scholarly journals The GTPase-activating protein n-chimaerin cooperates with Rac1 and Cdc42Hs to induce the formation of lamellipodia and filopodia.

1996 ◽  
Vol 16 (9) ◽  
pp. 5069-5080 ◽  
Author(s):  
R Kozma ◽  
S Ahmed ◽  
A Best ◽  
L Lim
Keyword(s):  
Mammalian Cells ◽  
Phorbol Esters ◽  
Morphological Changes ◽  
Neuroblastoma Cells ◽  
Leading Edge ◽  
Dominant Negative ◽  
Gtpase Activating Protein ◽  
Simultaneous Formation ◽  
Gdp Dissociation Inhibitor

n-Chimaerin is a GTPase-activating protein (GAP) mainly for Rac1 and less so for Cdc42Hs in vitro. The GAP activity of n-chimaerin is regulated by phospholipids and phorbol esters. Microinjection of Rac1 and Cdc42Hs into mammalian cells induces formation of the actin-based structures lamellipodia and filopodia, respectively, with the former being prevented by coinjection of the chimaerin GAP domain. Strikingly, microinjection of the full-length n-chimaerin into fibroblasts and neuroblastoma cells induces the simultaneous formation of lamellipodia and filopodia. These structures undergo cycles of dissolution and formation, resembling natural morphological events occurring at the leading edge of fibroblasts and neuronal growth cones. The effects of n-chimaerin on formation of lamellipodia and filopodia were inhibited by dominant negative Rac1(T17N) and Cdc42Hs(T17N), respectively. n-Chimaerin's effects were also inhibited by coinjection with Rho GDP dissociation inhibitor or by treatment with phorbol ester. A mutant n-chimaerin with no GAP activity and impaired p21 binding was ineffective in inducing morphological changes, while a mutant lacking GAP activity alone was effective. Microinjected n-chimaerin colocalized in situ with F-actin. Taken together, these results suggest that n-chimaerin acts synergistically with Rac1 and Cdc42Hs to induce actin-based morphological changes and that this action involves Rac1 and Cdc42Hs binding but not GAP activity. Thus, GAPs may have morphological functions in addition to downregulation of GTPases.

Species-specific Effects of Nitromethylene Heterocycle Insecticides on Nicotinic Receptors in Locust Neurons and Mouse N1E-115 and BC3H1 Cells

1994 ◽  
Vol 22 (6) ◽  
pp. 454-461
Author(s):  
Marga Oortgiesen ◽  
Ruud Zwart ◽  
Henk P.M. Vijverberg
Keyword(s):  
Mammalian Cells ◽  
Neuroblastoma Cells ◽  
Receptor Subtypes ◽  
Inward Current ◽  
Specific Effects ◽  
Blocking Effects ◽  
Inward Currents ◽  
Species Specific ◽  
Ganglion Neurons

The effects of nitromethylene heterocycle (NMH) insecticides on subtypes of nicotinic acetylcholine (nACh) receptors were investigated in locust thoracic ganglion neurons, mouse N1E-115 neuroblastoma cells, and mouse BC3H1 muscle cells by using electrophysiological techniques. In locust neurons, all of the six NMH insecticides tested induced transient inward currents resembling nicotinic ACh-induced inward currents, while, in the continued presence of the NMH compounds, the ACh-induced inward current was blocked. The amplitude of the inward current and the blocking effects of the NMH insecticides were enhanced by concentrations between 0.1 and 10μM. Cross-desensitisation with the ACh-induced inward current confirmed that the NMH-induced inward current was governed by the activation of nACh receptors. Mammalian endplate type nACh receptors in BC3H1 cells and mammalian neuronal type nACh receptors in N1E-115 cells were much less sensitive to the NMH insecticides than the locust neuronal nACh receptors. At a concentration of 10μM, which blocked 80–100% of the ACh-induced inward current in locust neurons, NMH insecticides only partially blocked the ACh-induced inward currents mediated by the two subtypes of mammalian nACh receptors. NMH insecticides also failed to induce significant agonist effects in the mammalian cells at this concentration. The results provide a possible explanation for the selectively greater toxicity of NMH insecticides to insects than to vertebrates, at the level of nACh receptor subtypes and, hence, demonstrate that this in vitro approach is valuable for the investigation of species-specific interactions of compounds at their target site.

scholarly journals Intercellular transmission of a bacterial cytotoxic prion-like protein in mammalian cells

2019 ◽  
Author(s):  
Aida Revilla-García ◽  
Cristina Fernández ◽  
María Moreno-del Álamo ◽  
Vivian de los Ríos ◽  
Ina M. Vorberg ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Protein Quality ◽  
Horizontal Cell ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Neuroblastoma Cell Line ◽  
Human Neuroblastoma ◽  
Intracellular Traffic ◽  
First Time

AbstractRepA is a bacterial protein that builds intracellular amyloid oligomers acting as inhibitory complexes of plasmid DNA replication. When carrying a mutation enhancing its amyloidogenesis (A31V), the N-terminal domain (WH1) generates cytosolic amyloid particles that are inheritable within a bacterial lineage. Such amyloids trigger in bacteria a lethal cascade reminiscent to mitochondria impairment in human cells affected by neurodegeneration. To fulfil all the features of a prion-like protein, horizontal (intercellular) transmissibility remains to be demonstrated for RepA-WH1. Since this is experimentally intractable in bacteria, here we transiently expressed in a murine neuroblastoma cell line the soluble, barely cytotoxic RepA-WH1(WT) and assayed its response to co-incubation with in vitro assembled RepA-WH1(A31V) amyloid fibres. In parallel, cells releasing RepA-WH1(A31V) aggregates were co-cultured with human neuroblastoma cells expressing RepA-WH1(WT). Both the assembled fibres and the extracellular RepA-WH1(A31V) aggregates induce, in the cytosol of recipient cells, the formation of cytotoxic amyloid particles. Mass spectrometry analyses of the proteomes of both types of injured cells point to alterations in mitochondria, protein quality triage, signalling and intracellular traffic.Summary blurbThe horizontal, cell-to-cell spread of a bacterial prion-like protein is shown for the first time in mammalian cells. Amyloid cross-aggregation of distinct variants, and their associated toxicities, follow the same trend found in bacteria, underlining the universality of prion biology.

scholarly journals Crystal structure of MICU2 and comparison with MICU1 reveal insights into the uniporter gating mechanism

2019 ◽  
Vol 116 (9) ◽  
pp. 3546-3555 ◽  
Author(s):  
Kimberli J. Kamer ◽  
Wei Jiang ◽  
Virendar K. Kaushik ◽  
Vamsi K. Mootha ◽  
Zenon Grabarek
Keyword(s):  
Binding Sites ◽  
Mammalian Cells ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Functional Coupling ◽  
Gating Mechanism ◽  
Ef Hand ◽  
Negative Effect ◽  
Oligomeric Complex

The mitochondrial uniporter is a Ca2+-channel complex resident within the organelle’s inner membrane. In mammalian cells the uniporter’s activity is regulated by Ca2+ due to concerted action of MICU1 and MICU2, two paralogous, but functionally distinct, EF-hand Ca2+-binding proteins. Here we present the X-ray structure of the apo form of Mus musculus MICU2 at 2.5-Å resolution. The core structure of MICU2 is very similar to that of MICU1. It consists of two lobes, each containing one canonical Ca2+-binding EF-hand (EF1, EF4) and one structural EF-hand (EF2, EF3). Two molecules of MICU2 form a symmetrical dimer stabilized by highly conserved hydrophobic contacts between exposed residues of EF1 of one monomer and EF3 of another. Similar interactions stabilize MICU1 dimers, allowing exchange between homo- and heterodimers. The tight EF1–EF3 interface likely accounts for the structural and functional coupling between the Ca2+-binding sites in MICU1, MICU2, and their complex that leads to the previously reported Ca2+-binding cooperativity and dominant negative effect of mutation of the Ca2+-binding sites in either protein. The N- and C-terminal segments of the two proteins are distinctly different. In MICU2 the C-terminal helix is significantly longer than in MICU1, and it adopts a more rigid structure. MICU2’s C-terminal helix is dispensable in vitro for its interaction with MICU1 but required for MICU2’s function in cells. We propose that in the MICU1–MICU2 oligomeric complex the C-terminal helices of both proteins form a central semiautonomous assembly which contributes to the gating mechanism of the uniporter.

scholarly journals βcap73-ARF6 Interactions Modulate Cell Shape and Motility after Injury In Vitro

2003 ◽  
Vol 14 (10) ◽  
pp. 4155-4161 ◽  
Author(s):  
Kathleen N. Riley ◽  
Angel E. Maldonado ◽  
Patrice Tellier ◽  
Crislyn D'Souza-Schorey ◽  
Ira M. Herman
Keyword(s):  
Western Blot ◽  
Molecular Mechanisms ◽  
Active Form ◽  
Leading Edge ◽  
Dominant Negative ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Capping Protein ◽  
Actin Capping Protein

To understand the role that ARF6 plays in regulating isoactin dynamics and cell motility, we transfected endothelial cells (EC) with HA-tagged ARF6: the wild-type form (WT), a constitutively-active form unable to hydrolyze GTP (Q67L), and two dominant-negative forms, which are either unable to release GDP (T27N) or fail to bind nucleotide (N122I). Motility was assessed by digital imaging microscopy before Western blot analysis, coimmunoprecipitation, or colocalization studies using ARF6, β-actin, or β-actin-binding protein-specific antibodies. EC expressing ARF6-Q67L spread and close in vitro wounds at twice the control rates. EC expressing dominant-negative ARF6 fail to develop a leading edge, are unable to ruffle their membranes (N122I), and possess arborized processes. Colocalization studies reveal that the Q67L and WT ARF6-HA are enriched at the leading edge with β-actin; but T27N and N122I ARF6-HA are localized on endosomes together with the β-actin capping protein, βcap73. Coimmunoprecipitation and Western blot analyses reveal the direct association of ARF6-HA with βcap73, defining a role for ARF6 in signaling cytoskeletal remodeling during motility. Knowledge of the role that ARF6 plays in orchestrating membrane and β-actin dynamics will help to reveal molecular mechanisms regulating actin-based motility during development and disease.

scholarly journals Conserved function of the lysine-based KXD/E motif in Golgi retention for endomembrane proteins among different organisms

2015 ◽  
Vol 26 (23) ◽  
pp. 4280-4293 ◽  
Author(s):  
Cheuk Hang Woo ◽  
Caiji Gao ◽  
Ping Yu ◽  
Linna Tu ◽  
Zhaoyue Meng ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Transgenic Arabidopsis ◽  
Morphological Changes ◽  
Retention Mechanism ◽  
Loss Of Function ◽  
Expression Systems ◽  
Conserved Motif ◽  
Ultrastructural Studies ◽  
Golgi Retention

We recently identified a new COPI-interacting KXD/E motif in the C-terminal cytosolic tail (CT) of Arabidopsis endomembrane protein 12 (AtEMP12) as being a crucial Golgi retention mechanism for AtEMP12. This KXD/E motif is conserved in CTs of all EMPs found in plants, yeast, and humans and is also present in hundreds of other membrane proteins. Here, by cloning selective EMP isoforms from plants, yeast, and mammals, we study the localizations of EMPs in different expression systems, since there are contradictory reports on the localizations of EMPs. We show that the N-terminal and C-terminal GFP-tagged EMP fusions are localized to Golgi and post-Golgi compartments, respectively, in plant, yeast, and mammalian cells. In vitro pull-down assay further proves the interaction of the KXD/E motif with COPI coatomer in yeast. COPI loss of function in yeast and plants causes mislocalization of EMPs or KXD/E motif–containing proteins to vacuole. Ultrastructural studies further show that RNA interference (RNAi) knockdown of coatomer expression in transgenic Arabidopsis plants causes severe morphological changes in the Golgi. Taken together, our results demonstrate that N-terminal GFP fusions reflect the real localization of EMPs, and KXD/E is a conserved motif in COPI interaction and Golgi retention in eukaryotes.

scholarly journals A dominant-negative cyclin D1 mutant prevents nuclear import of cyclin-dependent kinase 4 (CDK4) and its phosphorylation by CDK-activating kinase.

1997 ◽  
Vol 17 (12) ◽  
pp. 7362-7374 ◽  
Author(s):  
J A Diehl ◽  
C J Sherr
Keyword(s):  
Cyclin D1 ◽  
Nuclear Localization ◽  
Mammalian Cells ◽  
Phosphorylation Site ◽  
Dominant Negative ◽  
Cyclin Dependent Kinase ◽  
Linker Peptide ◽  
Cdk Activating Kinase

Cyclins contain two characteristic cyclin folds, each consisting of five alpha-helical bundles, which are connected to one another by a short linker peptide. The first repeat makes direct contact with cyclin-dependent kinase (CDK) subunits in assembled holoenzyme complexes, whereas the second does not contribute directly to the CDK interface. Although threonine 156 in mouse cyclin D1 is predicted to lie at the carboxyl terminus of the linker peptide that separates the two cyclin folds and is buried within the cyclin subunit, mutation of this residue to alanine has profound effects on the behavior of the derived cyclin D1-CDK4 complexes. CDK4 in complexes with mutant cyclin D1 (T156A or T156E but not T156S) is not phosphorylated by recombinant CDK-activating kinase (CAK) in vitro, fails to undergo activating T-loop phosphorylation in vivo, and remains catalytically inactive and unable to phosphorylate the retinoblastoma protein. Moreover, when it is ectopically overexpressed in mammalian cells, cyclin D1 (T156A) assembles with CDK4 in the cytoplasm but is not imported into the cell nucleus. CAK phosphorylation is not required for nuclear transport of cyclin D1-CDK4 complexes, because complexes containing wild-type cyclin D1 and a CDK4 (T172A) mutant lacking the CAK phosphorylation site are efficiently imported. In contrast, enforced overexpression of the CDK inhibitor p21Cip1 together with mutant cyclin D1 (T156A)-CDK4 complexes enhanced their nuclear localization. These results suggest that cyclin D1 (T156A or T156E) forms abortive complexes with CDK4 that prevent recognition by CAK and by other cellular factors that are required for their nuclear localization. These properties enable ectopically overexpressed cyclin D1 (T156A), or a more stable T156A/T286A double mutant that is resistant to ubiquitination, to compete with endogenous cyclin D1 in mammalian cells, thereby mobilizing CDK4 into cytoplasmic, catalytically inactive complexes and dominantly inhibiting the ability of transfected NIH 3T3 fibroblasts to enter S phase.

scholarly journals Characterization of the Rac-GAP (Rac-GTPase-activating protein) activity of β2-chimaerin, a ‘non-protein kinase C’ phorbol ester receptor

2003 ◽  
Vol 375 (2) ◽  
pp. 313-321 ◽  
Author(s):  
Maria Jose CALOCA ◽  
HongBin WANG ◽  
Marcelo G. KAZANIETZ
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Phorbol Esters ◽  
Gtpase Activating Protein ◽  
Protein Activity ◽  
Rac Gtpase ◽  
Kinase C ◽  
Phorbol Ester Receptor

The regulation and function of β2-chimaerin, a novel receptor for the phorbol ester tumour promoters and the second messenger DAG (diacylglycerol), is largely unknown. As with PKC (protein kinase C) isoenzymes, phorbol esters bind to β2-chimaerin with high affinity and promote its subcellular distribution. β2-Chimaerin has GAP (GTPase-activating protein) activity for the small GTP-binding protein Rac1, but for not Cdc42 or RhoA. We show that acidic phospholipids enhanced its catalytic activity markedly in vitro, but the phorbol ester PMA had no effect. β2-Chimaerin and other chimaerin isoforms decreased cellular levels of Rac-GTP markedly in COS-1 cells and impaired GTP loading on to Rac upon EGF (epidermal growth factor) receptor stimulation. Deletional and mutagenesis analysis determined that the β2-chimaerin GAP domain is essential for this effect. Interestingly, PMA has a dual effect on Rac-GTP levels in COS-1 cells. PMA increased Rac-GTP levels in the absence of a PKC inhibitor, whereas under conditions in which PKC activity is inhibited, PMA markedly decreased Rac-GTP levels and potentiated the effect of β2-chimaerin. Chimaerin isoforms co-localize at the plasma membrane with active Rac, and these results were substantiated by co-immunoprecipitation assays. In summary, the novel phorbol ester receptor β2-chimaerin regulates the activity of the Rac GTPase through its GAP domain, leading to Rac inactivation. These results strongly emphasize the high complexity of DAG signalling due to the activation of PKC-independent pathways, and cast doubts regarding the selectivity of phorbol esters and DAG analogues as selective PKC activators.

scholarly journals Dominant-negative Gα subunits are a mechanism of dysregulated heterotrimeric G protein signaling in human disease

2016 ◽  
Vol 9 (423) ◽  
pp. ra37-ra37 ◽  
Author(s):  
Arthur Marivin ◽  
Anthony Leyme ◽  
Kshitij Parag-Sharma ◽  
Vincent DiGiacomo ◽  
Anthony Y. Cheung ◽  
...  
Keyword(s):  
G Protein ◽  
Mammalian Cells ◽  
Neural Crest Cell ◽  
Craniofacial Development ◽  
Dominant Negative ◽  
Protein Subunit ◽  
Protein Activity ◽  
Guanine Nucleotide Binding Protein ◽  
Heterotrimeric G Protein

Auriculo-condylar syndrome (ACS), a rare condition that impairs craniofacial development, is caused by mutations in a G protein–coupled receptor (GPCR) signaling pathway. In mice, disruption of signaling by the endothelin type A receptor (ETAR), which is mediated by the G protein (heterotrimeric guanine nucleotide–binding protein) subunit Gαq/11 and subsequently phospholipase C (PLC), impairs neural crest cell differentiation that is required for normal craniofacial development. Some ACS patients have mutations in GNAI3, which encodes Gαi3, but it is unknown whether this G protein has a role within the ETAR pathway. We used a Xenopus model of vertebrate development, in vitro biochemistry, and biosensors of G protein activity in mammalian cells to systematically characterize the phenotype and function of all known ACS-associated Gαi3 mutants. We found that ACS-associated mutations in GNAI3 produce dominant-negative Gαi3 mutant proteins that couple to ETAR but cannot bind and hydrolyze guanosine triphosphate, resulting in the prevention of endothelin-mediated activation of Gαq/11 and PLC. Thus, ACS is caused by functionally dominant-negative mutations in a heterotrimeric G protein subunit.

scholarly journals Elevated Endosomal Cholesterol Levels in Niemann-Pick Cells Inhibit Rab4 and Perturb Membrane Recycling

2004 ◽  
Vol 15 (10) ◽  
pp. 4500-4511 ◽  
Author(s):  
Amit Choudhury ◽  
Deepak K. Sharma ◽  
David L. Marks ◽  
Richard E. Pagano
Keyword(s):  
Dominant Negative ◽  
Cholesterol Depletion ◽  
Membrane Recycling ◽  
Normal Human Skin ◽  
Lipid Storage Disease ◽  
Cholesterol Levels ◽  
Early Endosomes ◽  
Niemann Pick ◽  
Gdp Dissociation Inhibitor

In normal human skin fibroblasts (HSFs), fluorescent glycosphingolipid analogues are endocytosed and sorted into two pools, one that is recycled to the plasma membrane and one that is transported to the Golgi complex. Here, we investigated glycosphingolipid recycling in Niemann-Pick type A and C lipid storage disease fibroblasts (NPFs). Cells were incubated with a fluorescent analogue of lactosylceramide (LacCer) at 16°C to label early endosomes (EEs), shifted to 37°C, and lipid recycling was quantified. Using dominant negative rabs, we showed that, in normal HSFs, LacCer recycling was rapid (t1/2 ∼8 min) and mainly rab4-dependent. In NPFs, LacCer recycling was delayed (t1/2 ∼30–40 min), and rab4-dependent recycling was absent, whereas rab11-dependent recycling predominated. Transferrin recycling via the rab4 pathway was similarly perturbed in NPFs. Compared with normal HSFs, EEs in NPFs showed high cholesterol levels and an altered organization of rab4. In vitro extraction of rab4 (but not rab11) with GDP dissociation inhibitor was severely attenuated in NPF endosomal fractions. This impairment was reversed with cholesterol depletion of isolated endosomes or with high-salt treatment of endosomes. These data suggest that abnormal membrane recycling in NPFs results from specific inhibition of rab4 function by excess cholesterol in EEs.

scholarly journals Antileishmanial Activity of a Linalool-Rich Essential Oil from Croton cajucara

2003 ◽  
Vol 47 (6) ◽  
pp. 1895-1901 ◽  
Author(s):  
Maria do Socorro S. Rosa ◽  
Ricardo R. Mendonça-Filho ◽  
Humberto R. Bizzo ◽  
Igor de Almeida Rodrigues ◽  
Rosangela Maria A. Soares ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Essential Oil ◽  
Mammalian Cells ◽  
Morphological Changes ◽  
Peritoneal Macrophages ◽  
Transmission Electron ◽  
Low Concentrations ◽  
Mouse Peritoneal Macrophages ◽  
Croton Cajucara

ABSTRACT The in vitro leishmanicidal effects of a linalool-rich essential oil from the leaves of Croton cajucara against Leishmania amazonensis were investigated. Morphological changes in L. amazonensis promastigotes treated with 15 ng of essential oil per ml were observed by transmission electron microscopy; leishmanial nuclear and kinetoplast chromatin destruction, followed by cell lysis, was observed within 1 h. Pretreatment of mouse peritoneal macrophages with 15 ng of essential oil per ml reduced by 50% the interaction between these macrophages and L. amazonensis, with a concomitant increase by 220% in the level of nitric oxide production by the infected macrophages. Treatment of preinfected macrophages with 15 ng of essential oil per ml reduced by 50% the interaction between these cells and the parasites, which led to a 60% increase in the amount of nitric oxide produced by the preinfected macrophages. These results provide new perspectives on the development of drugs with activities against Leishmania, as linalool-rich essential oil is a strikingly potent leishmanicidal plant extract (50% lethal doses, 8.3 ng/ml for promastigotes and 8.7 ng/ml for amastigotes) which inhibited the growth of L. amazonensis promastigotes at very low concentrations (MIC, 85.0 pg/ml) and which presented no cytotoxic effects against mammalian cells.

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