scholarly journals Functional Overlap Between the mec-8 Gene and Five sym Genes in Caenorhabditis elegans

Genetics ◽  
1999 ◽  
Vol 153 (1) ◽  
pp. 117-134
Author(s):  
Andrew G Davies ◽  
Caroline A Spike ◽  
Jocelyn E Shaw ◽  
Robert K Herman
Keyword(s):  
Caenorhabditis Elegans ◽  
Rna Splicing ◽  
Fluorescent Protein ◽  
Synthetic Lethality ◽  
Signal Sequence ◽  
Close Relative ◽  
Loss Of Function ◽  
Muscle Attachment ◽  
Body Muscle ◽  
Green Fluorescent

Abstract Earlier work showed that the Caenorhabditis elegans gene mec-8 encodes a regulator of alternative RNA splicing and that mec-8 null mutants have defects in sensory neurons and body muscle attachment but are generally viable and fertile. We have used a genetic screen to identify five mutations in four genes, sym-1–sym-4, that are synthetically lethal with mec-8 loss-of-function mutations. The phenotypes of sym single mutants are essentially wild type. mec-8; sym-1 embryos arrest during embryonic elongation and exhibit defects in the attachment of body muscle to extracellular cuticle. sym-1 can encode a protein containing a signal sequence and 15 contiguous leucine-rich repeats. A fusion of sym-1 and the gene for green fluorescent protein rescued the synthetic lethality of mec-8; sym-1 mutants; the fusion protein was secreted from the apical hypodermal surface of the embryo. We propose that SYM-1 helps to attach body muscle to the extracellular cuticle and that another gene that is dependent upon mec-8 for pre-mRNA processing overlaps functionally with sym-1. RNA-mediated interference experiments indicated that a close relative of sym-1 functionally overlaps both sym-1 and mec-8 in affecting muscle attachment. sym-2, sym-3, and sym-4 appear to provide additional functions that are essential in the absence of mec-8(+).

scholarly journals ACT-5 Is an Essential Caenorhabditis elegans Actin Required for Intestinal Microvilli Formation

2005 ◽  
Vol 16 (7) ◽  
pp. 3247-3259 ◽  
Author(s):  
A. J. MacQueen ◽  
J. J. Baggett ◽  
N. Perumov ◽  
R. A. Bauer ◽  
T. Januszewski ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Fluorescent Protein ◽  
Intestinal Cell ◽  
Loss Of Function ◽  
Normal Morphology ◽  
C Elegans ◽  
Immuno Electron Microscopy ◽  
Actin Isoform ◽  
Green Fluorescent ◽  
Fluorescent Protein Reporter

Investigation of Caenorhabditis elegans act-5 gene function revealed that intestinal microvillus formation requires a specific actin isoform. ACT-5 is the most diverged of the five C. elegans actins, sharing only 93% identity with the other four. Green fluorescent protein reporter and immunofluorescence analysis indicated that act-5 gene expression is limited to microvillus-containing cells within the intestine and excretory systems and that ACT-5 is apically localized within intestinal cells. Animals heterozygous for a dominant act-5 mutation looked clear and thin and grew slowly. Animals homozygous for either the dominant act-5 mutation, or a recessive loss of function mutant, exhibited normal morphology and intestinal cell polarity, but died during the first larval stage. Ultrastructural analysis revealed a complete loss of intestinal microvilli in homozygous act-5 mutants. Forced expression of ACT-1 under the control of the act-5 promoter did not rescue the lethality of the act-5 mutant. Together with immuno-electron microscopy experiments that indicated ACT-5 is enriched within microvilli themselves, these results suggest a microvillus-specific function for act-5, and further, they raise the possibility that specific actins may be specialized for building microvilli and related structures.

scholarly journals The Caenorhabditis elegans paxillin orthologue, PXL-1, is required for pharyngeal muscle contraction and for viability

2011 ◽  
Vol 22 (14) ◽  
pp. 2551-2563 ◽  
Author(s):  
Adam Warner ◽  
Hiroshi Qadota ◽  
Guy M. Benian ◽  
A. Wayne Vogl ◽  
Donald G. Moerman
Keyword(s):  
Caenorhabditis Elegans ◽  
Body Wall ◽  
Fluorescent Protein ◽  
Repeat Unit ◽  
Body Wall Muscle ◽  
Loss Of Function ◽  
Pharyngeal Muscle ◽  
Adhesion Sites ◽  
Pharyngeal Muscles ◽  
Green Fluorescent

We have identified the gene C28H8.6 (pxl-1) as the Caenorhabditis elegans orthologue of vertebrate paxillin. PXL-1 contains the four C-terminal LIM domains conserved in paxillin across all species and three of the five LD motifs found in the N-terminal half of most paxillins. In body wall muscle, PXL-1 antibodies and a full-length green fluorescent protein translational fusion localize to adhesion sites in the sarcomere, the functional repeat unit in muscle responsible for contraction. PXL-1 also localizes to ring-shaped structures near the sarcolemma in pharyngeal muscle corresponding to podosome-like sites of actin attachment. Our analysis of a loss-of-function allele of pxl-1, ok1483, shows that loss of paxillin leads to early larval arrested animals with paralyzed pharyngeal muscles and eventual lethality, presumably due to an inability to feed. We rescued the mutant phenotype by expressing paxillin solely in the pharynx and found that these animals survived and are essentially wild type in movement and body wall muscle structure. This indicates a differential requirement for paxillin in these two types of muscle. In pharyngeal muscle it is essential for contraction, whereas in body wall muscle it is dispensable for filament assembly, sarcomere stability, and ultimately movement.

scholarly journals Dynamic Regulation of Caveolin-1 Trafficking in the Germ Line and Embryo of Caenorhabditis elegans

2006 ◽  
Vol 17 (7) ◽  
pp. 3085-3094 ◽  
Author(s):  
Ken Sato ◽  
Miyuki Sato ◽  
Anjon Audhya ◽  
Karen Oegema ◽  
Peter Schweinsberg ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Plasma Membrane ◽  
Caenorhabditis Elegans ◽  
Fluorescent Protein ◽  
Germ Line ◽  
Cortical Granule ◽  
Major Protein ◽  
Dynamic Regulation ◽  
Caveolin 1 ◽  
Green Fluorescent

Caveolin is the major protein component required for the formation of caveolae on the plasma membrane. Here we show that trafficking of Caenorhabditis elegans caveolin-1 (CAV-1) is dynamically regulated during development of the germ line and embryo. In oocytes a CAV-1-green fluorescent protein (GFP) fusion protein is found on the plasma membrane and in large vesicles (CAV-1 bodies). After ovulation and fertilization the CAV-1 bodies fuse with the plasma membrane in a manner reminiscent of cortical granule exocytosis as described in other species. Fusion of CAV-1 bodies with the plasma membrane appears to be regulated by the advancing cell cycle, and not fertilization per se, because fusion can proceed in spe-9 fertilization mutants but is blocked by RNA interference–mediated knockdown of an anaphase-promoting complex component (EMB-27). After exocytosis, most CAV-1-GFP is rapidly endocytosed and degraded within one cell cycle. CAV-1 bodies in oocytes appear to be produced by the Golgi apparatus in an ARF-1–dependent, clathrin-independent, mechanism. Conversely endocytosis and degradation of CAV-1-GFP in embryos requires clathrin, dynamin, and RAB-5. Our results demonstrate that the distribution of CAV-1 is highly dynamic during development and provides new insights into the sorting mechanisms that regulate CAV-1 localization.

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 Double-Labeled Rabies Virus: Live Tracking of Enveloped Virus Transport

2007 ◽  
Vol 82 (1) ◽  
pp. 237-245 ◽  
Author(s):  
Yvonne Klingen ◽  
Karl-Klaus Conzelmann ◽  
Stefan Finke
Keyword(s):  
Cell Surface ◽  
Rabies Virus ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Surface Membrane ◽  
Fusion Construct ◽  
Virus Particles ◽  
Enveloped Virus ◽  
Green Fluorescent

ABSTRACT Here we describe a strategy to fluorescently label the envelope of rabies virus (RV), of the Rhabdoviridae family, in order to track the transport of single enveloped viruses in living cells. Red fluorescent proteins (tm-RFP) were engineered to comprise the N-terminal signal sequence and C-terminal transmembrane spanning and cytoplasmic domain sequences of the RV glycoprotein (G). Two variants of tm-RFP were transported to and anchored in the cell surface membrane, independent of glycosylation. As shown by confocal microscopy, tm-RFP colocalized at the cell surface with the RV matrix and G protein and was incorporated into G gene-deficient virus particles. Recombinant RV expressing the membrane-anchored tm-RFP in addition to G yielded infectious viruses with mosaic envelopes containing both tm-RFP and G. Viable double-labeled virus particles comprising a red fluorescent envelope and a green fluorescent ribonucleoprotein were generated by expressing in addition an enhanced green fluorescent protein-phosphoprotein fusion construct (S. Finke, K. Brzozka, and K. K. Conzelmann, J. Virol. 78:12333-12343, 2004). Individual enveloped virus particles were observed under live cell conditions as extracellular particles and inside endosomal vesicles. Importantly, double-labeled RVs were transported in the retrograde direction over long distances in neurites of in vitro-differentiated NS20Y neuroblastoma cells. This indicates that the typical retrograde axonal transport of RV to the central nervous system involves neuronal transport vesicles in which complete enveloped RV particles are carried as a cargo.

A conditional mouse line for lineage tracing of Sox9 loss-of-function cells using enhanced green fluorescent protein

2013 ◽  
Vol 35 (12) ◽  
pp. 1991-1996 ◽  
Author(s):  
Sumantra Chatterjee ◽  
Petra Kraus ◽  
V. Sivakamasundari ◽  
Xing Xing ◽  
Sook Peng Yap ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Lineage Tracing ◽  
Mouse Line ◽  
Loss Of Function ◽  
Green Fluorescent

scholarly journals Viral Preprotoxin Signal Sequence Allows Efficient Secretion of Green Fluorescent Protein by Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe

2004 ◽  
Vol 70 (2) ◽  
pp. 961-966 ◽  
Author(s):  
Antje Eiden-Plach ◽  
Tatjana Zagorc ◽  
Tanja Heintel ◽  
Yvonne Carius ◽  
Frank Breinig ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Green Fluorescent Protein ◽  
Pichia Pastoris ◽  
Schizosaccharomyces Pombe ◽  
Protein Secretion ◽  
Candida Glabrata ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Foreign Protein ◽  
Green Fluorescent

ABSTRACT Besides its importance as model organism in eukaryotic cell biology, yeast species have also developed into an attractive host for the expression, processing, and secretion of recombinant proteins. Here we investigated foreign protein secretion in four distantly related yeasts (Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe) by using green fluorescent protein (GFP) as a reporter and a viral secretion signal sequence derived from the K28 preprotoxin (pptox), the precursor of the yeast K28 virus toxin. In vivo expression of GFP fused to the N-terminal pptox leader sequence and/or expression of the entire pptox gene was driven either from constitutive (PGK1 and TPI1) or from inducible and/or repressible (GAL1, AOX1, and NMT1) yeast promoters. In each case, GFP entered the secretory pathway of the corresponding host cell; confocal fluorescence microscopy as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of cell-free culture supernatants confirmed that GFP was efficiently secreted into the culture medium. In addition to the results seen with GFP, the full-length viral pptox was correctly processed in all four yeast genera, leading to the secretion of a biologically active virus toxin. Taken together, our data indicate that the viral K28 pptox signal sequence has the potential for being used as a unique tool in recombinant protein production to ensure efficient protein secretion in yeast.

scholarly journals Analyzing Defects in the Caenorhabditis elegans Nervous System Using Organismal and Cell Biological Approaches

2002 ◽  
Vol 1 (1) ◽  
pp. 18-25 ◽  
Author(s):  
Megan Guziewicz ◽  
Toni Vitullo ◽  
Bethany Simmons ◽  
Rebecca Eustance Kohn
Keyword(s):  
Nervous System ◽  
Caenorhabditis Elegans ◽  
Synaptic Vesicle ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Vesicle Transport ◽  
Laboratory Exercise ◽  
Cellular Analysis ◽  
Green Fluorescent ◽  
The Impact

The goal of this laboratory exercise is to increase student understanding of the impact of nervous system function at both the organismal and cellular levels. This inquiry-based exercise is designed for an undergraduate course examining principles of cell biology. After observing the movement of Caenorhabditis elegans with defects in their nervous system, students examine the structure of the nervous system to categorize the type of defect. They distinguish between defects in synaptic vesicle transport and defects in synaptic vesicle fusion with membranes. The synaptic vesicles are tagged with green fluorescent protein (GFP), simplifying cellular analysis. The expected outcome of this experiment is that students will better understand the concepts of vesicle transport, neurotransmitter release, GFP, and the relation between the nervous system and behavior.

scholarly journals Ecological Basis of the Interaction betweenPseudozyma flocculosaand Powdery Mildew Fungi

2010 ◽  
Vol 77 (3) ◽  
pp. 926-933 ◽  
Author(s):  
Walid Hammami ◽  
Candy Quiroga Castro ◽  
Wilfried Rémus-Borel ◽  
Caroline Labbé ◽  
Richard R. Bélanger
Keyword(s):  
Powdery Mildew ◽  
Fluorescent Protein ◽  
Close Relative ◽  
Tomato Plants ◽  
Powdery Mildews ◽  
Biocontrol Activity ◽  
Cellobiose Lipids ◽  
Powdery Mildew Fungi ◽  
Green Fluorescent ◽  
Ecological Basis

ABSTRACTIn this work, we sought to understand how glycolipid production and the availability of nutrients could explain the ecology ofPseudozyma flocculosaand its biocontrol activity. For this purpose, we compared the development ofP. flocculosato that of a close relative, the plant pathogenUstilago maydis, under different environmental conditions. This approach was further supported by measuring the expression ofcyp1, a pivotal gene in the synthesis of unique antifungal cellobiose lipids of both fungi. On healthy cucumber and tomato plants, the expression ofcyp1remained unchanged over time inP. flocculosaand was undetected inU. maydis. At the same time, green fluorescent protein (GFP) strains of both fungi showed only limited green fluorescence on control leaves. On powdery mildew-infected cucumber leaves,P. flocculosainduced a complete collapse of the pathogen colonies, but glycolipid production, as studied bycyp1expression, was still comparable to that of controls. In complete contrast,cyp1was upregulated nine times whenP. flocculosawas applied toBotrytis cinerea-infected leaves, but the biocontrol fungus did not develop very well on the pathogen. Analysis of the possible nutrients that could stimulate the growth ofP. flocculosaon powdery mildew structures revealed that the complex Zn/Mn played a key role in the interaction. Other related fungi such asU. maydisdo not appear to have the same nutritional requirements and hence lack the ability to colonize powdery mildews. Whether production of antifungal glycolipids contributes to the release of nutrients from powdery mildew colonies is unclear, but the specificity of the biocontrol activity ofP. flocculosatoward Erysiphales does appear to be more complex than simple antibiosis.

scholarly journals Normal Formation of a Subset of Intestinal Granules in Caenorhabditis elegans Requires ATP-binding Cassette Transporters HAF-4 and HAF-9, Which Are Highly Homologous to Human Lysosomal Peptide Transporter TAP-Like

2009 ◽  
Vol 20 (12) ◽  
pp. 2979-2990 ◽  
Author(s):  
Hiromi Kawai ◽  
Takahiro Tanji ◽  
Hirohisa Shiraishi ◽  
Mitsuo Yamada ◽  
Ryoko Iijima ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Fluorescent Protein ◽  
Model Organism ◽  
Physiological Role ◽  
Peptide Transporter ◽  
Atp Binding ◽  
Loss Of Function ◽  
Specific Expression ◽  
Atp Binding Cassette

TAP-like (TAPL; ABCB9) is a half-type ATP-binding cassette (ABC) transporter that localizes in lysosome and putatively conveys peptides from cytosol to lysosome. However, the physiological role of this transporter remains to be elucidated. Comparison of genome databases reveals that TAPL is conserved in various species from a simple model organism, Caenorhabditis elegans, to mammals. C. elegans possesses homologous TAPL genes: haf-4 and haf-9. In this study, we examined the tissue-specific expression of these two genes and analyzed the phenotypes of the loss-of-function mutants for haf-4 and haf-9 to elucidate the in vivo function of these genes. Both HAF-4 and HAF-9 tagged with green fluorescent protein (GFP) were mainly localized on the membrane of nonacidic but lysosome-associated membrane protein homologue (LMP-1)-positive intestinal granules from larval to adult stage. The mutants for haf-4 and haf-9 exhibited granular defects in late larval and young adult intestinal cells, associated with decreased brood size, prolonged defecation cycle, and slow growth. The intestinal granular phenotype was rescued by the overexpression of the GFP-tagged wild-type protein, but not by the ATP-unbound form of HAF-4. These results demonstrate that two ABC transporters, HAF-4 and HAF-9, are related to intestinal granular formation and some other physiological aspects.

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