scholarly journals Reorganization of Actin Cytoskeleton by the Phosphoinositide Metabolite Glycerophosphoinositol 4-Phosphate

2003 ◽  
Vol 14 (2) ◽  
pp. 503-515 ◽  
Author(s):  
Raffaella Mancini ◽  
Enza Piccolo ◽  
Stefania Mariggio' ◽  
Beatrice Maria Filippi ◽  
Cristiano Iurisci ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Fluorescent Protein ◽  
Biological Activities ◽  
Small Gtpases ◽  
Fiber Formation ◽  
Water Soluble ◽  
Biologically Active ◽  
Intact Cells ◽  
Green Fluorescent ◽  
Phosphatidylinositol 4

Glycerophosphoinositol 4-phosphate (GroPIns-4P) is a biologically active, water-soluble phospholipase A metabolite derived from phosphatidylinositol 4-phosphate, whose cellular concentrations have been reported to increase in Ras-transformed cells. It is therefore important to understand its biological activities. Herein, we have examined whether GroPIns-4P can regulate the organization of the actin cytoskeleton, because this could be a Ras-related function involved in cell motility and metastatic invasion. We find that in serum-starved Swiss 3T3 cells, exogenously added GroPIns-4P rapidly and potently induces the formation of membrane ruffles, and, later, the formation of stress fibers. These actin structures can be regulated by the small GTPases Cdc42, Rac, and Rho. To analyze the mechanism of action of GroPIns-4P, we selectively inactivated each of these GTPases. GroPIns-4P requires active Rac and Rho, but not Cdc42, for ruffle and stress fiber formation, respectively. Moreover, GroPIns-4P induces a rapid translocation of the green fluorescent protein-tagged Rac into ruffles, and increases the fraction of GTP-bound Rac, in intact cells. The activation of Rac by GroPIns-4P was near maximal and long-lasting. Interestingly, this feature seems to be critical in the induction of actin ruffles by GroPIns-4P.

scholarly journals The Reporter System for GPCR Assay with the Fission YeastSchizosaccharomyces pombe

Scientifica ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Shintaro Sasuga ◽  
Toshiya Osada
Keyword(s):  
Fluorescent Protein ◽  
Signal To Noise Ratio ◽  
Biological Activities ◽  
Inducible Promoter ◽  
Upstream Region ◽  
Reporter Plasmid ◽  
Reporter System ◽  
High Background ◽  
Downstream Region ◽  
Green Fluorescent

G protein-coupled receptors (GPCRs) are associated with a great variety of biological activities. Yeasts are often utilized as a host for heterologous GPCR assay. We engineered the intense reporter plasmids for fission yeast to produce green fluorescent protein (GFP) through its endogenous GPCR pathway. As a control region of GFP expression on the reporter plasmid, we focused on seven endogenous genes specifically activated through the pathway. When upstream regions of these genes were used as an inducible promoter in combination with LPI terminator, themam2upstream region produced GFP most rapidly and intensely despite the high background. Subsequently, LPI terminator was replaced with the corresponding downstream regions. The SPBC4.01 downstream region enhanced the response with the low background. Furthermore, combining SPBC4.01 downstream region with the sxa2 upstream region, the signal to noise ratio was obviously better than those of other regions. We also evaluated the time- and dose-dependent GFP productions of the strains transformed with the reporter plasmids. Finally, we exhibited a model of simplified GPCR assay with the reporter plasmid by expressing endogenous GPCR under the control of the foreign promoter.

Human p63RhoGEF, a novel RhoA-specific guanine nucleotide exchange factor, is localized in cardiac sarcomere

2002 ◽  
Vol 115 (3) ◽  
pp. 629-640 ◽  
Author(s):  
Michel Souchet ◽  
Elodie Portales-Casamar ◽  
David Mazurais ◽  
Susanne Schmidt ◽  
Isabelle Léger ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factor ◽  
Small Gtpases ◽  
Fiber Formation ◽  
Pleckstrin Homology Domain ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Intact Cells ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

The Rho small GTPases are crucial proteins involved in regulation of signal transduction cascades from extracellular stimuli to cell nucleus and cytoskeleton. It has been reported that these GTPases are directly associated with cardiovascular disorders. In this context, we have searched for novel modulators of Rho GTPases, and here we describe p63RhoGEF a new Db1-like guanine nucleotide exchange factor (GEF). P63RhoGEF encodes a 63 kDa protein containing a Db1 homology domain in tandem with a pleckstrin homology domain and is most closely related to the second Rho GEF domain of Trio. Northern blot and in situ analysis have shown that p63RhoGEF is mainly expressed in heart and brain. In vitro guanine nucleotide exchange assays have shown that p63RhoGEF specifically acts on RhoA. Accordingly, p63RhoGEF expression induces RhoA-dependent stress fiber formation in fibroblasts and in H9C2 cardiac myoblasts. Moreover, we show that p63RhoGEF activation of RhoA in intact cells is dependent on the presence of the PH domain. Using a specific anti-p63RhoGEF antibody, we have detected the p63RhoGEF protein by immunocytochemistry in human heart and brain tissue sections. Confocal microscopy shows that p63RhoGEF is located in the sarcomeric I-band mainly constituted of cardiac sarcomeric actin. Together, these results show that p63RhoGEF is a RhoA-specific GEF that may play a key role in actin cytoskeleton reorganization in different tissues, especially in heart cellular morphology.

scholarly journals Nucleofection Efficiency of Sheep Primary Fibroblasts Established from Refrigerated Skin

2018 ◽  
Vol 10 (4) ◽  
pp. 12
Author(s):  
Mahipal Singh ◽  
Xiaoling Ma
Keyword(s):  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Genetically Engineered ◽  
Dermal Fibroblasts ◽  
Biologically Active ◽  
Cell Type ◽  
Gfp Expression ◽  
Primary Fibroblasts ◽  
Species Specific ◽  
Green Fluorescent

Dermal fibroblasts are useful for production of genetically engineered biologically active factors for development of cellular therapies and tissue engineering products for regenerative medicine. However, their transfection efficiencies using traditional non-viral methods are low and vary based on cell-type and species-specific differences. Using nucleofection technology, here we show that the transfection efficiency of primary fibroblasts established after 0-, 35-, and 65-days of postmortem storage of sheep skin tissues in a refrigerator was 59.49 % ± 9.66 %, 59.33 % ± 11.59 %, and 43.48 % ± 8.09 % respectively, as determined by analysis of green fluorescent protein (GFP) expression. 

scholarly journals Autophagy and the cvt Pathway Both Depend onAUT9

2000 ◽  
Vol 182 (8) ◽  
pp. 2125-2133 ◽  
Author(s):  
Thomas Lang ◽  
Steffen Reiche ◽  
Michael Straub ◽  
Monika Bredschneider ◽  
Michael Thumm
Keyword(s):  
Protein Turnover ◽  
Protein Transport ◽  
Fluorescent Protein ◽  
Genomic Library ◽  
Integral Membrane Protein ◽  
Biologically Active ◽  
Transport Pathways ◽  
Rich Media ◽  
Green Fluorescent ◽  
Mutant Cells

ABSTRACT In growing cells of the yeast Saccharomyces cerevisiae, proaminopeptidase I reaches the vacuole via the selective cytoplasm-to-vacuole targeting (cvt) pathway. During nutrient limitation, autophagy is also responsible for the transport of proaminopeptidase I. These two nonclassical protein transport pathways to the vacuole are distinct in their characteristics but in large part use identical components. We expanded our initial screen foraut − mutants and isolated aut9-1cells, which show a defect in both pathways, the vacuolar targeting of proaminopeptidase I and autophagy. By complementation of the sporulation defect of homocygous diploid aut9-1 mutant cells with a genomic library, in this study we identified and characterized the AUT9 gene, which is allelic withCVT7. aut9-deficient cells have no obvious defects in growth on rich media, vacuolar biogenesis, and acidification, but like other mutant cells with a defect in autophagy, they exhibit a reduced survival rate and reduced total protein turnover during starvation. Aut9p is the first putative integral membrane protein essential for autophagy. A biologically active green fluorescent protein-Aut9 fusion protein was visualized at punctate structures in the cytosol of growing cells.

scholarly journals G Protein β Subunit–null Mutants Are Impaired in Phagocytosis and Chemotaxis Due to Inappropriate Regulation of the Actin Cytoskeleton

1998 ◽  
Vol 141 (7) ◽  
pp. 1529-1537 ◽  
Author(s):  
Barbara Peracino ◽  
Jane Borleis ◽  
Tian Jin ◽  
Monika Westphal ◽  
Jean-Marc Schwartz ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
G Protein ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Constitutive Expression ◽  
Fluid Phase ◽  
Β Subunit ◽  
Wild Type ◽  
Fluid Phase Endocytosis ◽  
Green Fluorescent

Chemotaxis and phagocytosis are basically similar in cells of the immune system and in Dictyostelium amebae. Deletion of the unique G protein β subunit in D. discoideum impaired phagocytosis but had little effect on fluid-phase endocytosis, cytokinesis, or random motility. Constitutive expression of wild-type β subunit restored phagocytosis and normal development. Chemoattractants released by cells or bacteria trigger typical transient actin polymerization responses in wild-type cells. In β subunit–null cells, and in a series of β subunit point mutants, these responses were impaired to a degree that correlated with the defect in phagocytosis. Image analysis of green fluorescent protein–actin transfected cells showed that β subunit– null cells were defective in reshaping the actin network into a phagocytic cup, and eventually a phagosome, in response to particle attachment. Our results indicate that signaling through heterotrimeric G proteins is required for regulating the actin cytoskeleton during phagocytic uptake, as previously shown for chemotaxis. Inhibitors of phospholipase C and intracellular Ca2+ mobilization inhibited phagocytosis, suggesting the possible involvement of these effectors in the process.

Transfer of Bacillus cereus Spores from Packaging Paper into Food

2009 ◽  
Vol 72 (11) ◽  
pp. 2236-2242 ◽  
Author(s):  
JAAKKO EKMAN ◽  
IRINA TSITKO ◽  
ASSI WEBER ◽  
CHRISTINA NIELSEN-LEROUX ◽  
DIDIER LERECLUS ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Food Packaging ◽  
Fluorescent Protein ◽  
Raw Materials ◽  
Fiber Formation ◽  
Relative Air Humidity ◽  
Commercial Paper ◽  
Dry Food ◽  
Packaging Paper ◽  
Green Fluorescent

Food packaging papers are not sterile, as the manufacturing is an open process, and the raw materials contain bacteria. We modeled the potential transfer of the Bacillus cereus spores from packaging paper to food by using a green fluorescent protein–expressing construct of Bacillus thuringiensis Bt 407Cry− [pHT315ω (papha3-gfp)], abbreviated BT-1. Paper (260 g m−2) containing BT-1 was manufactured with equipment that allowed fiber formation similar to that of full-scale manufactured paper. BT-1 adhered to pulp during papermaking and survived similar to an authentic B. cereus. Rice and chocolate were exposed to the BT-1–containing paper for 10 or 30 days at 40 or 20°C at relative air humidity of 10 to 60%. The majority of the spores remained immobilized inside the fiber web; only 0.001 to 0.03% transferred to the foods. This amount is low compared with the process hygiene criteria and densities commonly found in food, and it does not endanger food safety. To measure this, we introduced BT- 1 spores into the paper in densities of 100 to 1,000 times higher than the amounts of the B. cereus group bacteria found in commercial paper. Of BT-1 spores, 0.03 to 0.1% transferred from the paper to fresh agar surface within 5 min of contact, which is more than to food during 10 to 30 days of exposure. The findings indicate that transfer from paper to dry food is restricted to those microbes that are exposed on the paper surface and readily detectable with a contact agar method.

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 The Actin-Driven Movement and Formation of Acetylcholine Receptor Clusters

2000 ◽  
Vol 150 (6) ◽  
pp. 1321-1334 ◽  
Author(s):  
Zhengshan Dai ◽  
Xiaoyan Luo ◽  
Hongbo Xie ◽  
H. Benjamin Peng
Keyword(s):  
Actin Cytoskeleton ◽  
Acetylcholine Receptor ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Actin Binding ◽  
Heparin Binding ◽  
Actin Assembly ◽  
Achr Cluster ◽  
Green Fluorescent ◽  
Receptor Clusters

A new method was devised to visualize actin polymerization induced by postsynaptic differentiation signals in cultured muscle cells. This entails masking myofibrillar filamentous (F)-actin with jasplakinolide, a cell-permeant F-actin–binding toxin, before synaptogenic stimulation, and then probing new actin assembly with fluorescent phalloidin. With this procedure, actin polymerization associated with newly induced acetylcholine receptor (AChR) clustering by heparin-binding growth-associated molecule–coated beads and by agrin was observed. The beads induced local F-actin assembly that colocalized with AChR clusters at bead–muscle contacts, whereas both the actin cytoskeleton and AChR clusters induced by bath agrin application were diffuse. By expressing a green fluorescent protein–coupled version of cortactin, a protein that binds to active F-actin, the dynamic nature of the actin cytoskeleton associated with new AChR clusters was revealed. In fact, the motive force generated by actin polymerization propelled the entire bead-induced AChR cluster with its attached bead to move in the plane of the membrane. In addition, actin polymerization is also necessary for the formation of both bead and agrin-induced AChR clusters as well as phosphotyrosine accumulation, as shown by their blockage by latrunculin A, a toxin that sequesters globular (G)-actin and prevents F-actin assembly. These results show that actin polymerization induced by synaptogenic signals is necessary for the movement and formation of AChR clusters and implicate a role of F-actin as a postsynaptic scaffold for the assembly of structural and signaling molecules in neuromuscular junction formation.

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.

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