scholarly journals The IQGAP-related protein DGAP1 interacts with Rac and is involved in the modulation of the F-actin cytoskeleton and control of cell motility

1998 ◽  
Vol 111 (20) ◽  
pp. 3059-3071 ◽  
Author(s):  
J. Faix ◽  
C. Clougherty ◽  
A. Konzok ◽  
U. Mintert ◽  
J. Murphy ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Cell Motility ◽  
Fluorescent Protein ◽  
Actin Dynamics ◽  
Cell Cortex ◽  
A Cell ◽  
Microfilament System ◽  
Green Fluorescent ◽  
And Control

DGAP1 of Dictyostelium discoideum is a cell cortex associated 95 kDa protein that shows homology to both RasGTPase-activating proteins (RasGAPs) and RasGAP-related proteins. When tested for RasGAP activity, recombinant DGAP1 protein did not promote the GTPase activity of human H-Ras or of Dictyostelium RasG in vitro. Instead, DGAP1 bound to Dictyostelium Rac1A and human Rac1, but not to human Cdc42. DGAP1 preferentially interacted with the activated GTP-bound forms of Rac1 and Rac1A, but did not affect the GTPase activities. Since Rho-type GTPases are implicated in the formation of specific F-actin structures and in the control of cell morphology, the microfilament system of mutants that either lack or overexpress DGAP1 has been analysed. DGAP1-null mutants showed elevated levels of F-actin that was organised in large leading edges, membrane ruffles or numerous large filopods. Expression of actin fused to green fluorescent protein (GFP) was used to monitor the actin dynamics in these cells, and revealed that the F-actin cytoskeleton of DGAP1-null cells was rapidly re-arranged to form ruffles and filopods. Conversely, in DGAP1-overexpressing cells, the formation of cellular projections containing F-actin was largely suppressed. Measurement of cell migration demonstrated that DGAP1 expression is inversely correlated with the speed of cell motility.

scholarly journals Effect of Flumorph on F-Actin Dynamics in the Potato Late Blight Pathogen Phytophthora infestans

2015 ◽  
Vol 105 (4) ◽  
pp. 419-423 ◽  
Author(s):  
Chenlei Hua ◽  
Kiki Kots ◽  
Tijs Ketelaar ◽  
Francine Govers ◽  
Harold J. G. Meijer
Keyword(s):  
Actin Cytoskeleton ◽  
Phytophthora Infestans ◽  
Actin Filaments ◽  
Fluorescent Protein ◽  
Crop Protection ◽  
Hyphal Growth ◽  
Actin Dynamics ◽  
Protection Agent ◽  
Green Fluorescent

Oomycetes are fungal-like pathogens that cause notorious diseases. Protecting crops against oomycetes requires regular spraying with chemicals, many with an unknown mode of action. In the 1990s, flumorph was identified as a novel crop protection agent. It was shown to inhibit the growth of oomycete pathogens including Phytophthora spp., presumably by targeting actin. We recently generated transgenic Phytophthora infestans strains that express Lifeact-enhanced green fluorescent protein (eGFP), which enabled us to monitor the actin cytoskeleton during hyphal growth. For analyzing effects of oomicides on the actin cytoskeleton in vivo, the P. infestans Lifeact-eGFP strain is an excellent tool. Here, we confirm that flumorph is an oomicide with growth inhibitory activity. Microscopic analyses showed that low flumorph concentrations provoked hyphal tip swellings accompanied by accumulation of actin plaques in the apex, a feature reminiscent of tips of nongrowing hyphae. At higher concentrations, swelling was more pronounced and accompanied by an increase in hyphal bursting events. However, in hyphae that remained intact, actin filaments were indistinguishable from those in nontreated, nongrowing hyphae. In contrast, in hyphae treated with the actin depolymerizing drug latrunculin B, no hyphal bursting was observed but the actin filaments were completely disrupted. This difference demonstrates that actin is not the primary target of flumorph.

scholarly journals Actin dynamics in living mammalian cells

1998 ◽  
Vol 111 (12) ◽  
pp. 1649-1658 ◽  
Author(s):  
C. Ballestrem ◽  
B. Wehrle-Haller ◽  
B.A. Imhof
Keyword(s):  
Actin Cytoskeleton ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Leading Edge ◽  
Time Lapse ◽  
Living Cells ◽  
Actin Dynamics ◽  
Mammalian Cell Lines ◽  
Cytoskeletal Dynamics ◽  
Green Fluorescent

The actin cytoskeleton maintains the cellular architecture and mediates cell movements. To explore actin cytoskeletal dynamics, the enhanced green fluorescent protein (EGFP) was fused to human β-actin. The fusion protein was incorporated into actin fibers which became depolymerized upon cytochalasin B treatment. This functional EGFP-actin construct enabled observation of the actin cytoskeleton in living cells by time lapse fluorescence microscopy. Stable expression of the construct was obtained in mammalian cell lines of different tissue origins. In stationary cells, actin rich, ring-like structured ‘actin clouds’ were observed in addition to stress fibers. These ruffle-like structures were found to be involved in the reorganization of the actin cytoskeleton. In migratory cells, EGFP-actin was found in the advancing lamellipodium. Immobile actin spots developed in the lamellipodium and thin actin fibers formed parallel to the leading edge. Thus EGFP-actin expressed in living cells unveiled structures involved in the dynamics of the actin cytoskeleton.

Rac1 GTPases control filopodia formation, cell motility, endocytosis, cytokinesis and development in Dictyostelium

2000 ◽  
Vol 113 (12) ◽  
pp. 2253-2265 ◽  
Author(s):  
M. Dumontier ◽  
P. Hocht ◽  
U. Mintert ◽  
J. Faix
Keyword(s):  
Green Fluorescent Protein ◽  
Actin Cytoskeleton ◽  
Cell Motility ◽  
Fluorescent Protein ◽  
Colony Growth ◽  
Dominant Negative ◽  
Related Protein ◽  
Wild Type ◽  
Equal Capacity ◽  
Green Fluorescent

The function of the highly homologous Rac1A, Rac1B, and Rac1C GTPases of the Dictyostelium Rac1 group was investigated. All three GTPases bound with an equal capacity to the IQGAP-related protein DGAP1, with a preference for the activated GTP-bound form. Strong overexpression of wild-type Rac1 GTPases N-terminally tagged with green fluorescent protein (GFP), predominantly induced the formation of numerous long filopodia. Remarkably, expression of the constitutively-activated GTPases resulted in dominant-negative phenotypes: these Rac1-V12 mutants completely lacked filopodia but formed numerous crown shaped structures resembling macropinosomes. Moreover, these mutants were severely impaired in cell motility, colony growth, phagocytosis, pinocytosis, cytokinesis and development. Transformants expressing constitutively-inactivated Rac1-N17 proteins were similar to wild-type cells, but displayed abundant and short filopodia and exhibited a moderate defect in cytokinesis. Taken together, our results indicate that the three GTPases play an identical role in signaling pathways and are key regulators of cellular activities that depend on the re-organization of the actin cytoskeleton in Dictyostelium.

scholarly journals Safe and easy evaluation of tmRNA-SmpB-mediated trans-translation in ESKAPE pathogenic bacteria

2020 ◽  
Author(s):  
Marion Thépaut ◽  
Rodrigo Campos Da Silva ◽  
Eva Renard ◽  
Frédérique Barloy-Hubler ◽  
Eric Ennifar ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Pathogenic Bacteria ◽  
Fluorescent Protein ◽  
Translational Activity ◽  
Promising Target ◽  
A Cell ◽  
Bacterial Fitness ◽  
Green Fluorescent

AbstractBacteria cope with ribosome stalling thanks to trans-translation, a major quality control system of protein synthesis that is mediated by tmRNA, an hybrid RNA with properties of both a tRNA and an mRNA, and the small protein SmpB. Because trans-translation is absent in eukaryotes but necessary for bacterial fitness or survival, it is a promising target for the development of novel antibiotics. To facilitate screening of chemical libraries, various reliable in vitro and in vivo systems have been created for assessing trans-translational activity. However, none of these permits the safe and easy evaluation of trans-translation in pathogenic bacteria, which are obviously the ones we should be targeting. Based on green fluorescent protein (GFP) reassembly during active trans-translation, we have created a cell-free assay adapted to the rapid evaluation of trans-translation in ESKAPE bacteria, with 24 different possible combinations. It can be used for easy high-throughput screening of chemical compounds as well as for exploring the mechanism of trans-translation in these pathogens.

scholarly journals Infectious Hepatitis C Virus Pseudo-particles Containing Functional E1–E2 Envelope Protein Complexes

2003 ◽  
Vol 197 (5) ◽  
pp. 633-642 ◽  
Author(s):  
Birke Bartosch ◽  
Jean Dubuisson ◽  
François-Loïc Cosset
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Fluorescent Protein ◽  
Protein Complexes ◽  
Marker Gene ◽  
Infectious Hepatitis ◽  
Protein Marker ◽  
A Cell ◽  
Green Fluorescent

The study of hepatitis C virus (HCV), a major cause of chronic liver disease, has been hampered by the lack of a cell culture system supporting its replication. Here, we have successfully generated infectious pseudo-particles that were assembled by displaying unmodified and functional HCV glycoproteins onto retroviral and lentiviral core particles. The presence of a green fluorescent protein marker gene packaged within these HCV pseudo-particles allowed reliable and fast determination of infectivity mediated by the HCV glycoproteins. Primary hepatocytes as well as hepato-carcinoma cells were found to be the major targets of infection in vitro. High infectivity of the pseudo-particles required both E1 and E2 HCV glycoproteins, and was neutralized by sera from HCV-infected patients and by some anti-E2 monoclonal antibodies. In addition, these pseudo-particles allowed investigation of the role of putative HCV receptors. Although our results tend to confirm their involvement, they provide evidence that neither LDLr nor CD81 is sufficient to mediate HCV cell entry. Altogether, these studies indicate that these pseudo-particles may mimic the early infection steps of parental HCV and will be suitable for the development of much needed new antiviral therapies.

scholarly journals Supervillin Reorganizes the Actin Cytoskeleton and Increases Invadopodial Efficiency

2009 ◽  
Vol 20 (3) ◽  
pp. 948-962 ◽  
Author(s):  
Jessica L. Crowley ◽  
Tara C. Smith ◽  
Zhiyou Fang ◽  
Norio Takizawa ◽  
Elizabeth J. Luna
Keyword(s):  
Extracellular Matrix ◽  
Actin Cytoskeleton ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Myosin Ii ◽  
Cell Periphery ◽  
Related Protein ◽  
Peripheral Membrane Protein ◽  
Green Fluorescent

Tumor cells use actin-rich protrusions called invadopodia to degrade extracellular matrix (ECM) and invade tissues; related structures, termed podosomes, are sites of dynamic ECM interaction. We show here that supervillin (SV), a peripheral membrane protein that binds F-actin and myosin II, reorganizes the actin cytoskeleton and potentiates invadopodial function. Overexpressed SV induces redistribution of lamellipodial cortactin and lamellipodin/RAPH1/PREL1 away from the cell periphery to internal sites and concomitantly increases the numbers of F-actin punctae. Most punctae are highly dynamic and colocalize with the podosome/invadopodial proteins, cortactin, Tks5, and cdc42. Cortactin binds SV sequences in vitro and contributes to the formation of enhanced green fluorescent protein (EGFP)-SV induced punctae. SV localizes to the cores of Src-generated podosomes in COS-7 cells and with invadopodia in MDA-MB-231 cells. EGFP-SV overexpression increases average numbers of ECM holes per cell; RNA interference-mediated knockdown of SV decreases these numbers. Although SV knockdown alone has no effect, simultaneous down-regulation of SV and the closely related protein gelsolin reduces invasion through ECM. Together, our results show that SV is a component of podosomes and invadopodia and that SV plays a role in invadopodial function, perhaps as a mediator of cortactin localization, activation state, and/or dynamics of metalloproteinases at the ventral cell surface.

scholarly journals Reducing the Risk of Adeno-Associated Virus (AAV) Vector Mobilization with AAV Type 5 Vectors

2009 ◽  
Vol 83 (8) ◽  
pp. 3919-3929 ◽  
Author(s):  
F. Curtis Hewitt ◽  
Chengwen Li ◽  
Steven J. Gray ◽  
Shelley Cockrell ◽  
Michael Washburn ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Helper Virus ◽  
Adeno Associated Virus ◽  
Vector Genome ◽  
Gene Therapy Vectors ◽  
A Cell ◽  
Green Fluorescent ◽  
Relative Prevalence

ABSTRACT Current adeno-associated virus (AAV) gene therapy vectors package a transgene flanked by the terminal repeats (TRs) of AAV type 2 (AAV2). Although these vectors are replication deficient, wild-type (wt) AAV2 prevalent in the human population could lead to replication and packaging of a type 2 TR (TR2)-flanked transgene in trans during superinfection by a helper virus, leading to “mobilization” of the vector genome from treated cells. More importantly, it appears likely that the majority of currently characterized AAV serotypes as well as the majority of new novel isolates are capable of rescuing and replicating AAV2 vector templates. To investigate this possibility, we flanked a green fluorescent protein transgene with type 2 and, the most divergent AAV serotype, type 5 TRs (TR2 or TR5). Consistent with AAV clades, AAV5 specifically replicated TR5 vectors, while AAV2 and AAV6 replicated TR2-flanked vectors. To exploit this specificity, we created a TR5 vector production system for Cap1 to Cap5. Next, we showed that persisting recombinant AAV genomes flanked by TR2s or TR5s were mobilized in vitro after addition of the cognate AAV Rep (as well as Rep6 for TR2) and adenoviral helper. Finally, we showed that a cell line containing a stably integrated wt AAV2 genome resulted in mobilization of a TR2-flanked vector but not a TR5-flanked vector upon adenoviral superinfection. Based on these data and the relative prevalence of wt AAV serotypes in the population, we propose that TR5 vectors have a significantly lower risk of mobilization and should be considered for clinical use.

scholarly journals Insight into the Antiadhesive Effect of Yeast Wall Protein 1 of Candida albicans

2012 ◽  
Vol 11 (6) ◽  
pp. 795-805 ◽  
Author(s):  
Bruce L. Granger
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Fluorescent Protein ◽  
Surrounding Medium ◽  
Yeast Cells ◽  
Content Type ◽  
A Cell ◽  
Opportunistic Fungal Pathogen ◽  
Green Fluorescent

ABSTRACTYwp1 is a prominent glycosylphosphatidylinositol (GPI)-anchored glycoprotein of the cell wall ofCandida albicans; it is present in the yeast form of this opportunistic fungal pathogen but absent from filamentous forms and chlamydospores. Yeast cells that lack Ywp1 are more adhesive and form thicker biofilms, implying an antiadhesive activity for Ywp1, with a possible role in yeast dispersal. The antiadhesive effect of Ywp1 is transplantable from yeast to hyphae, as hyphae that are forced to expressYWP1lose adhesion in anin vitroassay. Deletion of the GPI anchor results in loss of Ywp1 to the surrounding medium and reduction of the antiadhesive effect, implying an importance of time-dependent residency in the cell wall. Anchor-negative versions of Ywp1 possessing or lacking a C-terminal green fluorescent protein (GFP) tag were created inC. albicansand harvested from culture supernatants; in addition to serving as quantifiable markers for Ywp1 secretion, they revealed that the cleaved 11-kDa propeptide of Ywp1 remains strongly but noncovalently associated with the Ywp1 core. This association is resistant to highly acidic and basic solutions, 8 M urea, and 1% SDS (below 45°C). Above 50°C, SDS dissociates the isolated complex, but even higher temperatures are required to dissociate the propeptide from native Ywp1 that is anchored in a cell wall. This property has permitted detection, for the first time, of orthologs of Ywp1 in other members of theCandidaclade. The cleaved propeptide, which carries the sole N-glycan of Ywp1, must participate in the antiadhesive effect of Ywp1.

scholarly journals Analysis of PRA1 and Its Relationship to Candida albicans- Macrophage Interactions

2008 ◽  
Vol 76 (9) ◽  
pp. 4345-4358 ◽  
Author(s):  
A. Marcil ◽  
C. Gadoury ◽  
J. Ash ◽  
J. Zhang ◽  
A. Nantel ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Fluorescent Protein ◽  
Raw 264.7 Cells ◽  
Minor Role ◽  
Human Fibrinogen ◽  
Fibrinogen Binding ◽  
A Cell ◽  
Green Fluorescent ◽  
A Minor

ABSTRACT Phagocytosis of Candida albicans by either primary bone marrow-derived mouse macrophages or RAW 264.7 cells upregulated transcription of PRA1, which encodes a cell wall/membrane-associated antigen previously described as a fibrinogen binding protein. However, a pra1 null mutant was still able to bind fibrinogen, showing that Pra1p is not uniquely required for fibrinogen binding. As well, Pra1 tagged with green fluorescent protein did not colocalize with AlexaFluor 546-labeled human fibrinogen, and while PRA1 expression was inhibited when Candida was grown in fetal bovine serum-containing medium, Candida binding to fibrinogen was activated by these conditions. Therefore, it appears that Pra1p can play at most a minor role in fibrinogen binding to C. albicans. PRA1 gene expression is induced in vitro by alkaline pH, and therefore its activation in phagosomes suggested that phagosome maturation was suppressed by the presence of Candida cells. LysoTracker red-labeled organelles failed to fuse with phagosomes containing live Candida, while phagosomes containing dead Candida underwent a normal phagosome-to-phagolysosome maturation. Immunofluorescence staining with the early/recycling endosomal marker transferrin receptor (CD71) suggested that live Candida may escape macrophage destruction through the inhibition of phagolysosomal maturation.

scholarly journals Engineering of Recombinant Sheep Pox Viruses Expressing Foreign Antigens

2021 ◽  
Vol 9 (5) ◽  
pp. 1005
Author(s):  
Olga Chervyakova ◽  
Elmira Tailakova ◽  
Nurlan Kozhabergenov ◽  
Sandugash Sadikaliyeva ◽  
Kulyaisan Sultankulova ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Viral Vector ◽  
Foot And Mouth Disease ◽  
Open Reading Frames ◽  
Foreign Gene ◽  
Mouth Disease Virus ◽  
Foreign Genes ◽  
Green Fluorescent ◽  
Reading Frames

Capripoxviruses with a host range limited to ruminants have the great potential to be used as vaccine vectors. The aim of this work was to evaluate attenuated sheep pox virus (SPPV) vaccine strain NISKHI as a vector expressing several genes. Open reading frames SPPV020 (ribonucleotide kinase) and SPPV066 (thymidine kinase) were selected as sites for the insertion of foreign genes. Two integration plasmids with expression cassette were designed and constructed. Recombinant SPPVs expressing an enhanced green fluorescent protein (EGFP) (rSPPV(RRΔ)EGFP and rSPPV(TKΔ)EGFP), Foot-and-mouth disease virus capsid protein (VP1), and Brucella spp. outer membrane protein 25 (OMP25) (rSPPV(RRΔ)VP1A-(TKΔ)OMP25) were generated under the transient dominant selection method. The insertion of foreign genes into the SPPV020 and SPPV066 open reading frames did not influence the replication of the recombinant viruses in the cells. Successful foreign gene expression in vitro was assessed by luminescent microscopy (EGFP) and Western blot (VP1 and OMP25). Our results have shown that foreign genes were expressed by rSPPV both in permissive (lamb testicles) and non-permissive (bovine kidney, saiga kidney, porcine kidney) cells. Mice immunized with rSPPV(RRΔ)VP1A-(TKΔ)OMP25 elicited specific antibodies to both SPPV and foreign genes VP1 and OMP25. Thus, SPPV NISKHI may be used as a potential safe immunogenic viral vector for the development of polyvalent vaccines.

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