scholarly journals Expression of a dominant allele of human ARF1 inhibits membrane traffic in vivo

1994 ◽  
Vol 124 (3) ◽  
pp. 289-300 ◽  
Author(s):  
CJ Zhang ◽  
AG Rosenwald ◽  
MC Willingham ◽  
S Skuntz ◽  
J Clark ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Rat Kidney ◽  
Brefeldin A ◽  
Membrane Traffic ◽  
Immunoblot Analysis ◽  
In Vitro Assays ◽  
Wild Type ◽  
Discernible Effect

ADP-ribosylation factor (ARF) proteins and inhibitory peptides derived from ARFs have demonstrated activities in a number of in vitro assays that measure ER-to-Golgi and intra-Golgi transport and endosome fusion. To better understand the roles of ARF proteins in vivo, stable cell lines were obtained from normal rat kidney (NRK) cells transfected with either wild-type or a dominant activating allele ([Q71L]) of the human ARF1 gene under the control of the interferon-inducible mouse Mx1 promoter. Upon addition of interferon, expression of ARF1 proteins increased with a half-time of 7-8 h, as determined by immunoblot analysis. Induction of mutant ARF1, but not wild-type ARF1, led to an inhibition of protein secretion with kinetics similar to that observed for induction of protein expression. Examination of the Golgi apparatus and the ER by indirect immunofluorescence or transmission electron microscopy revealed that expression of low levels of mutant ARF1 protein correlated with a dramatic increase in vesiculation of the Golgi apparatus and expansion of the ER lumen, while expression of substantially higher levels of wild-type ARF1 had no discernible effect. Endocytosis was also inhibited by expression of mutant ARF1, but not by the wild-type protein. Finally, the expression of [Q71L]ARF1, but not wild-type ARF1, antagonized the actions of brefeldin A, as determined by the delayed loss of ARF and beta-COP from Golgi membranes and disruption of the Golgi apparatus. General models for the actions of ARF1 in membrane traffic events are discussed.

Ultrastructural analysis of Drosophila ovarian follicles differing in yolk polypeptide (yps) composition

Development ◽  
1993 ◽  
Vol 117 (1) ◽  
pp. 319-328
Author(s):  
F. Giorgi ◽  
P. Lucchesi ◽  
A. Morelli ◽  
M. Bownes
Keyword(s):  
Golgi Apparatus ◽  
Juvenile Hormone ◽  
Developmental Stages ◽  
Ovarian Follicles ◽  
Endocytic Pathway ◽  
Intermediate Cell ◽  
Wild Type ◽  
Vesicular Traffic

Drosophila ovarian follicles were examined ultrastructurally to study the vesicular traffic in the cortical ooplasm. The endocytic pathway leading to the production of yolk spheres was visualized following in vivo or in vitro exposure to peroxidase. The Golgi apparatus and the yolk spheres of wild-type ovarian follicles were preferentially labelled by fixation with osmium zinc iodide (OZI). Labelling of wild-type ovarian follicles was compared to that of several mutant follicles--L186/Basc, fs(2)A17 and ap4--which are defective in vitellogenesis. In these mutants, the Golgi apparatus and the vesicles nearby were either scantly labelled or not labelled at all. In oocytes from flies homozygous for the gene fs(1)1163, the Golgi apparatus was labelled as in the controls, but no yolk spheres appeared to be labelled with OZI at any of the developmental stages. In several Drosophila strains, the pattern of OZI label in the cortical ooplasm was seen to vary in relation to the number of yp structural genes. In starved Drosophila females, OZI labelling of the cortical ooplasm appeared restricted to the Golgi apparatus and to an extended tubular network. A similar labelling pattern was also detected in in vitro cultured vitellogenic follicles. Refeeding, topical application of juvenile hormone analogue to starved females or hormone addition to the culture medium, all caused the yolk spheres to become labelled with OZI and to incorporate peroxidase. These observations prove that impairing endocytic uptake by either mutation or lack of juvenile hormone prevents fusion of coated vesicles and tubules with the yolk spheres and leads them instead to form an intermediate cell compartment with Golgi-derived vesicles.

scholarly journals The anti-inflammatory peptide Ac-SDKP is released from thymosin-β4 by renal meprin-α and prolyl oligopeptidase

2016 ◽  
Vol 310 (10) ◽  
pp. F1026-F1034 ◽  
Author(s):  
Nitin Kumar ◽  
Pablo Nakagawa ◽  
Branislava Janic ◽  
Cesar A. Romero ◽  
Morel E. Worou ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rat Kidney ◽  
Plasma Concentrations ◽  
Potential Candidate ◽  
Thymosin Β4 ◽  
Wild Type ◽  
Prolyl Oligopeptidase ◽  
Anti Inflammatory

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a natural tetrapeptide with anti-inflammatory and antifibrotic properties. Previously, we have shown that prolyl oligopeptidase (POP) is involved in the Ac-SDKP release from thymosin-β4 (Tβ4). However, POP can only hydrolyze peptides shorter than 30 amino acids, and Tβ4 is 43 amino acids long. This indicates that before POP hydrolysis takes place, Tβ4 is hydrolyzed by another peptidase that releases NH2-terminal intermediate peptide(s) with fewer than 30 amino acids. Our peptidase database search pointed out meprin-α metalloprotease as a potential candidate. Therefore, we hypothesized that, prior to POP hydrolysis, Tβ4 is hydrolyzed by meprin-α. In vitro, we found that the incubation of Tβ4 with both meprin-α and POP released Ac-SDKP, whereas no Ac-SDKP was released when Tβ4 was incubated with either meprin-α or POP alone. Incubation of Tβ4 with rat kidney homogenates significantly released Ac-SDKP, which was blocked by the meprin-α inhibitor actinonin. In addition, kidneys from meprin-α knockout (KO) mice showed significantly lower basal Ac-SDKP amount, compared with wild-type mice. Kidney homogenates from meprin-α KO mice failed to release Ac-SDKP from Tβ4. In vivo, we observed that rats treated with the ACE inhibitor captopril increased plasma concentrations of Ac-SDKP, which was inhibited by the coadministration of actinonin (vehicle, 3.1 ± 0.2 nmol/l; captopril, 15.1 ± 0.7 nmol/l; captopril + actinonin, 6.1 ± 0.3 nmol/l; P < 0.005). Similar results were obtained with urinary Ac-SDKP after actinonin treatment. We conclude that release of Ac-SDKP from Tβ4 is mediated by successive hydrolysis involving meprin-α and POP.

scholarly journals Substitutions in the Periplasmic Domain of Low-Abundance Chemoreceptor Trg That Induce or Reduce Transmembrane Signaling: Kinase Activation and Context Effects

2001 ◽  
Vol 183 (2) ◽  
pp. 671-679 ◽  
Author(s):  
Bryan D. Beel ◽  
Gerald L. Hazelbauer
Keyword(s):  
Context Effects ◽  
High Abundance ◽  
In Vitro Assays ◽  
Wild Type ◽  
Kinase Activation ◽  
Interaction Sites ◽  
Natural Enzyme ◽  
Signaling Receptors

ABSTRACT We extended characterization of mutational substitutions in the ligand-binding region of Trg, a low-abundance chemoreceptor ofEscherichia coli. Previous investigations using patterns of adaptational methylation in vivo led to the suggestion that one class of substitutions made the receptor insensitive, reducing ligand-induced signaling, and another mimicked ligand occupancy, inducing signaling in the absence of ligand. We tested these deductions with in vitro assays of kinase activation and found that insensitive receptors activated the kinase as effectively as wild-type receptors and that induced-signaling receptors exhibited the low level of kinase activation characteristic of occupied receptors. Differential activation by the two mutant classes was not dependent on high-abundance receptors. Cellular context can affect the function of low-abundance receptors. Assays of chemotactic response and adaptational modification in vivo showed that increasing cellular dosage of mutant forms of Trg to a high-abundance level did not significantly alter phenotypes, nor did the presence of high-abundance receptors significantly correct phenotypic defects of reduced-signaling receptors. In contrast, defects of induced-signaling receptors were suppressed by the presence of high-abundance receptors. Grafting the interaction site for the adaptational-modification enzymes to the carboxyl terminus of induced-signaling receptors resulted in a similar suppression of phenotypic defects of induced-signaling receptors, implying that high-abundance receptors could suppress defects in induced-signaling receptors by providing their natural enzyme interaction sites intrans in clusters of suppressing and suppressed receptors. As in the case of cluster-related functional assistance provided by high-abundance receptors for wild-type low-abundance receptors, suppression by high-abundance receptors of phenotypic defects in induced-signaling forms of Trg involved assistance in adaptation, not signaling.

scholarly journals Cnu, a Novel oriC-Binding Protein of Escherichia coli

2005 ◽  
Vol 187 (20) ◽  
pp. 6998-7008 ◽  
Author(s):  
Myung Suk Kim ◽  
Sung-Hun Bae ◽  
Sang Hoon Yun ◽  
Hee Jung Lee ◽  
Sang Chun Ji ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid Identity ◽  
Replication Initiation ◽  
In Vitro Assays ◽  
Wild Type ◽  
Genetic Method ◽  
Dna Replication Initiation ◽  
Pair Sequence

ABSTRACT We have found, using a newly developed genetic method, a protein (named Cnu, for oriC-binding nucleoid-associated) that binds to a specific 26-base-pair sequence (named cnb) in the origin of replication of Escherichia coli, oriC. Cnu is composed of 71 amino acids (8.4 kDa) and shows extensive amino acid identity to a group of proteins belonging to the Hha/YmoA family. Cnu was previously discovered as a protein that, like Hha, complexes with H-NS in vitro. Our in vivo and in vitro assays confirm the results and further suggest that the complex formation with H-NS is involved in Cnu/Hha binding to cnb. Unlike the hns mutants, elimination of either the cnu or hha gene did not disturb the growth rate, origin content, and synchrony of DNA replication initiation of the mutants compared to the wild-type cells. However, the cnu hha double mutant was moderately reduced in origin content. The Cnu/Hha complex with H-NS thus could play a role in optimal activity of oriC.

scholarly journals De novo disease-associated mutations in KIF1A dominant negatively inhibit axonal transport of synaptic vesicle precursors

2021 ◽  
Author(s):  
Yuzu Anazawa ◽  
Tomoki Kita ◽  
Kumiko Hayashi ◽  
Shinsuke Niwa
Keyword(s):  
Synaptic Vesicle ◽  
Axonal Transport ◽  
De Novo ◽  
Molecular Motor ◽  
Model Systems ◽  
In Vitro Assays ◽  
In Vivo Model ◽  
Wild Type

KIF1A is a kinesin superfamily molecular motor that transports synaptic vesicle precursors in axons. Mutations in Kif1a lead to a group of neuronal diseases called KIF1A-associated neuronal disorder (KAND). KIF1A forms a homodimer and KAND mutations are mostly de novo and autosomal dominant; however, it is not known whether the function of wild-type KIF1A is inhibited by disease-associated KIF1A. No reliable in vivo model systems to analyze the molecular and cellular biology of KAND have been developed; therefore, here, we established Caenorhabditis elegans models for KAND using CRISPR/cas9 technology and analyzed defects in axonal transport. In the C. elegans models, heterozygotes and homozygotes exhibited reduced axonal transport phenotypes. In addition, we developed in vitro assays to analyze the motility of single heterodimers composed of wild-type KIF1A and disease-associated KIF1A. Disease-associated KIF1A significantly inhibited the motility of wild-type KIF1A when heterodimers were formed. These data indicate the molecular mechanism underlying the dominant nature of de novo KAND mutations.

scholarly journals SAGA–CORE subunit Spt7 is required for correct Ubp8 localization, chromatin association and deubiquitinase activity

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Carme Nuño-Cabanes ◽  
Varinia García-Molinero ◽  
Manuel Martín-Expósito ◽  
María-Eugenia Gas ◽  
Paula Oliete-Calvo ◽  
...  
Keyword(s):  
Eukaryotic Cells ◽  
In Vitro Assays ◽  
Saga Complex ◽  
Wild Type ◽  
Core Complex ◽  
Independent Manner ◽  
Tetrameric Structure ◽  
Core Components

Abstract Background Histone H2B deubiquitination is performed by numerous deubiquitinases in eukaryotic cells including Ubp8, the catalytic subunit of the tetrameric deubiquitination module (DUBm: Ubp8; Sus1; Sgf11; Sgf73) of the Spt-Ada-Gcn5 acetyltransferase (SAGA). Ubp8 is linked to the rest of SAGA through Sgf73 and is activated by the adaptors Sus1 and Sgf11. It is unknown if DUBm/Ubp8 might also work in a SAGA-independent manner. Results Here we report that a tetrameric DUBm is assembled independently of the SAGA–CORE components SPT7, ADA1 and SPT20. In the absence of SPT7, i.e., independent of the SAGA complex, Ubp8 and Sus1 are poorly recruited to SAGA-dependent genes and to chromatin. Notably, cells lacking Spt7 or Ada1, but not Spt20, show lower levels of nuclear Ubp8 than wild-type cells, suggesting a possible role for SAGA–CORE subunits in Ubp8 localization. Last, deletion of SPT7 leads to defects in Ubp8 deubiquitinase activity in in vivo and in vitro assays. Conclusions Collectively, our studies show that the DUBm tetrameric structure can form without a complete intact SAGA–CORE complex and that it includes full-length Sgf73. However, subunits of this SAGA–CORE influence DUBm association with chromatin, its localization and its activity.

scholarly journals α-2,3-Sialyltransferase Enhances Neisseria gonorrhoeae Survival during Experimental Murine Genital Tract Infection

2006 ◽  
Vol 74 (7) ◽  
pp. 4094-4103 ◽  
Author(s):  
Hong Wu ◽  
Ann E. Jerse
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Genital Tract ◽  
In Vitro Assays ◽  
In Vivo Model ◽  
Wild Type ◽  
Genital Tract Infection ◽  
Direct Demonstration ◽  
Increased Sensitivity

ABSTRACT The addition of host-derived sialic acid to Neisseria gonorrhoeae lipooligosaccharide is hypothesized to be an important mechanism by which gonococci evade host innate defenses. This hypothesis is based primarily on in vitro assays of complement-mediated and phagocytic killing. Here we report that a nonpolar α-2,3-sialyltransferase (lst) mutant of N. gonorrhoeae was significantly attenuated in its capacity to colonize the lower genital tract of 17-β estradiol-treated female BALB/c mice during competitive infection with the wild-type strain. Genetic complementation of the lst mutation restored recovery of the mutant to wild-type levels. Studies with B10.D2-HCoH2dH2-T18c/OSN (C5-deficient) mice showed that attenuation of the lst mutant was not due to increased sensitivity to complement-mediated bacteriolysis, a result that is consistent with recently reported host restrictions in the complement cascade. However, Lst-deficient gonococci were killed more rapidly than sialylated wild-type gonococci following intraperitoneal injection into normal mice, which is consistent with sialylation conferring protection against killing by polymorphonuclear leukocytes (PMNs). As reported for human PMNs, sialylated gonococci were more resistant to killing by murine PMNs, and sialylation led to reduced association with and induction of a weaker respiratory burst in PMNs from estradiol-treated mice. In summary, these studies suggest sialylation confers a survival advantage to N. gonorrhoeae in mice by increasing resistance to PMN killing. This report is the first direct demonstration that α-2,3-sialyltransferase contributes to N. gonorrhoeae pathogenesis in an in vivo model. This study also validates the use of experimental murine infection to study certain aspects of gonococcal pathogenesis.

scholarly journals A Dictyostelium myosin II lacking a proximal 58-kDa portion of the tail is functional in vitro and in vivo.

1992 ◽  
Vol 3 (12) ◽  
pp. 1455-1462 ◽  
Author(s):  
E W Kubalek ◽  
T Q Uyeda ◽  
J A Spudich
Keyword(s):  
Molecular Genetic ◽  
Myosin Ii ◽  
In Vitro Assays ◽  
Dependent Manner ◽  
Fruiting Bodies ◽  
Wild Type ◽  
In Vitro Motility Assay ◽  
Dictyostelium Myosin

We used molecular genetic approaches to delete 521 amino acid residues from the proximal portion of the Dictyostelium myosin II tail. The deletion encompasses approximately 40% of the tail, including the S2-LMM junction, a region that in muscle myosin II has been proposed to be important for contraction. The functions of the mutant myosin II are indistinguishable from the wild-type myosin II in our in vitro assays. It binds to actin in a typical rigor configuration in the absence of ATP and it forms filaments in a normal salt-dependent manner. In an in vitro motility assay, both monomeric and filamentous forms of the mutant myosin II translocate actin filaments at 2.4 microns/s at 30 degrees C, similar to that of wild-type myosin II. The mutant myosin II is also functional in vivo. Cells expressing the mutant myosin II in place of the native myosin II perform myosin II-dependent activities such as cytokinesis and formation of fruiting bodies, albeit inefficiently. Growth of the mutant cells in suspension gives rise to many large multinucleated cells, demonstrating that cytokinesis often fails. The majority of the fruiting bodies are also morphologically abnormal. These results demonstrate that this region of the myosin II tail is not required for motile activities but its presence is necessary for optimum function in vivo.

scholarly journals Identification and Analysis of a Hyperactive Mutant Form of Drosophila P-Element Transposase

Genetics ◽  
2002 ◽  
Vol 162 (1) ◽  
pp. 217-227 ◽  
Author(s):  
Eileen L Beall ◽  
Matthew B Mahoney ◽  
Donald C Rio
Keyword(s):  
Dna Damage ◽  
Dna Cleavage ◽  
P Element ◽  
In Vitro Assays ◽  
Mutant Form ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein

Abstract Transposition in many organisms is regulated to control the frequency of DNA damage caused by the DNA breakage and joining reactions. However, genetic studies in prokaryotic systems have led to the isolation of mutant transposase proteins with higher or novel activities compared to those of the wild-type protein. In the course of our study of the effects of mutating potential ATM-family DNA damage checkpoint protein kinase sites in the Drosophila P-element transposase protein, we found one mutation, S129A, that resulted in an elevated level of transposase activity using in vivo recombination assays, including P-element-mediated germline transformation. In vitro assays for P-element transposase activity indicate that the S129A mutant exhibits elevated donor DNA cleavage activity when compared to the wild-type protein, whereas the strand-transfer activity is similar to that of wild type. This difference may reflect the nature of the in vitro assays and that normally in vivo the two reactions may proceed in concert. The P-element transposase protein contains 10 potential consensus phosphorylation sites for the ATM family of PI3-related protein kinases. Of these 10 sites, 8 affect transposase activity either positively or negatively when substituted individually with alanine and tested in vivo. A mutant transposase protein that contains all eight N-terminal serine and threonine residues substituted with alanine is inactive and can be restored to full activity by substitution of wild-type amino acids back at only 3 of the 8 positions. These data suggest that the activity of P-element transposase may be regulated by phosphorylation and demonstrate that one mutation, S129A, results in hyperactive transposition.

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