scholarly journals Molecular signatures of cell migration in C. elegans Q neuroblasts

2009 ◽  
Vol 185 (1) ◽  
pp. 77-85 ◽  
Author(s):  
Guangshuo Ou ◽  
Ronald D. Vale
Keyword(s):  
Cell Migration ◽  
Fluorescent Protein ◽  
Cell Movement ◽  
Live Animal ◽  
Α Subunit ◽  
C Elegans ◽  
Protein Levels ◽  
Neuroblast Migration ◽  
Green Fluorescent

Metazoan cell movement has been studied extensively in vitro, but cell migration in living animals is much less well understood. In this report, we have studied the Caenorhabditis elegans Q neuroblast lineage during larval development, developing live animal imaging methods for following neuroblast migration with single cell resolution. We find that each of the Q descendants migrates at different speeds and for distinct distances. By quantitative green fluorescent protein imaging, we find that Q descendants that migrate faster and longer than their sisters up-regulate protein levels of MIG-2, a Rho family guanosine triphosphatase, and/or down-regulate INA-1, an integrin α subunit, during migration. We also show that Q neuroblasts bearing mutations in either MIG-2 or INA-1 migrate at reduced speeds. The migration defect of the mig-2 mutants, but not ina-1, appears to result from a lack of persistent polarization in the direction of cell migration. Thus, MIG-2 and INA-1 function distinctly to control Q neuroblast migration in living C. elegans.

scholarly journals A laminopathic mutation disrupting lamin filament assembly causes disease-like phenotypes in Caenorhabditis elegans

2011 ◽  
Vol 22 (15) ◽  
pp. 2716-2728 ◽  
Author(s):  
Erin M. Bank ◽  
Kfir Ben-Harush ◽  
Naama Wiesel-Motiuk ◽  
Rachel Barkan ◽  
Naomi Feinstein ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Fluorescent Protein ◽  
Electron Tomography ◽  
Nuclear Periphery ◽  
Nuclear Lamina ◽  
C Elegans ◽  
Filament Assembly ◽  
Filament Structure ◽  
Green Fluorescent

Mutations in the human LMNA gene underlie many laminopathic diseases, including Emery-Dreifuss muscular dystrophy (EDMD); however, a mechanistic link between the effect of mutations on lamin filament assembly and disease phenotypes has not been established. We studied the ΔK46 Caenorhabditis elegans lamin mutant, corresponding to EDMD-linked ΔK32 in human lamins A and C. Cryo-electron tomography of lamin ΔK46 filaments in vitro revealed alterations in the lateral assembly of dimeric head-to-tail polymers, which causes abnormal organization of tetrameric protofilaments. Green fluorescent protein (GFP):ΔK46 lamin expressed in C. elegans was found in nuclear aggregates in postembryonic stages along with LEM-2. GFP:ΔK46 also caused mislocalization of emerin away from the nuclear periphery, consistent with a decreased ability of purified emerin to associate with lamin ΔK46 filaments in vitro. GFP:ΔK46 animals had motility defects and muscle structure abnormalities. These results show that changes in lamin filament structure can translate into disease-like phenotypes via altering the localization of nuclear lamina proteins, and suggest a model for how the ΔK32 lamin mutation may cause EDMD in humans.

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.

scholarly journals Akt Phosphorylation of Serine 21 on Pak1 Modulates Nck Binding and Cell Migration

2003 ◽  
Vol 23 (22) ◽  
pp. 8058-8069 ◽  
Author(s):  
Guo-Lei Zhou ◽  
Ya Zhuo ◽  
Charles C. King ◽  
Benjamin H. Fryer ◽  
Gary M. Bokoch ◽  
...  
Keyword(s):  
Cell Migration ◽  
Protein Kinases ◽  
Cellular Proliferation ◽  
Fluorescent Protein ◽  
Focal Adhesions ◽  
Adaptor Protein ◽  
Small Gtpases ◽  
Akt Phosphorylation ◽  
Green Fluorescent

ABSTRACT The p21-activated protein kinases (Paks) regulate cellular proliferation, differentiation, transformation, and survival through multiple downstream signals. Paks are activated directly by the small GTPases Rac and Cdc42 and several protein kinases including Akt and PDK-1. We found that Akt phosphorylated and modestly activated Pak1 in vitro. The major site phosphorylated by Akt on Pak1 mapped to serine 21, a site originally shown to be weakly autophosphorylated on Pak1 when Cdc42 or Rac activates it. A peptide derived from the region surrounding serine 21 was a substrate for Akt but not Pak1 in vitro, and Akt stimulated serine 21 phosphorylation on the full-length Pak1 much better than Rac did. The adaptor protein Nck binds Pak near serine 21, and its association is regulated by phosphorylation of this site. We found that either treatment of Pak1 in vitro with Akt or coexpression of constitutively active Akt with Pak1 reduced Nck binding to Pak1. In HeLa cells, green fluorescent protein-tagged Pak1 was concentrated at focal adhesions and was released when Akt was cotransfected. A peptide containing the Nck binding site of Pak1 fused to a portion of human immunodeficiency virus Tat to allow it to enter cells was used to test the functional importance of Nck/Pak binding in Akt-stimulated cell migration. This Tat-Nck peptide reduced Akt-stimulated cell migration. Together, these data suggest that Akt modulates the association of Pak with Nck to regulate cell migration.

scholarly journals Expressional and functional interactions of two Apis cerana cerana olfactory receptors

2018 ◽  
Author(s):  
Lina Guo ◽  
Huiting Zhao ◽  
Yusuo Jiang
Keyword(s):  
Fluorescent Protein ◽  
Apis Cerana ◽  
Olfactory Receptors ◽  
Surface Expression ◽  
Apis Cerana Cerana ◽  
Cell Surface Expression ◽  
Protein Levels ◽  
Green Fluorescent ◽  
Insight Into

Apis cerana cerana relies on the sensitive olfactory system to perform the foraging activities in the surrounding environment. Olfactory receptors (ORs) are a primary requirement for odorant recognition and coding. However, the molecular recognition of volatile with olfactory receptor in Apis cerana cerana is still not clear. Hence, in the present study, we achieved transient transfection and cell surface expression of Apis cerana cerana ORs (AcerOr1 and AcerOr2; AcerOr2 is orthologous to the co-receptor) in Spodoptera frugiperda Sf9 cells. The results showed that both mRNA and protein levels of AcerOr1 and AcerOr2 were drastically reduced when treated with their respective double stranded (ds) RNA compared to those in the control and double-stranded green fluorescent protein (dsGFP)-treated cells. The response to Ca2+ using 33 volatile odorants indicated that the molecular receptive range of AcerOr2 narrowly responded to N-(4-ethylphenyl)-2-((4-ethyl-5-(3-pyridinyl)-4H-1, 2, 4- triazol-3-yl) thio) acetamide (VUAA1) whereas AcerOr1 was sensitive to eugenol, lauric acid, ocimene, 1-nonanol, linolenic acid, hexyl acetate, undecanoic acid, 1-octyl alcohol, and nerol, and it revealed distinct changes in the dose-response curve. We discovered ligands that were useful for probing receptor activity during odor stimulation and validated three of them using an electroantennography (EAG) assay. The response increased with the concentration of the odorant. Further, both AcerOr1 and AcerOr2 knockdowns exhibited significantly reduced intracellular Ca2+ levels in response to the corresponding ligands in vitro. Overall, the present study provides insight into the mechanism of olfactory discrimination in Apis cerana cerana.

scholarly journals Expressional and functional interactions of two Apis cerana cerana olfactory receptors

2018 ◽  
Author(s):  
Lina Guo ◽  
Huiting Zhao ◽  
Yusuo Jiang
Keyword(s):  
Fluorescent Protein ◽  
Apis Cerana ◽  
Olfactory Receptors ◽  
Surface Expression ◽  
Apis Cerana Cerana ◽  
Cell Surface Expression ◽  
Protein Levels ◽  
Green Fluorescent ◽  
Insight Into

Apis cerana cerana relies on the sensitive olfactory system to perform the foraging activities in the surrounding environment. Olfactory receptors (ORs) are a primary requirement for odorant recognition and coding. However, the molecular recognition of volatile with olfactory receptor in Apis cerana cerana is still not clear. Hence, in the present study, we achieved transient transfection and cell surface expression of Apis cerana cerana ORs (AcerOr1 and AcerOr2; AcerOr2 is orthologous to the co-receptor) in Spodoptera frugiperda Sf9 cells. The results showed that both mRNA and protein levels of AcerOr1 and AcerOr2 were drastically reduced when treated with their respective double stranded (ds) RNA compared to those in the control and double-stranded green fluorescent protein (dsGFP)-treated cells. The response to Ca2+ using 33 volatile odorants indicated that the molecular receptive range of AcerOr2 narrowly responded to N-(4-ethylphenyl)-2-((4-ethyl-5-(3-pyridinyl)-4H-1, 2, 4- triazol-3-yl) thio) acetamide (VUAA1) whereas AcerOr1 was sensitive to eugenol, lauric acid, ocimene, 1-nonanol, linolenic acid, hexyl acetate, undecanoic acid, 1-octyl alcohol, and nerol, and it revealed distinct changes in the dose-response curve. We discovered ligands that were useful for probing receptor activity during odor stimulation and validated three of them using an electroantennography (EAG) assay. The response increased with the concentration of the odorant. Further, both AcerOr1 and AcerOr2 knockdowns exhibited significantly reduced intracellular Ca2+ levels in response to the corresponding ligands in vitro. Overall, the present study provides insight into the mechanism of olfactory discrimination in Apis cerana cerana.

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.

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.

scholarly journals CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Peng-Fei Fu ◽  
Xuan Cheng ◽  
Bing-Qian Su ◽  
Li-Fang Duan ◽  
Cong-Rong Wang ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Antiviral Agents ◽  
Firefly Luciferase ◽  
Inhibitory Effect ◽  
Economic Losses ◽  
Green Fluorescent ◽  
Swine Herds

AbstractPseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.

scholarly journals Paracetamol modulates biofilm formation in Staphylococcus aureus clonal complex 8 strains

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Andi R. Sultan ◽  
Kirby R. Lattwein ◽  
Nicole A. Lemmens-den Toom ◽  
Susan V. Snijders ◽  
Klazina Kooiman ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Fluorescent Protein ◽  
Clonal Complex ◽  
Regulatory Pathways ◽  
Immune Modulator ◽  
Viability Assay ◽  
Analgesic Agents ◽  
Green Fluorescent

AbstractStaphylococcus aureus biofilms are a major problem in modern healthcare due to their resistance to immune system defenses and antibiotic treatments. Certain analgesic agents are able to modulate S. aureus biofilm formation, but currently no evidence exists if paracetamol, often combined with antibiotic treatment, also has this effect. Therefore, we aimed to investigate if paracetamol can modulate S. aureus biofilm formation. Considering that certain regulatory pathways for biofilm formation and virulence factor production by S. aureus are linked, we further investigated the effect of paracetamol on immune modulator production. The in vitro biofilm mass of 21 S. aureus strains from 9 genetic backgrounds was measured in the presence of paracetamol. Based on biofilm mass quantity, we further investigated paracetamol-induced biofilm alterations using a bacterial viability assay combined with N-Acetylglucosamine staining. Isothermal microcalorimetry was used to monitor the effect of paracetamol on bacterial metabolism within biofilms and green fluorescent protein (GFP) promoter fusion technology for transcription of staphylococcal complement inhibitor (SCIN). Clinically relevant concentrations of paracetamol enhanced biofilm formation particularly among strains belonging to clonal complex 8 (CC8), but had minimal effect on S. aureus planktonic growth. The increase of biofilm mass can be attributed to the marked increase of N-Acetylglucosamine containing components of the extracellular matrix, presumably polysaccharide intercellular adhesion. Biofilms of RN6390A (CC8) showed a significant increase in the immune modulator SCIN transcription during co-incubation with low concentrations of paracetamol. Our data indicate that paracetamol can enhance biofilm formation. The clinical relevance needs to be further investigated.

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