A novel WD40 protein, CHE-2, acts cell-autonomously in the formation of C. elegans sensory cilia

Development ◽  
1999 ◽  
Vol 126 (21) ◽  
pp. 4839-4848 ◽  
Author(s):  
M. Fujiwara ◽  
T. Ishihara ◽  
I. Katsura
Keyword(s):  
Green Fluorescent Protein ◽  
Sensory Neurons ◽  
Fluorescent Protein ◽  
Interaction Analysis ◽  
C Elegans ◽  
Protein Protein Interaction ◽  
Amino Terminal ◽  
Wd40 Domain ◽  
Green Fluorescent ◽  
Almost All

To elucidate the mechanism of sensory cilium formation, we analyzed mutants in the Caenorhabditis elegans che-2 gene. These mutants have extremely short cilia with an abnormal posterior projection, and show defects in behaviors that are mediated by ciliated sensory neurons. The che-2 gene encodes a new member of the WD40 protein family, suggesting that it acts in protein-protein interaction. Analysis of mutation sites showed that both the amino-terminal WD40 repeats and the carboxyl-terminal non-WD40 domain are necessary for the CHE-2 function. CHE-2-tagged green fluorescent protein is localized at the cilia of almost all the ciliated sensory neurons. Expression of che-2 in a subset of sensory neurons of a che-2 mutant by using a heterologous promoter resulted in restoration of the functions and cilium morphology of only the che-2-expressing neurons. Thus, che-2 acts cell-autonomously. This technique can be used in the future for determining the function of each type of che-2-expressing sensory neuron. Using green fluorescent protein, we found that the extension of cilia in wild-type animals took place at the late embryonic stage, whereas the cilia of che-2 mutant animals remained always short during development. Hence, the abnormal posterior projection is due to the inability of cilia to extend, rather than degeneration of cilia once correctly formed. Expression of che-2 in a che-2 mutant under a heat shock promoter showed that the extension of cilia, surprisingly, can occur even at the adult stage, and that such cilia can function apparently normally in behavior.

scholarly journals Robust genome editing with short single-stranded and long, partially single-stranded DNA donors in C. elegans

2018 ◽  
Author(s):  
Gregoriy A. Dokshin ◽  
Krishna S. Ghanta ◽  
Katherine M. Piscopo ◽  
Craig C. Mello
Keyword(s):  
Green Fluorescent Protein ◽  
Cost Effectiveness ◽  
Genome Editing ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
C Elegans ◽  
Multiple Loci ◽  
Green Fluorescent ◽  
Fluorescent Protein Tags ◽  
Protein Tags

AbstractCRISPR-based genome editing using ribonucleoprotein (RNP) complexes and synthetic single stranded oligodeoxynucleotide (ssODN) donors can be highly effective. However, reproducibility can vary, and precise, targeted integration of longer constructs – such as green fluorescent protein (GFP) tags remains challenging in many systems. Here we describe a streamlined and optimized editing protocol for the nematode C. elegans. We demonstrate its efficacy, flexibility, and cost-effectiveness by affinity-tagging all twelve of the Worm-specific Argonaute (WAGO) proteins in C. elegans using ssODN donors. In addition, we describe a novel PCR-based partially single-stranded “hybrid” donor design that yields high efficiency editing with large (kilobase-scale) constructs. We use these hybrid donors to introduce fluorescent protein tags into multiple loci achieving editing efficiencies that approach those previously obtained only with much shorter ssODN donors. The principals and strategies described here are likely to translate to other systems and should allow researchers to reproducibly and efficiently obtain both long and short precision genome edits.

scholarly journals Adenoviral Gene Transfer of a u-PA Receptor-binding Plasmin Inhibitor and Green Fluorescent Protein: Inhibition of Migration and Visualization of Expression

2000 ◽  
Vol 84 (09) ◽  
pp. 460-467 ◽  
Author(s):  
M. L. M. Lamfers ◽  
M. J. Wijnberg ◽  
J. M. Grimbergen ◽  
L. G. M. Huisman ◽  
M. C. Aalders ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Cell Migration ◽  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Receptor Binding ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Adenoviral Gene Transfer ◽  
Amino Terminal ◽  
Green Fluorescent

SummarySmooth muscle cell migration plays a role in the development of intimal hyperplasia. Given the established role of the plasminogen activation system in cell migration, an approach to therapy is to overexpress an inhibitor of plasmin. Therefore, an adenoviral vector was constructed encoding the hybrid protein ATF.BPTI, which contains the active domain of bovine pancreas trypsin inhibitor (BPTI), fused to ATF, the amino terminal fragment or receptor-binding domain of u-PA. Adenoviral vectors expressing ATF and BPTI individually were also constructed, and a fourth vector was constructed encoding ATF.BPTI linked by an internal ribosomal entry site to Green Fluorescent Protein (ABIG). Both the expression and functionality of the recombinant proteins were established in human vascular smooth muscle cells. Adenoviral gene transfer of ATF.BPTI inhibited SMC migration more efficiently than the expression of ATF or BPTI individually. Expression of ABIG resulted in the co-expression of ATF.BPTI and Green Fluorescent Protein, thereby providing a tool to monitor transfection efficiency and the behavior of the transfected cells.

Intracellular trafficking of emerin, the Emery-Dreifuss muscular dystrophy protein

1999 ◽  
Vol 112 (11) ◽  
pp. 1709-1719 ◽  
Author(s):  
C. Ostlund ◽  
J. Ellenberg ◽  
E. Hallberg ◽  
J. Lippincott-Schwartz ◽  
H.J. Worman
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Muscular Dystrophy ◽  
Nuclear Membrane ◽  
Fluorescent Protein ◽  
Chimeric Protein ◽  
Transmembrane Segment ◽  
Inner Nuclear Membrane ◽  
Amino Terminal ◽  
Green Fluorescent

Emerin is an integral protein of the inner nuclear membrane that is mutated or not expressed in patients with Emery-Dreifuss muscular dystrophy. Confocal immunofluorescence microscopy studies of the intracellular targeting of truncated forms of emerin, some of which are found in patients with Emery-Dreifuss muscular dystrophy, show that the nucleoplasmic, amino-terminal domain is necessary and sufficient for nuclear retention. When this domain is fused to a transmembrane segment of an integral membrane protein of the ER/plasma membrane, the chimeric protein is localized in the inner nuclear membrane. The transmembrane segment of emerin is not targeted to the inner nuclear membrane. Fluorescence photobleaching experiments of emerin fused to green fluorescent protein demonstrate that the diffusional mobility (D) of emerin is decreased in the inner nuclear membrane (D=0.10+/-0.01 microm2/second) compared to the ER membrane (D=0.32+/-0.01 microm2/second). This is in agreement with a model where integral proteins reach the inner nuclear membrane by lateral diffusion and are retained there by association with nucleoplasmic components. Some overexpressed emerin-green fluorescent protein also reaches the plasma membrane of transfected cells, where its diffusion is similar to that in the inner nuclear membrane, suggesting that emerin may also associate with non-nuclear structures.

scholarly journals eat-5 and unc-7 represent a multigene family in Caenorhabditis elegans involved in cell-cell coupling.

1996 ◽  
Vol 134 (2) ◽  
pp. 537-548 ◽  
Author(s):  
T A Starich ◽  
R Y Lee ◽  
C Panzarella ◽  
L Avery ◽  
J E Shaw
Keyword(s):  
Green Fluorescent Protein ◽  
Caenorhabditis Elegans ◽  
Gene Family ◽  
Plasma Membranes ◽  
Fluorescent Protein ◽  
Amino Acid Sequences ◽  
C Elegans ◽  
Pharyngeal Muscles ◽  
Green Fluorescent ◽  
Posterior Pharynx

The Drosophila melanogaster genes Passover and l(1)ogre and the Caenorhabditis elegans gene unc-7 define a gene family whose function is not known. We have isolated and characterized the C. elegans gene eat-5, which is required for synchronized pharyngeal muscle contractions, and find that it is a new member of this family. Simultaneous electrical and video recordings reveal that in eat-5 mutants, action potentials of muscles in the anterior and posterior pharynx are unsynchronized. Injection of carboxyfluorescein into muscles of the posterior pharynx demonstrates that all pharyngeal muscles are dye-coupled in wild-type animals; in eat-5 mutants, however, muscles of the anterior pharynx are no longer dye-coupled to posterior pharyngeal muscles. We show that a gene fusion of eat-5 to the green fluorescent protein is expressed in pharyngeal muscles. unc-7 and eat-5 are two of at least sixteen members of this family in C. elegans as determined by database searches and PCR-based screens. The amino acid sequences of five of these members in C. elegans have been deduced from cDNA sequences. Polypeptides of the family are predicted to have four transmembrane domains with cytoplasmic amino and carboxyl termini. We have constructed fusions of one of these polypeptides with beta-galactosidase and with green fluorescent protein. The fusion proteins appear to be localized in a punctate pattern at or near plasma membranes. We speculate that this gene family is required for the formation of gap junctions.

Immuno-EM Localization of GFP-tagged Yolk Proteins in C. Elegans Using Microwave Fixation

2001 ◽  
Vol 49 (8) ◽  
pp. 949-956 ◽  
Author(s):  
Marie-Christine Paupard ◽  
Agnes Miller ◽  
Barth Grant ◽  
David Hirsh ◽  
David H. Hall
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Cell Types ◽  
Protein Fusion ◽  
C Elegans ◽  
High Resolution Analysis ◽  
Resolution Analysis ◽  
Microwave Fixation ◽  
Green Fluorescent ◽  
Transgenic Strains

Because of the presence of a low-permeability cuticle covering the animal, fixation of C. elegans tissue for immunoelectron microscopy has proved very difficult. Here we applied a microwave fixation protocol to improve penetration of fixatives before postembedding immunogold labeling. Using this technique, we were able to successfully localize several components of yolk (YP170) trafficking in both wild-type and transgenic strains expressing a vitellogenin::green fluorescent protein fusion (YP170::GFP). Green fluorescent protein (GFP) and its variants are commonly used as markers to localize proteins in transgenic C. elegans using fluorescence microscopy. We have developed a robust method to localize GFP at the EM level. This procedure is applicable to the characterization of transgenic strains in which GFP is used to mark particular proteins or cell types and will undoubtedly be very useful for high-resolution analysis of marked structures. (J Histochem Cytochem 49:949–956, 2001)

Analysis of osm-6, a Gene That Affects Sensory Cilium Structure and Sensory Neuron Function in Caenorhabditis elegans

Genetics ◽  
1998 ◽  
Vol 148 (1) ◽  
pp. 187-200 ◽  
Author(s):  
Joan Collet ◽  
Caroline A Spike ◽  
Erik A Lundquist ◽  
Jocelyn E Shaw ◽  
Robert K Herman
Keyword(s):  
Green Fluorescent Protein ◽  
Caenorhabditis Elegans ◽  
Sensory Neurons ◽  
Fluorescent Protein ◽  
Fusion Gene ◽  
Genetic Mosaics ◽  
Sensory Cilia ◽  
Green Fluorescent ◽  
Neuron Function ◽  
Mammalian Protein

AbstractMutation in the Caenorhabditis elegans gene osm-6 was previously shown to result in defects in the ultrastructure of sensory cilia and defects in chemosensory and mechanosensory behaviors. We have cloned osm-6 by transposon tagging and transformation rescue and have identified molecular lesions associated with five osm-6 mutations. The osm-6 gene encodes a protein that is 40% identical in amino acid sequence to a predicted mammalian protein of unknown function. We fused osm-6 with the gene for green fluorescent protein (GFP); the fusion gene rescued the osm-6 mutant phenotype and showed accumulation of GFP in ciliated sensory neurons exclusively. The OSM-6::GFP protein was localized to cytoplasm, including processes and dendritic endings where sensory cilia are situated. Mutations in other genes known to cause ciliary defects led to changes in the appearance of OSM-6::GFP in dendritic endings or, in the case of daf-19, reduced OSM-6::GFP accumulation. We conclude from an analysis of genetic mosaics that osm-6 acts cell autonomously in affecting cilium structure.

scholarly journals A Short Peptide at the Amino Terminus of the Sendai Virus C Protein Acts as an Independent Element That Induces STAT1 Instability

2004 ◽  
Vol 78 (16) ◽  
pp. 8799-8811 ◽  
Author(s):  
Dominique Garcin ◽  
Jean-Baptiste Marq ◽  
Fréderic Iseni ◽  
Stephen Martin ◽  
Daniel Kolakofsky
Keyword(s):  
Protein C ◽  
Fluorescent Protein ◽  
Sendai Virus ◽  
C Protein ◽  
Protein Protein Interaction ◽  
Amino Terminal ◽  
Innate Antiviral Response ◽  
Α Helix ◽  
Virus C ◽  
Green Fluorescent

ABSTRACT The Sendai virus C protein acts to dismantle the interferon-induced cellular antiviral state in an MG132-sensitive manner, in part by inducing STAT1 instability. This activity of C maps to the first 23 amino acids (C1-23) of the 204-amino-acid (aa)-long protein (C1-204). C1-23 was found to act as an independent viral element that induces STAT1 instability, since this peptide fused to green fluorescent protein (C1-23/GFP) is at least as active as C1-204 in this respect. This peptide also induces the degradation of C1-23/GFP and other proteins to which it is fused. Most of C1-204, and particularly its amino-terminal half, is predicted to be structurally disordered. C1-23 as a peptide was found to be disordered by circular dichroism, and the first 11 aa have a strong potential to form an amphipathic α-helix in low concentrations of trifluoroethanol, which is thought to mimic protein-protein interaction. The critical degradation-determining sequence of C1-23 was mapped by mutation to eight residues near its N terminus: 4FLKKILKL11. All the large hydrophobic residues of 4FLKKILKL11, plus its ability to form an amphipathic α-helix, were found to be critical for STAT1 degradation. In contrast, C1-23/GFP self-degradation did not require 8ILKL11, nor the ability to form an α-helix throughout this region. Remarkably, C1-23/GFP also stimulated C1-204 degradation, and this degradation in trans required the same peptide determinants as for STAT1. Our results suggest that C1-204 coordinates its dual activities of regulating viral RNA synthesis and counteracting the host innate antiviral response by sensing both its own intracellular concentration and that of STAT1.

scholarly journals Subatomic resolution X-ray structures of green fluorescent protein

IUCrJ ◽  
2019 ◽  
Vol 6 (3) ◽  
pp. 387-400 ◽  
Author(s):  
Kiyofumi Takaba ◽  
Yang Tai ◽  
Haruhiko Eki ◽  
Hoang-Anh Dao ◽  
Yuya Hanazono ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Interaction Analysis ◽  
Lone Pair ◽  
Resonance State ◽  
Structural Features ◽  
Structural Polymorphism ◽  
Prosthetic Group ◽  
X Ray ◽  
Green Fluorescent

Green fluorescent protein (GFP) is a light-emitting protein that does not require a prosthetic group for its fluorescent activity. As such, GFP has become indispensable as a molecular tool in molecular biology. Nonetheless, there has been no subatomic elucidation of the GFP structure owing to the structural polymorphism around the chromophore. Here, subatomic resolution X-ray structures of GFP without the structural polymorphism are reported. The positions of H atoms, hydrogen-bonding network patterns and accurate geometric parameters were determined for the two protonated forms. Compared with previously determined crystal structures and theoretically optimized structures, the anionic chromophores of the structures represent the authentic resonance state of GFP. In addition, charge-density analysis based on atoms-in-molecules theory and noncovalent interaction analysis highlight weak but substantial interactions between the chromophore and the protein environment. Considered with the derived chemical indicators, the lone pair–π interactions between the chromophore and Thr62 should play a sufficient role in maintaining the electronic state of the chromophore. These results not only reveal the fine structural features that are critical to understanding the properties of GFP, but also highlight the limitations of current quantum-chemical calculations.

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