scholarly journals In Vivo Observations of Myosin II Dynamics Support a Role in Rear Retraction

1999 ◽  
Vol 10 (5) ◽  
pp. 1309-1323 ◽  
Author(s):  
Patricia A. Clow ◽  
James G. McNally
Keyword(s):  
Heavy Chain ◽  
Fluorescent Protein ◽  
Rotational Velocity ◽  
Myosin Ii ◽  
Cell Mass ◽  
Cell Movement ◽  
Posterior Cortex ◽  
Guidance Cues ◽  
Green Fluorescent

To investigate myosin II function in cell movement within a cell mass, we imaged green fluorescent protein-myosin heavy chain (GFP-MHC) cells moving within the tight mound of Dictyostelium discoideum. In the posterior cortex of cells undergoing rotational motion around the center of the mound, GFP-MHC cyclically formed a “C,” which converted to a spot as the cell retracted its rear. Consistent with an important role for myosin in rotation, cells failed to rotate when they lacked the myosin II heavy chain (MHC−) or when they contained predominantly monomeric myosin II (3xAsp). In cells lacking the myosin II regulatory light chain (RLC−), rotation was impaired and eventually ceased. These rotational defects reflect a mechanical problem in the 3xAsp and RLC− cells, because these mutants exhibited proper rotational guidance cues. MHC− cells exhibited disorganized and erratic rotational guidance cues, suggesting a requirement for the MHC in organizing these signals. However, the MHC− cells also exhibited mechanical defects in rotation, because they still moved aberrantly when seeded into wild-type mounds with proper rotational guidance cues. The mechanical defects in rotation may be mediated by the C-to-spot, because RLC− cells exhibited a defective C-to-spot, including a slower C-to-spot transition, consistent with this mutant’s slower rotational velocity.

scholarly journals Fluorescently Tagged Potato virus Y: A Versatile Tool for Functional Analysis of Plant-Virus Interactions

2015 ◽  
Vol 28 (7) ◽  
pp. 739-750 ◽  
Author(s):  
Matevz Rupar ◽  
Florence Faurez ◽  
Michel Tribodet ◽  
Ion Gutiérrez-Aguirre ◽  
Agnès Delaunay ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Potato Virus ◽  
Plant Virus ◽  
Potato Virus Y ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Cell Movement ◽  
Long Distance ◽  
Green Fluorescent

Potato virus Y (PVY) is an economically important plant virus that infects Solanaceous crops such as tobacco and potato. To date, studies into the localization and movement of PVY in plants have been limited to detection of viral RNA or proteins ex vivo. Here, a PVY N605 isolate was tagged with green fluorescent protein (GFP), characterized and used for in vivo tracking. In Nicotiana tabacum cv. Xanthi, PVY N605-GFP was biologically comparable to nontagged PVY N605, stable through three plant-to-plant passages and persisted for four months in infected plants. GFP was detected before symptoms and fluorescence intensity correlated with PVY RNA concentrations. PVY N605-GFP provided in vivo tracking of long-distance movement, allowing estimation of the cell-to-cell movement rate of PVY in N. tabacum cv. Xanthi (7.1 ± 1.5 cells per hour). PVY N605-GFP was adequately stable in Solanum tuberosum cvs. Désirée and NahG-Désirée and able to infect S. tuberosum cvs. Bintje and Bea, Nicotiana benthamiana, and wild potato relatives. PVY N605-GFP is therefore a powerful tool for future studies of PVY-host interactions, such as functional analysis of viral and plant genes involved in viral movement.

scholarly journals Neuroendocrine Plasticity in the Anterior Pituitary: Gonadotropin-Releasing Hormone-Mediated Movement in Vitro and in Vivo

Endocrinology ◽  
2007 ◽  
Vol 148 (4) ◽  
pp. 1736-1744 ◽  
Author(s):  
Amy M. Navratil ◽  
J. Gabriel Knoll ◽  
Jennifer D. Whitesell ◽  
Stuart A. Tobet ◽  
Colin M. Clay
Keyword(s):  
Pituitary Gland ◽  
Anterior Pituitary ◽  
Fluorescent Protein ◽  
Cell Movement ◽  
Pituitary Cells ◽  
Rous Sarcoma ◽  
Cytoskeleton Disruption ◽  
Green Fluorescent

The secretion of LH is cued by the hypothalamic neuropeptide, GnRH. After delivery to the anterior pituitary gland via the hypothalamic-pituitary portal vasculature, GnRH binds to specific high-affinity receptors on the surface of gonadotrope cells and stimulates synthesis and secretion of the gonadotropins, FSH, and LH. In the current study, GnRH caused acute and dramatic changes in cellular morphology in the gonadotrope-derived αT3-1 cell line, which appeared to be mediated by engagement of the actin cytoskeleton; disruption of actin with jasplakinolide abrogated cell movement and GnRH-induced activation of ERK. In live murine pituitary slices infected with an adenovirus-containing Rous sarcoma virus-green fluorescent protein, selected cells responded to GnRH by altering their cellular movements characterized by both formation and extension of cell processes and, surprisingly, spatial repositioning. Consistent with the latter observation, GnRH stimulation increased the migration of dissociated pituitary cells in transwell chambers. Our data using live pituitary slices are a striking example of neuropeptide-evoked movements of cells outside the central nervous system and in a mature peripheral endocrine organ. These findings call for a fundamental change in the current dogma of simple passive diffusion of LH from gonadotropes to capillaries in the pituitary gland.

Cell-sorting in aggregates of Dictyostelium discoideum

1999 ◽  
Vol 112 (22) ◽  
pp. 3923-3929 ◽  
Author(s):  
A. Nicol ◽  
W. Rappel ◽  
H. Levine ◽  
W.F. Loomis
Keyword(s):  
Cell Sorting ◽  
Fluorescent Protein ◽  
Cell Mass ◽  
Three Dimensional ◽  
Cell Movement ◽  
Time Lapse ◽  
Cell Types ◽  
Cell Type ◽  
Prespore Cell ◽  
Green Fluorescent

When Dictyostelium cells are induced to develop between a coverslip and a layer of agarose, they aggregate normally into groups containing up to a thousand cells but are then constrained to form disks only a few cells thick that appear to be equivalent to the three-dimensional mounds formed on top of agarose. Such vertically restricted aggregates frequently develop into elongated motile structures, the flattened equivalent of three-dimensional slugs. The advantage of using this system is that the restricted z-dimension enables direct microscopic visualization of most of the cells in the developing structure. We have used time lapse digital fluorescence microscopy of Dictyostelium strains expressing green fluorescent protein (GFP) under the control of either prestalk or prespore specific promoters to follow cell sorting in these flattened mounds. We find that prestalk and prespore cells expressing GFP arise randomly in early aggregates and then rotate rapidly around the disk mixed with the other cell type. After a few hours, the cell types sort out by a process which involves striking changes in relative cell movement. Once sorted, the cell types move independently of each other showing very little heterotypic adhesion. When a group of prestalk cells reaches the edge of the disk, it moves out and is followed by the prespore cell mass. We suggest that sorting may result from cell type specific changes in adhesion and the consequent disruption of movement in the files of cells that are held together by end-to-end adhesion.

scholarly journals Pituitary Corticotroph Ontogeny and Regulation in Transgenic Zebrafish

2003 ◽  
Vol 17 (5) ◽  
pp. 959-966 ◽  
Author(s):  
Ning-Ai Liu ◽  
Haigen Huang ◽  
Zhongan Yang ◽  
Wiebke Herzog ◽  
Matthias Hammerschmidt ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Cell Mass ◽  
Gene Promoter ◽  
Genetic System ◽  
Regulatory Elements ◽  
Transgenic Zebrafish ◽  
Posterior Pituitary ◽  
Gfp Expression ◽  
Green Fluorescent

Abstract We characterized zebrafish proopiomelanocortin (POMC) gene promoter, and sequence analysis revealed that the promoter contains regulatory elements conserved among vertebrate species. To monitor the ontogeny of the pituitary POMC lineage in living vertebrates, we generated transgenic zebrafish expressing green fluorescent protein (GFP) driven by the POMC promoter. Zebrafish POMC-GFP is first expressed asymmetrically as two bilateral groups of cells most anterior to the neural ridge midline at 18–20 h post fertilization (hpf). POMC-GFP-positive cells then fuse into a single-cell mass within the pituitary anlage after 24 hpf and subsequently organize as distinct anterior and posterior domains between 48 and 64 hpf. Immunohistochemical studies with ACTH and αMSH antisera showed that POMC-GFP was mainly targeted to both anterior and posterior pituitary corticotrophs, whereas posterior pituitary region melanotrophs did not express GFP. To determine in vivo zebrafish corticotroph responses, dexamethasone (10−5m) was added to live embryos, which selectively suppressed POMC-GFP expression in the anterior group of corticotrophs, suggesting a distinct domain that is responsive to glucocorticoid feedback. Transgenic zebrafish with specific POMC-GFP expression in pituitary corticotrophs offers a powerful genetic system for in vivo study of vertebrate corticotroph lineage development.

scholarly journals Sequential Assembly of Myosin II, an IQGAP-like Protein, and Filamentous Actin to a Ring Structure Involved in Budding Yeast Cytokinesis

1998 ◽  
Vol 140 (2) ◽  
pp. 355-366 ◽  
Author(s):  
John Lippincott ◽  
Rong Li
Keyword(s):  
Cell Cycle ◽  
Fluorescent Protein ◽  
Budding Yeast ◽  
Sequence Similarity ◽  
Myosin Ii ◽  
Ring Structure ◽  
Binding Activity ◽  
Filamentous Actin ◽  
Green Fluorescent

We have identified a Saccharomyces cerevisiae protein, Cyk1p, that exhibits sequence similarity to the mammalian IQGAPs. Gene disruption of Cyk1p results in a failure in cytokinesis without affecting other events in the cell cycle. Cyk1p is diffused throughout most of the cell cycle but localizes to a ring structure at the mother–bud junction after the initiation of anaphase. This ring contains filamentous actin and Myo1p, a myosin II homologue. In vivo observation with green fluorescent protein–tagged Myo1p showed that the ring decreases drastically in size during cell division and therefore may be contractile. These results indicate that cytokinesis in budding yeast is likely to involve an actomyosin-based contractile ring. The assembly of this ring occurs in temporally distinct steps: Myo1p localizes to a ring that overlaps the septins at the G1-S transition slightly before bud emergence; Cyk1p and actin then accumulate in this ring after the activation of the Cdc15 pathway late in mitosis. The localization of myosin is abolished by a mutation in Cdc12p, implicating a role for the septin filaments in the assembly of the actomyosin ring. The accumulation of actin in the cytokinetic ring was not observed in cells depleted of Cyk1p, suggesting that Cyk1p plays a role in the recruitment of actin filaments, perhaps through a filament-binding activity similar to that demonstrated for mammalian IQGAPs.

Recapitulating the Expression Pattern of Jak2 in Transgenic Zebrafish Using Jak2 Promoters and a GFP Reporter Gene.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1156-1156
Author(s):  
Jing Zhang ◽  
Hui-Feng Lin ◽  
Robert I. Handin
Keyword(s):  
Green Fluorescent Protein ◽  
Expression Pattern ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Cell Mass ◽  
Translation Initiation Site ◽  
Transgenic Zebrafish ◽  
Intermediate Cell ◽  
Green Fluorescent

Abstract The non-receptor tyrosine kinase Jak2 plays an important role in regulating erythro and thrombopoiesis. There has been intense interest in Jak2 since the observation that an activating mutation V617F is present in almost all patients with Polycythemia vera and many patients with Essential Thrombocytosis and Myelofibrosis. The analysis of Jak2 function in vivo has been limited as the murine jak2 knockout is lethal at day 10.5 of embryogenesis. Our laboratory has taken advantage of an ancestral partial duplication of the zebrafish genome, which has yielded two jak2 alleles --- jak2a and jak2b to study jak2 expression and function. Whole mount in situ hybridization studies confirm that the jak2a gene is only expressed in hematopoietic tissues, while jak2b is expressed in the developing lens and nephritic ducts. We have cloned and characterized the full-length jak2a and jak2b cDNAs and characterized the jak2a and 2b genomic loci. The jak2b gene has 24 exons spanning 79kb of genomic DNA. We amplified a 4kb zebrafish genomic fragment upstream of the first exon of the jak2b gene and linked it to the enhanced green fluorescent protein (EGFP) cDNA reporter and then microinjected the construct into single-cell zebrafish embryos. At 24 hours post fertilization (hpf), we observed fluorescence in the lens and nephritic ducts of developing embryos, with some expression in skin, muscle and notochord. The jak2a gene locus is complex as the jak2a gene is linked to a gene of unknown function, STARD4, in a head-to-tail manner with a small intergenic region of 1kb. As observed with jak2b, the first exon contains the jak2b 5′-UTR and the second exon contains the translation initiation site. We cloned a 1.9kb DNA fragment that included exons 1 and 2 the intervening first intron and an additional 800bp upstream of exon 1. This 1.9 kb promoter fragment was sufficient to drive expression of enhanced green fluorescent protein (EGFP) in injected embryos in a manner that recapitulated the native expression pattern of jak2a. In injected embryos 24hpf, GFP+ cells were present in the anterior intermediate cell mass (ICM) and the lens. Fluorescent circulating blood cells, largely erythrocytes, were detected in 12 of 124 microinjected embryos 48 hours after fertilization. The jak2-EGFP transgenic zebrafish strains should be useful in the study of normal and pathologic hematopoiesis and in future studies of the pathogenesis of the MPDs.

scholarly journals Attenuation of Phospholipid Signaling Provides a Novel Mechanism for the Action of Valproic Acid

2007 ◽  
Vol 6 (6) ◽  
pp. 899-906 ◽  
Author(s):  
Xuehua Xu ◽  
Annette Müller-Taubenberger ◽  
Kathryn E. Adley ◽  
Nadine Pawolleck ◽  
Vivian W. Y. Lee ◽  
...  
Keyword(s):  
Valproic Acid ◽  
Fluorescent Protein ◽  
Cell Movement ◽  
Acute Effect ◽  
Phosphatidylinositol 3 Kinase ◽  
Phospholipid Signaling ◽  
The Social ◽  
Green Fluorescent ◽  
Rat Nerve

ABSTRACT Valproic acid (VPA) is used to treat epilepsy and bipolar disorder and to prevent migraine. It is also undergoing trials for cancer therapy. However, the biochemical and molecular biological actions of VPA are poorly understood. Using the social amoeba Dictyostelium discoideum, we show that an acute effect of VPA is the inhibition of chemotactic cell movement, a process partially dependent upon phospholipid signaling. Analysis of this process shows that VPA attenuates the signal-induced translocation of PHCrac-green fluorescent protein from cytosol to membrane, suggesting the inhibition of phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) production. Direct labeling of lipids in vivo also shows a reduction in PIP and PIP2 phosphorylation following VPA treatment. We further show that VPA acutely reduces endocytosis and exocytosis—processes previously shown to be dependent upon PIP3 production. These results suggest that in Dictyostelium, VPA rapidly attenuates phospholipid signaling to reduce endocytic trafficking. To examine this effect in a mammalian model, we also tested depolarization-dependent neurotransmitter release in rat nerve terminals, and we show that this process is also suppressed upon application of VPA and an inhibitor of phosphatidylinositol 3-kinase. Although a more comprehensive analysis of the effect of VPA on lipid signaling will be necessary in mammalian systems, these results suggest that VPA may function to reduce phospholipid signaling processes and thus may provide a novel therapeutic effect for this drug.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

scholarly journals Development of a Fluorescent Tool for Studying Legionella bozemanae Intracellular Infection

2021 ◽  
Vol 9 (2) ◽  
pp. 379
Author(s):  
Breanne M. Head ◽  
Christopher I. Graham ◽  
Teassa MacMartin ◽  
Yoav Keynan ◽  
Ann Karen C. Brassinga
Keyword(s):  
Growth Kinetics ◽  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Disease Incidence ◽  
Acanthamoeba Castellanii ◽  
Infection Model ◽  
Intracellular Infection ◽  
Green Fluorescent

Legionnaires’ disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Legionella pneumophila. Nominally a parasite of protozoa, L. pneumophila can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. L. pneumophila pathogenesis has been well characterized; however, little is known about the >25 different Legionella spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an L. bozemanae clinical isolate using approaches previously established for L. pneumophila investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the L. bozemanae presence in the Acanthamoeba castellanii protozoan infection model. As comparative controls, L. pneumophila strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the Legionella parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing Legionella spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating Legionella non-pneumophila spp. in real time.

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