacking coronin grow and migrate more slowly than wild-type cells. When these cor- cells grow in liquid medium they become multinucleate, indicating a role of coronin in cytokinesis. To explore this role, coronin has been localized in mitotic wild-type cells by immunofluorescence labeling. During separation of the daughter cells, coronin is strongly accumulated at their distal portions including the leading edges. This contrasts with the localization of myosin II in the cleavage furrow and suggests that coronin functions independently of the conventional myosin in facilitating cytokinesis.

Analysis of the function of the 70-kilodalton cyclase-associated protein (CAP) by using mutants of yeast adenylyl cyclase defective in CAP binding

1993 ◽  
Vol 13 (7) ◽  
pp. 4087-4097
Author(s):  
J Wang ◽  
N Suzuki ◽  
Y Nishida ◽  
T Kataoka
Keyword(s):  
Heat Shock ◽  
Adenylyl Cyclase ◽  
Gene Replacement ◽  
Yeast Cells ◽  
Cyclase Activity ◽  
In Vitro Mutagenesis ◽  
Wild Type ◽  
Adenylyl Cyclase Activity

In Saccharomyces cerevisiae, adenylyl cyclase forms a complex with the 70-kDa cyclase-associated protein (CAP). By in vitro mutagenesis, we assigned a CAP-binding site of adenylyl cyclase to a small segment near its C terminus and created mutants which lost the ability to bind CAP. CAP binding was assessed first by observing the ability of the overproduced C-terminal 150 residues of adenylyl cyclase to sequester CAP, thereby suppressing the heat shock sensitivity of yeast cells bearing the activated RAS2 gene (RAS2Val-19), and then by immunoprecipitability of adenylyl cyclase activity with anti-CAP antibody and by direct measurement of the amount of CAP bound. Yeast cells whose chromosomal adenylyl cyclase genes were replaced by the CAP-nonbinding mutants possessed adenylyl cyclase activity fully responsive to RAS2 protein in vitro. However, they did not exhibit sensitivity to heat shock in the RAS2Val-19 background. When glucose-induced accumulation of cyclic AMP (cAMP) was measured in these mutants carrying RAS2Val-19, a rapid transient rise indistinguishable from that of wild-type cells was observed and a high peak level and following persistent elevation of the cAMP concentration characteristic of RAS2Val-19 were abolished. In contrast, in the wild-type RAS2 background, similar cyclase gene replacement did not affect the glucose-induced cAMP response. These results suggest that the association with CAP, although not involved in the in vivo response to the wild-type RAS2 protein, is somehow required for the exaggerated response of adenylyl cyclase to activated RAS2.

scholarly journals Mutations in RNA Polymerase II and Elongation Factor SII Severely Reduce mRNA Levels in Saccharomyces cerevisiae

1998 ◽  
Vol 18 (10) ◽  
pp. 5771-5779 ◽  
Author(s):  
J. Cale Lennon ◽  
Megan Wind ◽  
Laura Saunders ◽  
M. Benjamin Hock ◽  
Daniel Reines
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Genetic Interaction ◽  
Elongation Factor ◽  
Mrna Levels ◽  
Wild Type ◽  
Mutant Cells ◽  
Rna Levels

ABSTRACT Elongation factor SII interacts with RNA polymerase II and enables it to transcribe through arrest sites in vitro. The set of genes dependent upon SII function in vivo and the effects on RNA levels of mutations in different components of the elongation machinery are poorly understood. Using yeast lacking SII and bearing a conditional allele of RPB2, the gene encoding the second largest subunit of RNA polymerase II, we describe a genetic interaction between SII and RPB2. An SII gene disruption or therpb2-10 mutation, which yields an arrest-prone enzyme in vitro, confers sensitivity to 6-azauracil (6AU), a drug that depresses cellular nucleoside triphosphates. Cells with both mutations had reduced levels of total poly(A)+ RNA and specific mRNAs and displayed a synergistic level of drug hypersensitivity. In cells in which the SII gene was inactivated, rpb2-10 became dominant, as if template-associated mutant RNA polymerase II hindered the ability of wild-type polymerase to transcribe. Interestingly, while 6AU depressed RNA levels in both wild-type and mutant cells, wild-type cells reestablished normal RNA levels, whereas double-mutant cells could not. This work shows the importance of an optimally functioning elongation machinery for in vivo RNA synthesis and identifies an initial set of candidate genes with which SII-dependent transcription can be studied.

scholarly journals The Nonessential H2A N-Terminal Tail Can Function as an Essential Charge Patch on the H2A.Z Variant N-Terminal Tail

2003 ◽  
Vol 23 (8) ◽  
pp. 2778-2789 ◽  
Author(s):  
Qinghu Ren ◽  
Martin A. Gorovsky
Keyword(s):  
Tetrahymena Thermophila ◽  
Gene Replacement ◽  
In Vitro Mutagenesis ◽  
Wild Type ◽  
Wild Type Protein ◽  
Lysine Residues ◽  
In The Wild ◽  
Essential Function

ABSTRACT Tetrahymena thermophila cells contain three forms of H2A: major H2A.1 and H2A.2, which make up ∼80% of total H2A, and a conserved variant, H2A.Z. We showed previously that acetylation of H2A.Z was essential (Q. Ren and M. A. Gorovsky, Mol. Cell 7:1329-1335, 2001). Here we used in vitro mutagenesis of lysine residues, coupled with gene replacement, to identify the sites of acetylation of the N-terminal tail of the major H2A and to analyze its function in vivo. Tetrahymena cells survived with all five acetylatable lysines replaced by arginines plus a mutation that abolished acetylation of the N-terminal serine normally found in the wild-type protein. Thus, neither posttranslational nor cotranslational acetylation of major H2A is essential. Surprisingly, the nonacetylatable N-terminal tail of the major H2A was able to replace the essential function of the acetylation of the H2A.Z N-terminal tail. Tail-swapping experiments between H2A.1 and H2A.Z revealed that the nonessential acetylation of the major H2A N-terminal tail can be made to function as an essential charge patch in place of the H2A.Z N-terminal tail and that while the pattern of acetylation of an H2A N-terminal tail is determined by the tail sequence, the effects of acetylation on viability are determined by properties of the H2A core and not those of the N-terminal tail itself.

scholarly journals Involvement of the AcrAB-TolC Efflux Pump in the Resistance, Fitness, and Virulence of Enterobacter cloacae

2012 ◽  
Vol 56 (4) ◽  
pp. 2084-2090 ◽  
Author(s):  
Astrid Pérez ◽  
Margarita Poza ◽  
Ana Fernández ◽  
Maria del Carmen Fernández ◽  
Susana Mallo ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Pathogenic Bacteria ◽  
Efflux Pump ◽  
Enterobacter Cloacae ◽  
Efflux Pumps ◽  
Clinical Isolates ◽  
Wild Type ◽  
Content Type

ABSTRACTMultidrug efflux pumps have emerged as important mechanisms of antimicrobial resistance in bacterial pathogens. In order to cause infection, pathogenic bacteria require mechanisms to avoid the effects of host-produced compounds, and express efflux pumps may accomplish this task. In this study, we evaluated the effect of the inactivation of AcrAB-TolC on antimicrobial resistance, fitness, and virulence inEnterobacter cloacae, an opportunistic pathogen usually involved in nosocomial infections. Two different clinical isolates ofE. cloacaewere used, EcDC64 (multidrug resistance overexpressing the AcrAB-TolC efflux pump) and Jc194 (basal AcrAB-TolC expression). TheacrAandtolCgenes were deleted in strains EcDC64 and Jc194 to produce, respectively, EcΔacrAand EcΔtolCand JcΔacrAand JcΔtolCknockout (KO) derivatives. Antibiotic susceptibility testing was performed with all isolates, and we discovered that these mechanisms are involved in the resistance ofE. cloacaeto several antibiotics. Competition experiments were also performed with wild-type and isogenic KO strains. The competition index (CI), defined as the mutant/wild-type ratio, revealed that theacrAandtolCgenes both affect the fitness ofE. cloacae, as fitness was clearly reduced in theacrAandtolCKO strains. The median CI values obtainedin vitroandin vivowere, respectively, 0.42 and 0.3 for EcDC64/EcΔacrA, 0.24 and 0.38 for EcDC64/EcΔtolC, 0.15 and 0.11 for Jc194/JcΔacrA, and 0.38 and 0.39 for Jc194/JcΔtolC. Use of an intraperitoneal mouse model of systemic infection revealed reduced virulence in bothE. cloacaeclinical strains when either theacrAortolCgene was inactivated. In conclusion, the structural components of the AcrAB-TolC efflux pump appear to play a role in antibiotic resistance as well as environmental adaptation and host virulence in clinical isolates ofE. cloacae.

scholarly journals A stealth adhesion factor contributes toVibrio vulnificuspathogenicity: Flp pili play roles in host invasion, survival in the blood stream and resistance to complement activation

2019 ◽  
Author(s):  
Tra–My Duong–Nu ◽  
Kwangjoon Jeong ◽  
Soo Young Kim ◽  
Wenzhi Tan ◽  
Sao Puth ◽  
...  
Keyword(s):  
Host Cell ◽  
Complement Activation ◽  
Vibrio Vulnificus ◽  
Alternative Pathway ◽  
Molecular Genetic ◽  
Wild Type ◽  
Triple Mutant ◽  
Mutant Cells

AbstractThe tad operons encode the machinery required for adhesive Flp (fimbrial low-molecular-weight protein) pili biogenesis.Vibrio vulnificus, an opportunistic pathogen, harbors three distincttadloci. Among them, onlytad1locus was highly upregulated inin vivogrowing bacteria compared toin vitroculture condition. To understand the pathogenic roles of the threetadloci during infection, we constructed single, double and triple tad loci deletion mutants. Interestingly, only theΔtad123triple mutant cells exhibited significantly decreased lethality in mice. Ultrastructural observations revealed short, thin filamentous projections disappeared on theΔtad123mutant cells. Since the pilin was paradoxically non-immunogenic, a V5 tag was fused to Flp to visualize the pilin protein by using immunogold EM and immunofluorescence microscopy. TheΔtad123mutant cells showed attenuated host cell adhesion, delayed RtxA1 exotoxin secretion and subsequently impaired translocation across the intestinal epithelium compared to wild type, which could be partially complemented with each wild type operon. TheΔtad123mutant was susceptible to complement-mediated bacteriolysis, predominantly via the alternative pathway, suggesting stealth hiding role of the Tad pili. Taken together, all threetadloci cooperate to confer successful invasion ofV. vulnificusinto deeper tissue and evasion from host defense mechanisms, ultimately resulting in septicemia.Author SummaryTo understand the roles of the three Tad operons in the pathogenesis ofV. vulnificusinfection, we constructed mutant strain with single, double and triple Tad loci deletions. Employing a variety of mouse infection models coupled with molecular genetic analyses, we demonstrate here that all three Tad operons are required forV. vulnificuspathogenicity as the cryptic pili contribute to host cell and tissue invasion, survival in the blood, and resistance to complement activation.

scholarly journals Analysis of the function of the 70-kilodalton cyclase-associated protein (CAP) by using mutants of yeast adenylyl cyclase defective in CAP binding.

1993 ◽  
Vol 13 (7) ◽  
pp. 4087-4097 ◽  
Author(s):  
J Wang ◽  
N Suzuki ◽  
Y Nishida ◽  
T Kataoka
Keyword(s):  
Heat Shock ◽  
Adenylyl Cyclase ◽  
Gene Replacement ◽  
Yeast Cells ◽  
Cyclase Activity ◽  
In Vitro Mutagenesis ◽  
Wild Type ◽  
Adenylyl Cyclase Activity

In Saccharomyces cerevisiae, adenylyl cyclase forms a complex with the 70-kDa cyclase-associated protein (CAP). By in vitro mutagenesis, we assigned a CAP-binding site of adenylyl cyclase to a small segment near its C terminus and created mutants which lost the ability to bind CAP. CAP binding was assessed first by observing the ability of the overproduced C-terminal 150 residues of adenylyl cyclase to sequester CAP, thereby suppressing the heat shock sensitivity of yeast cells bearing the activated RAS2 gene (RAS2Val-19), and then by immunoprecipitability of adenylyl cyclase activity with anti-CAP antibody and by direct measurement of the amount of CAP bound. Yeast cells whose chromosomal adenylyl cyclase genes were replaced by the CAP-nonbinding mutants possessed adenylyl cyclase activity fully responsive to RAS2 protein in vitro. However, they did not exhibit sensitivity to heat shock in the RAS2Val-19 background. When glucose-induced accumulation of cyclic AMP (cAMP) was measured in these mutants carrying RAS2Val-19, a rapid transient rise indistinguishable from that of wild-type cells was observed and a high peak level and following persistent elevation of the cAMP concentration characteristic of RAS2Val-19 were abolished. In contrast, in the wild-type RAS2 background, similar cyclase gene replacement did not affect the glucose-induced cAMP response. These results suggest that the association with CAP, although not involved in the in vivo response to the wild-type RAS2 protein, is somehow required for the exaggerated response of adenylyl cyclase to activated RAS2.

scholarly journals SEC62 encodes a putative membrane protein required for protein translocation into the yeast endoplasmic reticulum.

1989 ◽  
Vol 109 (6) ◽  
pp. 2653-2664 ◽  
Author(s):  
R J Deshaies ◽  
R Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Alpha Helix ◽  
Wild Type ◽  
Cytosol Fraction ◽  
Arginine Residues ◽  
Mutant Cells

Yeast sec62 mutant cells are defective in the translocation of several secretory precursor proteins into the lumen of the endoplasmic reticulum (Rothblatt et al., 1989). The deficiency, which is most restrictive for alpha-factor precursor (pp alpha F) and preprocarboxypeptidase Y, has been reproduced in vitro. Membranes isolated from mutant cells display low and labile translocation activity with pp alpha F translated in a wild-type cytosol fraction. The defect is unique to the membrane fraction because cytosol from mutant cells supports translocation into membranes from wild-type yeast. Invertase assembly is only partly affected by the sec62 mutation in vivo and is nearly normal with mutant membranes in vitro. A potential membrane location for the SEC62 gene product is supported by evaluation of the molecular clone. DNA sequence analysis reveals a 32-kD protein with no obvious NH2-terminal signal sequence but with two domains of sufficient length and hydrophobicity to span a lipid bilayer. Sec62p is predicted to display significant NH2- and COOH-terminal hydrophilic domains on the cytoplasmic surface of the ER membrane. The last 30 amino acids of the COOH terminus may form an alpha-helix with 14 lysine and arginine residues arranged uniformly about the helix. This domain may allow Sec62p to interact with other proteins of the putative translocation complex.

scholarly journals Cell competition between wild-type and JAK2V617F mutant cells in a murine model of a myeloproliferative neoplasm

2020 ◽  
Author(s):  
Melissa Castiglione ◽  
Haotian Zhang ◽  
Huichun Zhan
Keyword(s):  
Stem Cell ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
List Type ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Increased Risk ◽  
Mutant Cells

AbstractThe myeloproliferative neoplasms (MPNs) are clonal stem cell disorders characterized by overproduction of mature blood cells and increased risk of transformation to frank leukemia. The acquired kinase mutation JAK2V617F plays a central role in a majority of these disorders. The hematopoietic stem cell (HSC) compartment in MPN is heterogeneous with the presence of both JAK2 wild-type and JAK2V617F mutant cells in most patients with MPN. Utilizing in vitro co-culture assays and in vivo competitive transplantation assays, we found that the presence of wild-type cells altered the behavior of co-existing JAK2V617F mutant cells, and a mutant microenvironment (niche) could overcome the competition between wild-type and mutant cells, leading to mutant clonal expansion and overt MPN. We also demonstrated that competition between wild-type and JAK2V617F mutant cells triggered a significant immune response, and there was a dynamic PD-L1 deregulation in the mutant stem/progenitor cells caused by their interactions with the neighboring wild-type cells and the microenvironment. Therefore, while accumulation of oncogenic mutations is unavoidable during aging, our data suggest that, if we could therapeutically enhance normal cells’ ability to compete, we might be better able to control neoplastic cell expansion and prevent the development of a full-blown malignancy.Key PointsThe presence of wild-type cells alters the behavior of co-existing JAK2V617F mutant cellsA mutant microenvironment overcomes the competition between wild-type and JAK2V617F mutant cells, leading to the development of a MPN

scholarly journals Interplay between Flavodiiron Proteins and Photorespiration in Synechocystis sp. PCC 6803

2011 ◽  
Vol 286 (27) ◽  
pp. 24007-24014 ◽  
Author(s):  
Yagut Allahverdiyeva ◽  
Maria Ermakova ◽  
Marion Eisenhut ◽  
Pengpeng Zhang ◽  
Pierre Richaud ◽  
...  
Keyword(s):  
Anaerobic Bacteria ◽  
Transcript Abundance ◽  
Wild Type ◽  
Mehler Reaction ◽  
Homologous Proteins ◽  
Mutant Cells ◽  
Pcc 6803

Flavodiiron (Flv) proteins are involved in detoxification of O2 and NO in anaerobic bacteria and archaea. Cyanobacterial Flv proteins, on the contrary, function in oxygenic environment and possess an extra NAD(P)H:flavin oxidoreductase module. Synechocystis sp. PCC 6803 has four genes (sll1521, sll0219, sll0550, and sll0217) encoding Flv proteins (Flv1, Flv2, Flv3, and Flv4). Previous in vitro studies with recombinant Flv3 protein from Synechocystis provided evidence that it functions as a NAD(P)H:oxygen oxidoreductase, and subsequent in vivo studies with Synechocystis confirmed the role of Flv1 and Flv3 proteins in the Mehler reaction (photoreduction of O2 to H2O). Interestingly, homologous proteins to Flv1 and Flv3 can be found also in green algae, mosses, and Selaginella. Here, we addressed the function of Flv1 and Flv3 in Synechocystis using the Δflv1, Δflv3, and Δflv1/Δflv3 mutants and applying inorganic carbon (Ci)-deprivation conditions. We propose that only the Flv1/Flv3 heterodimer form is functional in the Mehler reaction in vivo. 18O2 labeling was used to discriminate between O2 evolution in photosynthetic water splitting and O2 consumption. In wild type, ∼20% of electrons originated from water was targeted to O2 under air level CO2 conditions but increased up to 60% in severe limitation of Ci. Gas exchange experiments with Δflv1, Δflv3, and Δflv1/Δflv3 mutants demonstrated that a considerable amount of electrons in these mutants is directed to photorespiration under Ci deprivation. This assumption is in line with increased transcript abundance of photorespiratory genes and accumulation of photorespiratory intermediates in the WT and to a higher extent in mutant cells under Ci deprivation.

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