scholarly journals Identification of Miro as a mitochondrial receptor for myosin XIX

2018 ◽  
Author(s):  
Stefanie J. Oeding ◽  
Katarzyna Majstrowicz ◽  
Xiao-Ping Hu ◽  
Vera Schwarz ◽  
Angelika Freitag ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Specific Receptor ◽  
Tail Region ◽  
Gtpase Domain ◽  
Mitochondrial Movement ◽  
Binding Partners ◽  
Potential Binding ◽  
Mitochondrial Distribution ◽  
In Cells

ABSTRACTMitochondrial distribution in cells is critical for cellular function and proper inheritance during cell division. In mammalian cells, mitochondria are transported predominantly along microtubules by kinesin and dynein and along actin filaments by myosin. Myosin XIX (Myo19) associates with the outer mitochondrial membrane, but no specific receptor has been identified. Using proximity BioID labeling, we identified Miro-1 and Miro-2 as potential binding partners of Myo19. Interaction studies show that Miro-1 binds to a C-terminal fragment of the Myo19 tail region and that Miro recruits the Myo19 tail in vivo. This recruitment is regulated by the nucleotide-state of the N-terminal Rho-like GTPase domain of Miro. Notably, Myo19 protein stability in cells depends on its association with Miro. Finally, Myo19 regulates the subcellular distribution of mitochondria. Downregulation, as well as overexpression, of Myo19 induces perinuclear collapse of mitochondria, phenocopying the loss of kinesin KIF5 or its mitochondrial receptor Miro. These results suggest that Miro coordinates microtubule- and actin-based mitochondrial movement.

scholarly journals A 1H/15N n.m.r. study of nitrogen metabolism in cultured mammalian cells

1993 ◽  
Vol 291 (2) ◽  
pp. 485-492 ◽  
Author(s):  
J C Street ◽  
A M Delort ◽  
P S H Braddock ◽  
K M Brindle
Keyword(s):  
Glutamine Synthetase ◽  
Glutamate Dehydrogenase ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Cho Cell ◽  
15N Labelling ◽  
Hela Cell Lines ◽  
Proton Resonances ◽  
In Cells

1. Heteronuclear 1H/15N n.m.r. experiments are described in which 15N labelling of cellular metabolites is detected via their proton resonances. 2. These n.m.r. experiments have been used to monitor label redistribution amongst extracellular metabolites in cultures of mammalian cells incubated with L-[2-15N]glutamine, L-[5-15N]glutamine and 15NH4Cl. Label redistribution was monitored in two HeLa cell lines and in two CHO cell lines which showed a range of extractable activities of glutamate dehydrogenase, glutaminase and glutamine synthetase. 3. In cells incubated with L-[2-15N]glutamine the 15N label was subsequently found in a number of metabolites including alanine, aspartate, glycine and pyrrolidone-5-carboxylic acid. There was no detectable production of 15NH4+, showing that most of the glutamate formed in the reaction catalysed by glutaminase was subsequently transaminated rather than oxidatively deaminated by glutamate dehydrogenase. 4. Incubation of cells with L-[5-15N]glutamine showed that the ammonia in the cultures was derived predominantly from the amide group of glutamine. 5. The rate of formation of L-[5-15N]glutamine in cells incubated with 15NH4Cl was used to estimate glutamine synthetase flux in vivo. Flux in this reaction was only observable in the two CHO cell lines which express relatively high levels of the enzyme.

scholarly journals FtsA Regulates Z-Ring Morphology and Cell Wall Metabolism in an FtsZ C-Terminal Linker-Dependent Manner in Caulobacter crescentus

2020 ◽  
Vol 202 (7) ◽  
Author(s):  
Jordan M. Barrows ◽  
Kousik Sundararajan ◽  
Anant Bhargava ◽  
Erin D. Goley
Keyword(s):  
Cell Wall ◽  
Caulobacter Crescentus ◽  
Bacterial Cell Division ◽  
Cell Wall Metabolism ◽  
Gtpase Domain ◽  
Content Type ◽  
Binding Partners ◽  
Z Ring ◽  
Membrane Anchoring

ABSTRACT Bacterial cell division requires the assembly of a multiprotein division machinery, or divisome, that remodels the cell envelope to cause constriction. The cytoskeletal protein FtsZ forms a ringlike scaffold for the divisome at the incipient division site. FtsZ has three major regions: a conserved GTPase domain that polymerizes into protofilaments on binding GTP, a C-terminal conserved peptide (CTC) required for binding membrane-anchoring proteins, and a C-terminal linker (CTL) region of varied length and low sequence conservation. Recently, we demonstrated that the CTL regulates FtsZ polymerization properties in vitro and Z-ring structure and cell wall metabolism in vivo. In Caulobacter crescentus, an FtsZ variant lacking the CTL (designated ΔCTL) can recruit all known divisome members and drive local cell wall synthesis but has dominant lethal effects on cell wall metabolism. To understand the underlying mechanism of the CTL-dependent regulation of cell wall metabolism, we expressed chimeras of FtsZ domains from C. crescentus and Escherichia coli and observed that the E. coli GTPase domain fused to the C. crescentus CTC phenocopies C. crescentus ΔCTL. By investigating the contributions of FtsZ-binding partners, we identified variants of FtsA, a known membrane anchor for FtsZ, that delay or exacerbate the ΔCTL phenotype. Additionally, we observed that the ΔCTL protein forms extended helical structures in vivo upon FtsA overproduction. We propose that misregulation downstream of defective ΔCTL assembly is propagated through the interaction between the CTC and FtsA. Overall, our study provides mechanistic insights into the CTL-dependent regulation of cell wall enzymes downstream of FtsZ polymerization. IMPORTANCE Bacterial cell division is essential and requires the recruitment and regulation of a complex network of proteins needed to initiate and guide constriction and cytokinesis. FtsZ serves as a master regulator for this process, and its function is highly dependent on both its assembly into the canonical Z ring and interactions with protein binding partners, all of which results in the activation of enzymes that remodel the cell wall to drive constriction. Using mutants of FtsZ, we have elaborated on the role of its C-terminal linker domain in regulating Z-ring stability and dynamics, as well as the requirement for its conserved C-terminal domain and interaction with the membrane-anchoring protein FtsA for regulating the process of cell wall remodeling for constriction.

scholarly journals Determinants of FtsZ C-terminal linker-dependent regulation of cell wall metabolism in Caulobacter crescentus

2019 ◽  
Author(s):  
Kousik Sundararajan ◽  
Jordan M. Barrows ◽  
Erin D. Goley
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Caulobacter Crescentus ◽  
Bacterial Cell Division ◽  
Cell Wall Metabolism ◽  
Gtpase Domain ◽  
Binding Partners ◽  
Z Ring ◽  
Membrane Anchoring

AbstractBacterial cell division requires assembly of a multi-protein machinery or “divisome” that remodels the cell envelope to cause constriction. The cytoskeletal protein FtsZ forms a ring-like scaffold for the divisome at the incipient division site. FtsZ has three major regions – a conserved, polymerizing GTPase domain; a C-terminal conserved (CTC) peptide required for binding membrane-anchoring proteins; and a C-terminal linker (CTL) of poor length and sequence conservation. We previously demonstrated that, in Caulobacter crescentus, the CTL regulates FtsZ polymerization in vitro and cell wall metabolism in vivo. To understand the mechanism of CTL-dependent regulation of cell wall metabolism, here we investigated the impact of the CTL on Z-ring structure in cells and employed genetics to identify molecular determinants of the dominant lethal effects of ΔCTL. Deleting the CTL specifically resulted in formation of dense, asymmetric, non-ring FtsZ assemblies in vivo. Moreover, we observed that production of an FtsZ variant with the GTPase domain of Escherichia coli FtsZ fused to the CTC of C. crescentus FtsZ phenocopied the effects of C. crescentus ΔCTL, suggesting the CTC mediates signaling to cell wall metabolism. Finally, whereas overproduction of ZapA, FzlC, or FtsEX had slight protective effects against ΔCTL, depletion of FtsA partially suppressed the effects of ΔCTL. From these results, we propose that the cell wall misregulation downstream of ΔCTL results from its aberrant assembly properties and is propagated through the interaction between the CTC of FtsZ and FtsA. Our study provides mechanistic insights into CTL-dependent regulation of cell wall enzymes downstream of FtsZ.ImportanceBacterial cell division is essential and requires the recruitment and regulation of a complex network of proteins needed to initiate and guide constriction and cytokinesis. FtsZ serves as a master regulator for this process, and its function is highly dependent on both its self-assembly into a canonical “Z-ring” and interaction with protein binding partners, which results in the activation of enzymes that remodel the cell wall to drive constriction. Using mutants of FtsZ and its binding partners, we have established the role of its C-terminal linker domain in regulating Z-ring organization, as well as the requirement for its C-terminal conserved peptide and interaction with the membrane-anchoring protein FtsA for regulating cell wall remodeling for constriction.

scholarly journals Positive Regulation of IκB Kinase Signaling by Protein Serine/Threonine Phosphatase 2A

2005 ◽  
Vol 280 (43) ◽  
pp. 35974-35982 ◽  
Author(s):  
Arlene E. Kray ◽  
Robert S. Carter ◽  
Kevin N. Pennington ◽  
Rey J. Gomez ◽  
Laura E. Sanders ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cellular Response ◽  
Kinase Activity ◽  
Nuclear Translocation ◽  
Regulatory Role ◽  
Regulatory Subunit ◽  
Iκb Kinase ◽  
Phosphatase 2A ◽  
In Cells

Transcription factor NF-κB plays a key regulatory role in the cellular response to pro-inflammatory cytokines such as tumor necrosis factor-α (TNF). In the absence of TNF, NF-κB is sequestered in the cytoplasm by inhibitory IκB proteins. Phosphorylation of IκBby the β-catalytic subunit of IKK, a multicomponent IκB kinase, targets the inhibitor for proteolytic destruction and facilitates nuclear translocation of NF-κB. This pathway is initiated by TNF-dependent phosphorylation of T loop serines in IKKβ, which greatly stimulates IκB kinase activity. Prior in vitro mixing experiments indicate that protein serine/threonine phosphatase 2A (PP2A) can dephosphorylate these T loop serines and inactivate IKK, suggesting a negative regulatory role for PP2A in IKK signaling. Here we provided several in vivo lines of evidence indicating that PP2A plays a positive rather than a negative role in the regulation of IKK. First, TNF-induced degradation of IκB is attenuated in cells treated with okadaic acid or fostriecin, two potent inhibitors of PP2A. Second, PP2A forms stable complexes with IKK in untransfected mammalian cells. This interaction is critically dependent on amino acid residues 121–179 of the IKKγ regulatory subunit. Third, deletion of the PP2A-binding site in IKKγ attenuates T loop phosphorylation and catalytic activation of IKKβ in cells treated with TNF. Taken together, these data provide strong evidence that the formation of IKK·PP2A complexes is required for the proper induction of IκB kinase activity in vivo.

Effect of Some Metabolic Inhibitors on Vinblastine-Induced Ribosomal-Granular Material Complexes

1971 ◽  
Vol 29 ◽  
pp. 548-549
Author(s):  
Awtar Krishan ◽  
Dora Hsu
Keyword(s):  
Granular Material ◽  
Rna Synthesis ◽  
Culture Medium ◽  
Actinomycin D ◽  
Metabolic Inhibitors ◽  
Protein And Rna Synthesis ◽  
Apparent Reduction ◽  
Plant Alkaloids ◽  
In Cells

Cells exposed to antitumor plant alkaloids, vinblastine and vincristine sulfate have large proteinacious crystals and complexes of ribosomes, helical polyribosomes and electron-dense granular material (ribosomal complexes) in their cytoplasm, Binding of H3-colchicine by the in vivo crystals shows that they contain microtubular proteins. Association of ribosomal complexes with the crystals suggests that these structures may be interrelated.In the present study cultured human leukemic lymphoblasts (CCRF-CEM), were incubated with protein and RNA-synthesis inhibitors, p. fluorophenylalanine, puromycin, cycloheximide or actinomycin-D before the addition of crystal-inducing doses of vinblastine to the culture medium. None of these compounds could completely prevent the formation of the ribosomal complexes or the crystals. However, in cells pre-incubated with puromycin, cycloheximide, or actinomycin-D, a reduction in the number and size of the ribosomal complexes was seen. Large helical polyribosomes were absent in the ribosomal complexes of cells treated with puromycin, while in cells exposed to cycloheximide, there was an apparent reduction in the number of ribosomes associated with the ribosomal complexes (Fig. 2).

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

Cytological changes induced by platinum-thymine in sarcoma-180 cells: Electron microscopy study

1990 ◽  
Vol 48 (3) ◽  
pp. 290-291
Author(s):  
Gustav Ofosu
Keyword(s):  
Mammalian Cells ◽  
Antitumor Agent ◽  
Microscopy Study ◽  
Electron Microscopy Study ◽  
Limiting Factor ◽  
Sarcoma 180 ◽  
Electron Microscopic ◽  
Cytological Changes

Platinum-thymine has been found to be a potent antitumor agent, which is quite soluble in water, and lack nephrotoxicity as the dose-limiting factor. The drug has been shown to interact with DNA and inhibits DNA, RNA and protein synthesis in mammalian cells in vitro. This investigation was undertaken to elucidate the cytotoxic effects of piatinum-thymine on sarcoma-180 cells in vitro ultrastructurally, Sarcoma-180 tumor bearing mice were treated with intraperitoneal injection of platinum-thymine 40mg/kg. A concentration of 60μg/ml dose of platinum-thymine was used in in vitro experiments. Treatments were at varying time intervals of 3, 7 and 21 days for in vivo experiments, and 30, 60 and 120 min., 6, 12, and 24th in vitro. Controls were not treated with platinum-thymine.Electron microscopic analyses of the treated cells in vivo and in vitro showed drastic cytotoxic effect.

DEGRADATION OF MESSENGER RNA IN MAMMALIAN CELLS

1972 ◽  
Vol 71 (2_Suppla) ◽  
pp. S369-S380 ◽  
Author(s):  
Francis T. Kenney ◽  
Kai-Lin Lee ◽  
Charles D. Stiles
Keyword(s):  
Messenger Rna ◽  
Mammalian Cells ◽  
Messenger Rnas ◽  
Tyrosine Transaminase

ABSTRACT Analyses of the response of hydrocortisone-induced tyrosine transaminase in cultured H-35 cells to inhibitors of translation (cycloheximide, puromycin) suggest: (1) that bound ribosomes stabilize messenger RNA in vivo; (2) that messenger is degraded at a rate determined by the rate of translation. Since specific messenger RNAs of mammalian cells are degraded at quite different rates, there may be extensive heterogeneity either in the rate at which ribosomes traverse different messengers or in the number of ribosomes which translate specific messenger RNAs.

Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

2018 ◽  
Author(s):  
Noor H. Dashti ◽  
Rufika S. Abidin ◽  
Frank Sainsbury
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Cell Engineering ◽  
Particle Analysis ◽  
Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both <i>in vitro</i> and <i>in vivo</i> cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for <i>in vivo</i> self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by <i>in vitro</i> self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

The Role of nitro (NO2-), chloro (Cl), and fluoro (F) Substitution in the Design of Antileishmanial and Antichagasic Compounds

2020 ◽  
Vol 21 ◽  
Author(s):  
Boniface Pone ◽  
Ferreira Igne Elizabeth
Keyword(s):  
Chagas Disease ◽  
Mammalian Cells ◽  
Neglected Tropical Diseases ◽  
New Drugs ◽  
Pharmacokinetic Studies ◽  
Scientific Publications ◽  
Antitrypanosomal Activity ◽  
Chemical Groups

: Neglected tropical diseases (NTDs) are responsible for over 500,000 deaths annually and are characterized by multiple disabilities. Leishmaniasis and Chagas disease are among the most severe NTDs, and are caused by the Leishmania sp, and Trypanosoma cruzi, respectively. Glucantime, pentamidine and miltefosine are commonly used to treat leishmaniasis, whereas nifurtimox, benznidazole are current treatments for Chagas disease. However, these treatments are associated with drug resistance, and severe side effects. Hence, the development of synthetic products, especially those containing N02, F, or Cl, which chemical groups are known to improve the biological activity. The present work summarizes the information on the antileishmanial and antitrypanosomal activity of nitro-, chloro-, and fluoro-synthetic derivatives. Scientific publications referring to halogenated derivatives in relation to antileishmanial and antitrypanosomal activities were hand searched in databases such as SciFinder, Wiley, Science Direct, PubMed, ACS, Springer, Scielo, and so on. According to the literature information, more than 90 compounds were predicted as lead molecules with reference to their IC50/EC50 values in in vitro studies. It is worth to mention that only active compounds with known cytotoxic effects against mammalian cells were considered in the present study. The observed activity was attributed to the presence of nitro-, fluoro- and chloro-groups in the compound backbone. All in all, nitro and h0alogenated derivatives are active antileishmanial and antitrypanosomal compounds and can serve as baseline for the development of new drugs against leishmaniasis and Chagas disease. However, efforts on in vitro and in vivo toxicity studies of the active synthetic compounds is still needed. Pharmacokinetic studies, and the mechanism of action of the promising compounds need to be explored. The use of new catalysts and chemical transformation can afford unexplored halogenated compounds with improved antileishmanial and antitrypanosomal activity.

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