scholarly journals The Intercalated Disc Protein, mXinα, Is Capable of Interacting with β-Catenin and Bundling Actin Filaments

2007 ◽  
Vol 282 (49) ◽  
pp. 36024-36036 ◽  
Author(s):  
Sunju Choi ◽  
Elisabeth A. Gustafson-Wagner ◽  
Qinchuan Wang ◽  
Shannon M. Harlan ◽  
Haley W. Sinn ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Adherens Junction ◽  
Full Length ◽  
Terminal Deletion ◽  
Actin Binding ◽  
Electron Microscopic ◽  
Microscopic Studies ◽  
Green Fluorescent

Targeted deletion of mXinα results in cardiac hypertrophy and cardiomyopathy with conduction defects (Gustafson-Wagner, E., Sinn, H. W., Chen, Y.-L., Wang, D.-Z., Reiter, R. S., Lin, J. L.-C., Yang, B., Williamson, R. A., Chen, J. N., Lin, C.-I., and Lin, J. J.-C. (2007) Am. J. Physiol. 293, H2680-H2692). To understand the underlying mechanisms leading to such cardiac defects, the functional domains of mXinα and its interacting proteins were investigated. Interaction studies using co-immunoprecipitation, pull-down, and yeast two-hybrid assays revealed that mXinα directly interacts with β-catenin. The β-catenin-binding site on mXinα was mapped to amino acids 535-636, which overlaps with the known actin-binding domains composed of the Xin repeats. The overlapping nature of these domains provides insight into the molecular mechanism for mXinα localization and function. Purified recombinant glutathione S-transferase- or His-tagged mXinα proteins are capable of binding and bundling actin filaments, as determined by co-sedimentation and electron microscopic studies. The binding to actin was saturated at an approximate stoichiometry of nine actin monomers to one mXinα. A stronger interaction was observed between mXinα C-terminal deletion and actin as compared with the interaction between full-length mXinα and actin. Furthermore, force expression of green fluorescent protein fused to an mXinα C-terminal deletion in cultured cells showed greater stress fiber localization compared with force-expressed GFP-mXinα. These results suggest a model whereby the C terminus of mXinα may prevent the full-length molecule from binding to actin, until the β-catenin-binding domain is occupied by β-catenin. The binding of mXinα to β-catenin at the adherens junction would then facilitate actin binding. In support of this model, we found that the actin binding and bundling activity of mXinα was enhanced in the presence of β-catenin.

scholarly journals Toxofilin, a Novel Actin-binding Protein from Toxoplasma gondii, Sequesters Actin Monomers and Caps Actin Filaments

2000 ◽  
Vol 11 (1) ◽  
pp. 355-368 ◽  
Author(s):  
Olivier Poupel ◽  
Haralabia Boleti ◽  
Sophie Axisa ◽  
Evelyne Couture-Tosi ◽  
Isabelle Tardieux
Keyword(s):  
Green Fluorescent Protein ◽  
Toxoplasma Gondii ◽  
Host Cell ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Green Fluorescent

Toxoplasma gondii relies on its actin cytoskeleton to glide and enter its host cell. However, T. gondii tachyzoites are known to display a strikingly low amount of actin filaments, which suggests that sequestration of actin monomers could play a key role in parasite actin dynamics. We isolated a 27-kDa tachyzoite protein on the basis of its ability to bind muscle G-actin and demonstrated that it interacts with parasite G-actin. Cloning and sequence analysis of the gene coding for this protein, which we named Toxofilin, showed that it is a novel actin-binding protein. In in vitro assays, Toxofilin not only bound to G-actin and inhibited actin polymerization as an actin-sequestering protein but also slowed down F-actin disassembly through a filament end capping activity. In addition, when green fluorescent protein-tagged Toxofilin was overexpressed in mammalian nonmuscle cells, the dynamics of actin stress fibers was drastically impaired, whereas green fluorescent protein-Toxofilin copurified with G-actin. Finally, in motile parasites, during gliding or host cell entry, Toxofilin was localized in the entire cytoplasm, including the rear end of the parasite, whereas in intracellular tachyzoites, especially before they exit from the parasitophorous vacuole of their host cell, Toxofilin was found to be restricted to the apical end.

An Improved Chemical Fixation Method Suitable for Immunogold Localization of Green Fluorescent Protein in the Golgi Apparatus of Tobacco Bright Yellow (BY-2) Cells

2003 ◽  
Vol 51 (7) ◽  
pp. 931-940 ◽  
Author(s):  
Marie-Laure Follet-Gueye ◽  
Sophie Pagny ◽  
Loïc Faye ◽  
Véronique Gomord ◽  
Azeddine Driouich
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Electron Microscopic Level ◽  
Fixation Method ◽  
Chemical Fixation ◽  
Electron Microscopic ◽  
One Step ◽  
Green Fluorescent ◽  
Structural Preservation

In plant systems, the green fluorescent protein (GFP) is increasingly used as a marker to study dynamics of the secretory apparatus using fluorescence microscopy. The purpose of this study was to immunogold localize the GFP, at the electron microscopic level, in a line of tobacco BY-2-cultured cells, expressing a GFP-tagged Golgi glycosyltransferase. To this end we have developed a simple, one-step chemical fixation method that allow good structural preservation and specific labeling with anti-GFP antibodies. Using this method, we have been able to show that an N-glycan GFP-tagged xylosyltransferase is specifically associated with Golgi stacks of BY-2 transformed cells and is preferentially located in medial cisternae. As an alternative to cryofixation methods, such as high-pressure freezing, which requires specialized and expensive equipment not available in most laboratories, this method offers researchers the opportunity to investigate GFP-tagged proteins of the endomembrane system in tobacco BY-2 cells.

scholarly journals The Equine Herpesvirus 1 UL20 Product Interacts with Glycoprotein K and Promotes Egress of Mature Particles

2006 ◽  
Vol 80 (1) ◽  
pp. 95-107 ◽  
Author(s):  
Simone Guggemoos ◽  
Frank T. Just ◽  
Antonie Neubauer
Keyword(s):  
Fluorescent Protein ◽  
Equine Herpesvirus ◽  
Electron Microscopic ◽  
Equine Herpesvirus 1 ◽  
Transport Vesicles ◽  
Glycoprotein K ◽  
Microscopic Studies ◽  
Herpesvirus 1 ◽  
Green Fluorescent ◽  
Golgi Network

ABSTRACT The aim of the present study was to identify and functionally characterize the equine herpesvirus 1 (EHV-1) UL20 protein (UL20p). Using a specific antiserum, UL20p was shown to be associated with membranes of infected cells, as well as with envelopes of purified virions. By Western blot analysis, UL20p was detected in two main forms exhibiting M rs of 25,000 and 75,000. Both moieties did not enter the separating gel after heating of protein samples to 99°C. The slower-migrating form of UL20p contains N-linked carbohydrates, and its presence is dependent of that of other viral proteins. Infection of cells that either constitutively express UL20p or a gK-green fluorescent protein (GFP) fusion protein with various EHV-1 deletion mutants revealed a relatively stable hetero-oligomer containing gK and UL20p with an apparent M r of 75,000. As demonstrated by confocal microscopy, UL20p distribution in Rk13 cells changed from a diffuse granular or netlike appearance to a pattern confined to the Golgi network when gK was coexpressed. Analysis of a UL20 deletion mutant of EHV-1 strain RacL11 indicated an involvement of UL20p in cell-to-cell spread, as well as in very late events in virus egress. Based on these and electron microscopic studies we suggest that the EHV-1 UL20 protein might be necessary to avoid fusion of mature virions with membranes of their transport vesicles.

scholarly journals MinCD Proteins Control the Septation Process during Sporulation of Bacillus subtilis

1998 ◽  
Vol 180 (20) ◽  
pp. 5327-5333 ◽  
Author(s):  
Imrich Barák ◽  
Peter Prepiak ◽  
Falko Schmeisser
Keyword(s):  
Bacillus Subtilis ◽  
Fluorescent Protein ◽  
Open Reading Frames ◽  
Protein Fusion ◽  
Electron Microscopic ◽  
Septum Formation ◽  
Microscopic Studies ◽  
Green Fluorescent ◽  
Mutant Cells ◽  
Reading Frames

ABSTRACT Mutation of the divIVB locus in Bacillus subtilis causes misplacement of the septum during cell division and allows the formation of anucleate minicells. The divIVBlocus contains five open reading frames (ORFs). The last two ORFs (minCD) are homologous to minC andminD of Escherichia coli but a minEhomolog is lacking in B. subtilis. There is some similarity between minicell formation and the asymmetric septation that normally occurs during sporulation in terms of polar septum localization. However, it has been proposed that MinCD has no essential role in sporulation septum formation. We have used electron microscopic studies to show septation events during sporulation in some minDstrains. We have observed an unusually thin septum at the midcell position in minD and also in minD spoIIE71mutant cells. Fluorescence microscopy also localized a SpoIIE-green fluorescent protein fusion protein at the midcell site inminD cells. We propose that the MinCD complex plays an important role in asymmetric septum formation during sporulation ofB. subtilis cells.

Barrier role of actin filaments in regulated mucin secretion from airway goblet cells

2005 ◽  
Vol 288 (1) ◽  
pp. C46-C56 ◽  
Author(s):  
Camille Ehre ◽  
Andrea H. Rossi ◽  
Lubna H. Abdullah ◽  
Kathleen De Pestel ◽  
Sandra Hill ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Yellow Fluorescent Protein ◽  
Goblet Cells ◽  
Actin Binding ◽  
Secretory Cells ◽  
Cortical Actin ◽  
Mucin Secretion

Airway goblet cells secrete mucin onto mucosal surfaces under the regulation of an apical, phospholipase C/Gq-coupled P2Y2receptor. We tested whether cortical actin filaments negatively regulate exocytosis in goblet cells by forming a barrier between secretory granules and plasma membrane docking sites as postulated for other secretory cells. Immunostaining of human lung tissues and SPOC1 cells (an epithelial, mucin-secreting cell line) revealed an apical distribution of β- and γ-actin in ciliated and goblet cells. In goblet cells, actin appeared as a prominent subplasmalemmal sheet lying between granules and the apical membrane, and it disappeared from SPOC1 cells activated by purinergic agonist. Disruption of actin filaments with latrunculin A stimulated SPOC1 cell mucin secretion under basal and agonist-activated conditions, whereas stabilization with jasplakinolide or overexpression of β- or γ-actin conjugated to yellow fluorescent protein (YFP) inhibited secretion. Myristoylated alanine-rich C kinase substrate, a PKC-activated actin-plasma membrane tethering protein, was phosphorylated after agonist stimulation, suggesting a translocation to the cytosol. Scinderin (or adseverin), a Ca2+-activated actin filament severing and capping protein was cloned from human airway and SPOC1 cells, and synthetic peptides corresponding to its actin-binding domains inhibited mucin secretion. We conclude that actin filaments negatively regulate mucin secretion basally in airway goblet cells and are dynamically remodeled in agonist-stimulated cells to promote exocytosis.

scholarly journals Impact of Heterogeneity within Cultured Cells on Bacterial Invasion: Analysis of Pseudomonas aeruginosa andSalmonella enterica Serovar Typhi Entry into MDCK cells by Using a Green Fluorescent Protein-Labelled Cystic Fibrosis Transmembrane Conductance Regulator Receptor

2000 ◽  
Vol 68 (2) ◽  
pp. 861-870 ◽  
Author(s):  
A. Alev Gerçeker ◽  
Tanweer Zaidi ◽  
Peter Marks ◽  
David E. Golan ◽  
Gerald B. Pier
Keyword(s):  
Epithelial Cells ◽  
Protein Expression ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Mdck Cells ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Bacterial Uptake ◽  
Cftr Protein ◽  
Green Fluorescent

ABSTRACT The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel that also serves as a receptor for entry ofPseudomonas aeruginosa and Salmonella entericaserovar Typhi into epithelial cells. To evaluate heterogeneity in CFTR protein expression in cultured cells and the effect of heterogeneity on internalization of different P. aeruginosa and serovar Typhi strains, we used two-color flow cytometry and confocal laser microscopy to study bacterial uptake by Madin-Darby canine kidney (MDCK) type I epithelial cells stably expressing a green fluorescent protein (GFP)-CFTR fusion construct (MDCK–GFP-CFTR cells). We found a strong correlation between cell size and GFP-CFTR protein expression, with 60 to 70% of cells expressing low levels of GFP-CFTR protein, 20 to 30% expressing intermediate levels, and <10% expressing high levels. The cells were sorted into low-, intermediate-, or high-level producers of CFTR protein; in vitro growth of each sorted population yielded the same distribution of CFTR protein expression as that in the original population. Cells expressing either low or high levels of CFTR protein internalized bacteria poorly; maximal bacterial uptake occurred in the cells expressing intermediate levels of CFTR protein. Treatment of MDCK cells with sodium butyrate markedly enhanced the production of CFTR protein without increasing cell size; butyrate treatment also increased the proportion of cells with internalized bacteria. However, there were fewer bacteria per butyrate-treated cell and, for P. aeruginosa, there was an overall decrease in the total level of bacterial uptake. The most highly ingested bacterial strains were internalized by fewer total MDCK–GFP-CFTR cells, indicating preferential bacterial uptake by a minority of epithelial cells within a given culture. Confocal fluorescence microscopy showed that P. aeruginosa and serovar Typhi induced cytoplasmic accumulation of CFTR protein close to the plasma membrane where the bacteria were adherent. These results show that within a population of MDCK–GFP-CFTR cells, there are cells with markedly different abilities to ingest bacteria via CFTR, the majority of the P. aeruginosa and serovar Typhi cells are ingested by the one-fourth to one-third of the cells that exhibit an intermediate size and level of CFTR protein expression, and overexpression of the CFTR receptor does not increase total bacterial uptake but rather allows more epithelial cells to ingest fewer total bacteria.

Initiation and maturation of I-Z-I bodies in the growth tips of transfected myotubes

1999 ◽  
Vol 112 (22) ◽  
pp. 4101-4112 ◽  
Author(s):  
K. Ojima ◽  
Z.X. Lin ◽  
Z.Q. Zhang ◽  
T. Hijikata ◽  
S. Holtzer ◽  
...  
Keyword(s):  
Binding Sites ◽  
Thin Filament ◽  
Fluorescent Protein ◽  
De Novo ◽  
Actin Binding ◽  
Double Staining ◽  
Green Fluorescent ◽  
Short Time ◽  
Alpha Actin ◽  
De Novo Assembling

While over a dozen I-Z-I proteins are expressed in postmitotic myoblasts and myotubes it is unclear how, when, or where these first assemble into transitory I-Z-I bodies (thin filament/Z-band precursors) and, a short time later, into definitive I-Z-I bands. By double-staining the growth tips of transfected myotubes expressing (a) MYC-tagged s-alpha-actinins (MYC/s-alpha-actinins) or (b) green fluorescent protein-tagged titin cap (GFP/T-cap) with antibodies against MYC and I-Z-I band proteins, we found that the de novo assembly of I-Z-I bodies and their maturation into I-Z-I bands involved relatively concurrent, cooperative binding and reconfiguration of, at a minimum, 5 integral Z-band molecules. These included s-alpha-actinin, nebulin, titin, T-cap and alpha-actin. Resolution of the approximately 1.0 microm polarized alpha-actin/nebulin/tropomyosin/troponin thin filament complexes occurred subsequent to the maturation of Z-bands into a dense tetragonal configuration. Of particular interest is finding that mutant MYC/s-alpha-actinin peptides (a) lacking spectrin-like repeats 1–4, or consisting of spectrin-like repeats 1–4 only, as well as (b) mutants/fragments lacking titin or alpha-actin binding sites, were promptly and exclusively incorporated into de novo assembling I-Z-I bodies and definitive I-Z-I bands as was exogenous full length MYC/s-alpha-actinin or GFP/T-cap.

Disruption of dynamic cell surface architecture of NIH3T3 fibroblasts by the N-terminal domains of moesin and ezrin: in vivo imaging with GFP fusion proteins

1999 ◽  
Vol 112 (1) ◽  
pp. 111-125 ◽  
Author(s):  
M.R. Amieva ◽  
P. Litman ◽  
L. Huang ◽  
E. Ichimaru ◽  
H. Furthmayr
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Full Length ◽  
Cell Behavior ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal

Lamellipodia, filopodia, microspikes and retraction fibers are characteristic features of a dynamic and continuously changing cell surface architecture and moesin, ezrin and radixin are thought to function in these microextensions as reversible links between plasma membrane proteins and actin microfilaments. Full-length and truncated domains of the three proteins were fused to green fluorescent protein (GFP), expressed in NIH3T3 cells, and distribution and behaviour of cells were analysed by using digitally enhanced differential interference contrast (DIC) and fluorescence video microscopy. The amino-terminal (N-)domains of all three proteins localize to the plasma membrane and fluorescence recordings parallel the dynamic changes in cell surface morphology observed by DIC microscopy of cultured cells. Expression of this domain, however, significantly affects cell surface architecture by the formation of abnormally long and fragile filopodia that poorly attach and retract abnormally. Even more striking are abundant irregular, branched and motionless membraneous structures that accumulate during retraction of lamellipodia. These are devoid of actin, endogenous moesin, ezrin and radixin, but contain the GFP-labeled domain. While a large proportion of endogenous proteins can be extracted with non-ionic detergents as in untransfected control cells, &gt;90% of N-moesin and &gt;60% of N-ezrin and N-radixin remain insoluble. The minimal size of the domain of moesin required for membrane localization and change in behavior includes residues 1–320. Deletions of amino acid residues from either end result in diffuse intracellular distribution, but also in normal cell behavior. Expression of GFP-fusions of full-length moesin or its carboxy-terminal domain has no effect on cell behavior during the observation period of 6–8 hours. The data suggest that, in the absence of the carboxy-terminal domain, N-moesin, -ezrin and -radixin interact tightly with the plasma membrane and interfere with normal functions of endogeneous proteins mainly during retraction.

Quantitative measurement of mammalian chromosome mitotic loss rates using the green fluorescent protein

1999 ◽  
Vol 112 (16) ◽  
pp. 2705-2714
Author(s):  
E.M. Burns ◽  
L. Christopoulou ◽  
P. Corish ◽  
C. Tyler-Smith
Keyword(s):  
Green Fluorescent Protein ◽  
Mammalian Cells ◽  
Quantitative Measurement ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Chromosome Loss ◽  
Facs Analysis ◽  
Fluorescent Cells ◽  
Green Fluorescent ◽  
Loss Rates

We have measured the mitotic loss rates of mammalian chromosomes in cultured cells. The green fluorescent protein (GFP) gene was incorporated into a non-essential chromosome so that cells containing the chromosome fluoresced green, while those lacking it did not. The proportions of fluorescent and non-fluorescent cells were measured by fluorescence activated cell sorter (FACS) analysis. Loss rates ranged from 0.005% to 0.20% per cell division in mouse LA-9 cells, and from 0.02% to 0.40% in human HeLa cells. The rate of loss was elevated by treatment with aneugens, demonstrating that the system rapidly identifies agents which induce chromosome loss in mammalian cells.

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