scholarly journals Calreticulin modulates cell adhesiveness via regulation of vinculin expression.

1996 ◽  
Vol 135 (6) ◽  
pp. 1913-1923 ◽  
Author(s):  
M Opas ◽  
M Szewczenko-Pawlikowski ◽  
G K Jass ◽  
N Mesaeli ◽  
M Michalak
Keyword(s):  
Fluorescent Protein ◽  
High Capacity ◽  
Fibroblast Cell ◽  
Cell Junctions ◽  
Mrna Levels ◽  
Regulation Of Expression ◽  
Low Concentrations ◽  
Green Fluorescent ◽  
In Cells ◽  
Cell Cell

Calreticulin is an ubiquitous and highly conserved high capacity Ca(2+)-binding protein that plays a major role in Ca2+ storage within the lumen of the ER. Here, using L fibroblast cell lines expressing different levels of calreticulin, we show that calreticulin plays a role in the control of cell adhesiveness via regulation of expression of vinculin, a cytoskeletal protein essential for cell-substratum and cell-cell attachments. Both vinculin protein and mRNA levels are increased in cells overexpressing calreticulin and are downregulated in cells expressing reduced level of calreticulin. Abundance of actin, talin, alpha 5 and beta 1 integrins, pp125 focal adhesion kinase, and alpha-catenin is not affected by the differential calreticulin expression. Overexpression of calreticulin increases both cell-substratum and cell-cell adhesiveness of L fibroblasts that, most surprisingly, establish vinculin-rich cell-cell junctions. Upregulation of calreticulin also affects adhesion-dependent phenomena such as cell motility (which decreases) and cell spreading (which increases). Downregulation of calreticulin brings about inverse effects. Cell adhesiveness is Ca2+ regulated. The level of calreticulin expression, however, has no effect on either the resting cytoplasmic Ca2+ concentration or the magnitude of FGF-induced Ca2+ transients. Calreticulin, however, participates in Ca2+ homeostasis as its level of expression affects cell viability at low concentrations of extracellular Ca2+. Consequently, we infer that it is not the Ca2+ storage function of calreticulin that affects cell adhesiveness. Neither endogenous calreticulin nor overexpressed green fluorescent protein-calreticulin construct can be detected outside of the ER. Since all of the adhesion-related effects of differential calreticulin expression can be explained by its regulation of vinculin expression, we conclude that it is the ER-resident calreticulin that affects cellular adhesiveness.

scholarly journals Asc Modulates the Function of NLRC4 in Response to Infection of Macrophages by Legionella pneumophila

mBio ◽  
2011 ◽  
Vol 2 (4) ◽  
Author(s):  
Christopher L. Case ◽  
Craig R. Roy
Keyword(s):  
Cell Death ◽  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Microbial Infection ◽  
Content Type ◽  
Caspase 1 ◽  
Cell Death Pathway ◽  
Dependent Cell ◽  
Green Fluorescent ◽  
In Cells

ABSTRACTNucleotide-binding domain, leucine-rich repeat containing proteins (NLRs) activate caspase-1 in response to a variety of bacterium-derived signals in macrophages. NLR-mediated activation of caspase-1 byLegionella pneumophilaoccurs through both an NLRC4/NAIP5-dependent pathway and a pathway requiring the adapter protein Asc. Both pathways are needed for maximal activation of caspase-1 and for the release of the cytokines interleukin-1β (IL-1β) and IL-18. Asc is not required for caspase-1-dependent pore formation and cell death induced upon infection of macrophages byL. pneumophila. Here, temporal and spatial localization of caspase-1-dependent processes was examined to better define the roles of Asc and NLRC4 during infection. Imaging studies revealed that caspase-1 localized to a single punctate structure in infected cells containing Asc but not in cells lacking this adapter. Both endogenous Asc and ectopically produced NLRC4 tagged with green fluorescent protein (GFP) were found to localize to caspase-1 puncta followingL. pneumophilainfection, suggesting that NLRC4 and Asc coordinate signaling through this complex during caspase-1 activation. Formation of caspase-1-containing puncta correlated with caspase-1 processing, suggesting a role for the Asc/NLRC4/caspase-1 complex in caspase-1 cleavage. In cells deficient for Asc, NLRC4 did not assemble into discrete puncta, and pyroptosis occurred at an accelerated rate. These data indicate that Asc mediates integration of NLR components into caspase-1 processing platforms and that recruitment of NLR components into an Asc complex can dampen pyroptotic responses. Thus, a negative feedback role of complexes containing Asc may be important for regulating caspase-1-mediated responses during microbial infection.IMPORTANCECaspase-1 is a protease activated during infection that is central to the regulation of several innate immune pathways. Studies examining the macromolecular complexes containing this protein, known as inflammasomes, have provided insight into the regulation of this protease. This work demonstrates that the intracellular bacteriumLegionella pneumophilainduces formation of complexes containing caspase-1 by multiple mechanisms and illustrates that an adapter molecule called Asc integrates signals from multiple independent upstream caspase-1 activators in order to assemble a spatially distinct complex in the macrophage. There were caspase-1-associated activities such as cytokine processing and secretion that were controlled by Asc. Importantly, this work uncovered a new role for Asc in dampening a caspase-1-dependent cell death pathway called pyroptosis. These findings suggest that Asc plays a central role in controlling a distinct subset of caspase-1-dependent activities by both assembling complexes that are important for cytokine processing and suppressing processes that mediate pyroptosis.

scholarly journals Protease-activatable biosensors of SARS-CoV-2 infection for cell-based drug, neutralisation and virological assays

2021 ◽  
Author(s):  
Pehuen Pereyra Gerber ◽  
Lidia M Duncan ◽  
Edward JD Greenwood ◽  
Sara Marelli ◽  
Adi Naamati ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Firefly Luciferase ◽  
Neutralising Antibodies ◽  
Antiviral Therapies ◽  
Viral Proteases ◽  
Green Fluorescent ◽  
High Throughput Screens ◽  
Protease Expression ◽  
In Cells

The world is in the grip of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, and there is an urgent unmet clinical need for effective antiviral therapies. Many inhibitors of viral enzymes identified in vitro have limited efficacy against viral replication in cells, but conventional plaque assays are impractical for high-throughput screens. In this study, we therefore engineer cell-based biosensors of SARS-CoV-2 infection. Our assays exploit the cleavage of specific oligopeptide linkers by SARS-CoV-2 Main or Papain-like proteases, leading to the activation of green fluorescent protein (GFP) or firefly luciferase-based reporters. First, we characterise these biosensors in cells using recombinant viral proteases. Next, we confirm their ability to detect endogenous viral protease expression during infection with wildtype SARS-CoV-2. Finally, we develop a sensitive luminescent reporter cell line, confirm that it accurately quantitates infectious SARS-CoV-2 virus, and demonstrate its utility for drug screening and titration of neutralising antibodies.

Imaging myosin 10 in cells

2004 ◽  
Vol 32 (5) ◽  
pp. 689-693 ◽  
Author(s):  
D. Tacon ◽  
P.J. Knight ◽  
M. Peckham
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Major Task ◽  
Green Fluorescent ◽  
In Cells

Cellular motors (kinesin, dynein and myosin) are ubiquitous. A major task in cell biology is to determine how they function in cells. Here we focus on myosin 10, an intrafilopodial motor, and show how imaging green fluorescent protein fused to myosin 10 or its tail domains can help us understand the function of this myosin.

scholarly journals Complexes between Herpes Simplex Virus Glycoproteins gD, gB, and gH Detected in Cells by Complementation of Split Enhanced Green Fluorescent Protein

2007 ◽  
Vol 81 (20) ◽  
pp. 11532-11537 ◽  
Author(s):  
Elisa Avitabile ◽  
Cristina Forghieri ◽  
Gabriella Campadelli-Fiume
Keyword(s):  
Herpes Simplex Virus ◽  
Green Fluorescent Protein ◽  
Herpes Simplex ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Split Egfp ◽  
Green Fluorescent ◽  
Simplex Virus ◽  
In Cells

ABSTRACT The interactions between herpes simplex virus gD and its nectin1 receptor or between gD, gB, and gH were analyzed by complementation of the N and C portions of split enhanced green fluorescent protein (EGFP) fused to the glycoproteins. The gDN-NectC complex was readily detected; the gDN-gCC complex was undetectable, highlighting the specificity of the assay. Split EGFP complementation was detected between proteins designated gDN+gHC, gDN+gBC, and gHN+gBC+wtgD (gB was deleted of endocytosis motifs), both in cells transfected with two-tree glycoproteins and in syncytia. The in situ assay provides evidence that gD interacts with gH and gB independently of each other and supports a model whereby gH and gB in complex exert their activities to gD.

Relative Strengths and Regulation of Different Promoter-Associated Sequences for Various blaSHV Genes and Their Relationships to β-Lactam Resistance

2017 ◽  
Vol 27 (2) ◽  
pp. 91-101 ◽  
Author(s):  
Yao Zhai ◽  
Zhao Zhang ◽  
Zhanwei Wang ◽  
Yusheng Chen ◽  
Qi Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Mrna Expression ◽  
Inhibitory Concentration ◽  
Fluorescent Protein ◽  
Mrna Levels ◽  
Associated Sequences ◽  
Regulation Function ◽  
Green Fluorescent ◽  
Mrna Expression Levels

<b><i>Aims:</i></b> This work investigated the relative strengths of different <i>bla</i><sub>SHV</sub> promoter-associated sequences and their regulation function in <i>bla</i><sub>SHV</sub> expression and β-lactam resistance. <b><i>Methods:</i></b> Recombinant plasmids with the promoter-associated sequences (P-W, P-S, P-IS, and P-WPD), <i>tac</i> promoter, and combined fragments of promoter and <i>bla</i><sub>SHV</sub> were separately constructed and transformed into <i>Escherichia coli</i> DH5α. The relative strengths of the promoters indicated by the intensities of green fluorescent protein and the mRNA expression levels of <i>bla</i><sub>SHV</sub> were compared. The minimum inhibitory concentration and extended spectrum β-lactamase phenotypes were evaluated. <b><i>Results:</i></b> The relative strengths were ranked as P-<i>tac</i> > P-WPD > P-IS > P-S > P-W. The mRNA expression and β-lactam resistance levels of the different promoter-associated sequence groups were generally consistent with the strength rank, but the extent of <i>gfp</i> and <i>bla</i><sub>SHV</sub> mRNA levels varied significantly in each group. The β-lactam resistance levels were inconsistent with the strength rank in certain <i>bla</i><sub>SHV</sub> groups. In relation to the different promoter-associated sequences,<i> bla</i><sub>SHV-ESBLs</sub> displayed significantly different change modes of β-lactam resistance compared with <i>bla</i><sub>SHV-non-ESBLs</sub>. <b><i>Conclusion:</i></b> The mRNA expression and β-lactam resistance of the <i>bla</i><sub>SHV</sub> showed consistencies and inconsistencies with the strengths of the promoter-associated sequences. The mechanisms accounting for these discrepancies need further investigation.

Involvement of a Rab8-like protein of Dictyostelium discoideum, Sas1, in the formation of membrane extensions, secretion and adhesion during development

Microbiology ◽  
2004 ◽  
Vol 150 (8) ◽  
pp. 2513-2525 ◽  
Author(s):  
Rhonda R. Powell ◽  
Lesly A. Temesvari
Keyword(s):  
Cell Adhesion ◽  
Dictyostelium Discoideum ◽  
Fluorescent Protein ◽  
Aggregate Size ◽  
Developmental Context ◽  
Cellular Events ◽  
Membrane Protrusions ◽  
A Cell ◽  
Green Fluorescent ◽  
Cell Cell

Establishment of cell–cell adhesions, regulation of actin, and secretion are critical during development. Rab8-like GTPases have been shown to modulate these cellular events, suggesting an involvement in developmental processes. To further elucidate the function of Rab8-like GTPases in a developmental context, a Rab8-related protein (Sas1) of Dictyostelium discoideum was examined, the expression of which increases at the onset of development. Dictyostelium cell lines expressing inactive (N128I mutant) and constitutively active (Q74L mutant) Sas1 as green fluorescent protein (GFP)-Sas1 chimeras were generated. Cells expressing Sas1Q74L displayed numerous actin-rich membrane protrusions, increased secretion, and were unable to complete development. In particular, these cells demonstrated a reduction in adhesion as well as in the levels of a cell adhesion molecule, gp24 (DdCAD-1). In contrast, cells expressing Sas1N128I exhibited increased cell–cell adhesion and increased levels of gp24. Counting factor is a multisubunit signalling complex that is secreted in early development and controls aggregate size by negatively regulating the levels of cell adhesion molecules, including gp24. Interestingly, the Sas1Q74L mutant demonstrated increased levels of extracellular countin, a subunit of counting factor, suggesting that Sas1 may regulate trafficking of counting factor components. Together, the data suggest that Sas1 may be a key regulator of actin, adhesion and secretion during development.

scholarly journals Enhanced Survival of Salmonella enterica in Vesicles Released by a Soilborne Tetrahymena Species

2005 ◽  
Vol 71 (3) ◽  
pp. 1562-1569 ◽  
Author(s):  
M. T. Brandl ◽  
B. M. Rosenthal ◽  
A. F. Haxo ◽  
S. G. Berk
Keyword(s):  
Public Health ◽  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Survival Rates ◽  
Human Pathogen ◽  
Initial Ratio ◽  
Calcium Hypochlorite ◽  
Human Macrophages ◽  
Low Concentrations ◽  
Green Fluorescent

ABSTRACT Nondestructive ingestion by soilborne protozoa may enhance the environmental resiliency of important bacterial pathogens and may model how such bacteria evade destruction in human macrophages. Here, the interaction of Salmonella enterica serovar Thompson with a soilborne Tetrahymena sp. isolate was examined using serovar Thompson cells labeled with the green fluorescent protein. The bacteria were mixed in solution with cells of Tetrahymena at several ratios. During incubation with serovar Thompson, Tetrahymena cells released a large number of vesicles containing green fluorescent serovar Thompson cells. In comparison, grazing on Listeria monocytogenes cells resulted in their digestion and thus the infrequent release of this pathogen in vesicles. The number of serovar Thompson cells per vesicle increased significantly as the initial ratio of serovar Thompson to Tetrahymena cells increased from 500:1 to 5,000:1. The density of serovar Thompson was as high as 50 cells per vesicle. Staining with propidium iodide revealed that a significantly higher proportion of serovar Thompson cells remained viable when enclosed in vesicles than when free in solution. Enhanced survival rates were observed in vesicles that were secreted by both starved (F = 28.3, P < 0.001) and unstarved (F = 14.09, P < 0.005) Tetrahymena cells. Sequestration in vesicles also provided greater protection from low concentrations of calcium hypochlorite. Thus, the release of this human pathogen from Tetrahymena cells in high-density clusters enclosed in a membrane may have important implications for public health.

scholarly journals Interaction of a Host Protein with Core Complexes of Bacteriophage Φ6 To Control Transcription

2010 ◽  
Vol 84 (9) ◽  
pp. 4821-4825 ◽  
Author(s):  
Xueying Qiao ◽  
Yang Sun ◽  
Jian Qiao ◽  
Leonard Mindich
Keyword(s):  
Green Fluorescent Protein ◽  
Rna Polymerase ◽  
Fluorescent Protein ◽  
Host Protein ◽  
Genomic Segment ◽  
Double Stranded Rna ◽  
Infection Period ◽  
The Family ◽  
Green Fluorescent ◽  
In Cells

ABSTRACT Bacteriophages of the family Cystoviridae have genomes consisting of three double-stranded RNA (dsRNA) segments, L, S, and M, packaged within a polyhedral capsid along with RNA polymerase. Transcription of genomic segment L is activated by the interaction of host protein YajQ with the capsid structure. Segment L codes for the proteins of the inner capsid, which are expressed early in infection. Green fluorescent protein (GFP) fusions with YajQ produce uniform fluorescence in uninfected cells and in cells infected with viruses not dependent on YajQ. Punctate fluorescence develops when cells are infected with YajQ-dependent viruses. It appears that the host protein binds to the infecting particles and remains with them during the entire infection period.

Sign in / Sign up

Export Citation Format

Share Document