scholarly journals Caenorhabditis elegans UNC-98, a C2H2 Zn Finger Protein, Is a Novel Partner of UNC-97/PINCH in Muscle Adhesion Complexes

2003 ◽  
Vol 14 (6) ◽  
pp. 2492-2507 ◽  
Author(s):  
Kristina B. Mercer ◽  
Denise B. Flaherty ◽  
Rachel K. Miller ◽  
Hiroshi Qadota ◽  
Tina L. Tinley ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Nuclear Localization ◽  
Nuclear Export ◽  
Fluorescent Protein ◽  
Nuclear Export Signal ◽  
Focal Adhesions ◽  
Cell Nuclei ◽  
C Elegans ◽  
Dense Bodies ◽  
Green Fluorescent

To further understand the assembly and maintenance of the muscle contractile apparatus, we have identified a new protein, UNC-98, in the muscle of Caenorhabditis elegans. unc-98 mutants display reduced motility and a characteristic defect in muscle structure. We show that the major defect in the mutant muscle is in the M-lines and dense bodies (Z-line analogs). Both functionally and compositionally, nematode M-lines and dense bodies are analogous to focal adhesions of nonmuscle cells. UNC-98 is a novel 310-residue polypeptide consisting of four C2H2 Zn fingers and several possible nuclear localization signal and nuclear export signal sequences. By use of UNC-98 antibodies and green fluorescent protein fusions (to full-length UNC-98 and UNC-98 fragments), we have shown that UNC-98 resides at M-lines, muscle cell nuclei, and possibly at dense bodies. Furthermore, we demonstrated that 1) the N-terminal 106 amino acids are both necessary and sufficient for nuclear localization, and 2) the C-terminal (fourth) Zn finger is required for localization to M-lines and dense bodies. UNC-98 interacts with UNC-97, a C. elegans homolog of PINCH. We propose that UNC-98 is both a structural component of muscle focal adhesions and a nuclear protein that influences gene expression.

The Saccharomyces cerevisiae cyclin Clb2p is targeted to multiple subcellular locations by cis- and trans-acting determinants

2001 ◽  
Vol 114 (3) ◽  
pp. 589-597 ◽  
Author(s):  
J.K. Hood ◽  
W.W. Hwang ◽  
P.A. Silver
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Localization ◽  
Nuclear Export ◽  
Fluorescent Protein ◽  
Nuclear Export Signal ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cell Cycle Regulatory Proteins ◽  
Localization Signal ◽  
Green Fluorescent

The cyclin-dependent kinase Cdc28p associates with the cyclin Clb2p to induce mitosis in the yeast Saccharomyces cerevisiae. Several cell cycle regulatory proteins have been shown to require specific nuclear transport events to exert their regulatory functions. Therefore, we investigated the subcellular localization of wild-type Clb2p and several mutant versions of the protein using green fluorescent protein (GFP) fusion constructs. Wild-type Clb2p is primarily nuclear at all points of the cell. A point mutation in a potential leucine-rich nuclear export signal (NES) enhances the nuclear localization of the protein, and delta-yrb2 cells exhibit an apparent Clb2p nuclear export defect. Clb2p contains a bipartite nuclear localization signal (NLS), and its nuclear localization requires the alpha and beta importins (Srp1p and Kap95p), as well as the yeast Ran GTPase and its regulators. Deletion of the Clb2p NLS causes increased cytoplasmic localization of the protein, as well as accumulation at the bud neck. These data indicate that Clb2p exists in multiple places in the yeast cell, possibly allowing Cdc28p to locally phosphorylate substrates at distinct subcellular sites.

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 NEUROPHYSIOLOGICAL AND BEHAVIOURAL PERTURBATIONS IN Caenorhabditis elegans EXPOSED TO ORGANOPHOSPHATE PESTICIDES

2021 ◽  
Vol 9 (3) ◽  
pp. 343-352
Author(s):  
Rajul Jain ◽  
◽  
Priyanka Gautam ◽  
Keyword(s):  
Caenorhabditis Elegans ◽  
Fluorescent Protein ◽  
Developmental Toxicity ◽  
Yellow Fluorescent Protein ◽  
Model Organism ◽  
Egg Laying ◽  
High Exposure ◽  
C Elegans ◽  
Significant Difference ◽  
Green Fluorescent

The ubiquitous use of pesticides all over the world leads to adverse effects on both targets as well as non-target species. The extensive and uncontrolled use of organophosphates (OPs), a large group of pesticidal compounds in agricultural and household products are resulting in high exposure to humans. This research has been carried out to study the adverse effect of OPs i.e., chlorpyrifos, trichlorfon, and disulfoton on model organism Caenorhabditis elegans to evaluate their behavioural as well as developmental toxicity at different time intervals i.e., 4, 24, 48, and 72 hours (hrs) of exposure. A significant difference was observed in all the behavioural endpoints like locomotion, egg-laying, offspring count, and learning along with developmental parameters like mortality, paralysis, and growth rendering from moderate to high toxic effects. Based on the above screening, trichlorfon resulted in glutamatergic and cholinergic neurodegeneration along with elevated autofluorescence. Loss in Yellow fluorescent Protein (YFP) and Green Fluorescent Protein (GFP) was recorded by 57.96% and 30.52% using transgenic strains OH11124 (otIs388 [eat-4(fosmid)::SL2::YFP::H2B + (pBX)pha-1(+)] III) and OH13083 (otIs576 [unc-17(fosmid)::GFP + lin-44::YFP]). These results have shown the biological potency of toxicants in C. elegans and pave the way forward to provide insight into various neurogenerative diseases in humans.

abf-1 and abf-2, ASABF-type antimicrobial peptide genes in Caenorhabditis elegans

2002 ◽  
Vol 361 (2) ◽  
pp. 221-230 ◽  
Author(s):  
Yusuke KATO ◽  
Tomoyasu AIZAWA ◽  
Hirokazu HOSHINO ◽  
Keiichi KAWANO ◽  
Katsutoshi NITTA ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Antimicrobial Peptide ◽  
Fluorescent Protein ◽  
Fusion Gene ◽  
C Elegans ◽  
Specific Distribution ◽  
Genes Encoding ◽  
Precursor Rna ◽  
Factor Type ◽  
Green Fluorescent

Two genes encoding the ASABF (Ascarissuumantibacterial factor)-type antimicrobial peptide, abf-1 and abf-2, were identified in Caenorhabditis elegans. Recombinant ABF-2 exhibited potent microbicidal activity against Gram-positive and Gram-negative bacteria, and yeasts. The tissue-specific distribution estimated by immunofluorescence staining and transgenic analysis of a gfp fusion gene (where GFP corresponds to green fluorescent protein) suggested that ABF-2 contributes to surface defence in the pharynx. abf-1 contains a single intron at a conserved position, suggesting that asabf and abf originated from a common ancestor. Both transcripts for abf-1 and abf-2 were detected as two distinct forms, i.e. spliced leader (SL)1-trans-spliced with a long 5′-untranslated region (UTR) and SL-less with a short 5′-UTR. A polycistronic precursor RNA encoding ABF-1 and ABF-2 was detected, suggesting that these genes form an operon. An ‘opportunistic operon’ model for regulation of abf genes, including the generation of short SL-less transcripts, is proposed. In conclusion, C. elegans should have an immune defence system due to the antimicrobial peptides. C. elegans can be a novel model for innate immunity. Furthermore, the combination of biochemical identification in Ascaris suum and homologue hunting in C. elegans should be a powerful method of finding rapidly evolved proteins, such as some immune-related molecules in C. elegans.

scholarly journals LZTS2 Is a Novel β-Catenin-Interacting Protein and Regulates the Nuclear Export of β-Catenin

2006 ◽  
Vol 26 (23) ◽  
pp. 8857-8867 ◽  
Author(s):  
Gregory Thyssen ◽  
Tzu-Huey Li ◽  
Lynn Lehmann ◽  
Ming Zhuo ◽  
Manju Sharma ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Glycogen Synthase ◽  
Nuclear Export Signal ◽  
Point Mutations ◽  
Cellular Level ◽  
Interacting Protein ◽  
Nuclear Exclusion ◽  
Green Fluorescent ◽  
Wnt Signal

ABSTRACT β-Catenin plays multiple roles in cell-cell adhesion and Wnt signal transduction. Through the Wnt signal, the cellular level of β-catenin is constitutively regulated by the multicomponent destruction complex containing glycogen synthase kinase 3β, axin, and adenomatous polyposis coli. Here, we present multiple lines of evidence to demonstrate that LZTS2 (lucine zipper tumor suppressor 2) interacts with β-catenin, represses the transactivation of β-catenin, and affects the subcellular localization of β-catenin. The LZTS2 gene is located at 10q24.3, which is frequently lost in a variety of human tumors. A functional nuclear export signal (NES) was identified in the C terminus of the protein (amino acids 631 to 641). Appending this motif to green fluorescent protein (GFP) induced nuclear exclusion of the GFP fusion protein. However, introducing point mutations in either one or two leucine residues of this NES sequence abolished the nuclear exclusion of the LZTS2 protein. The nuclear export of LZTS2 can be blocked by leptomycin B (LMB), an inhibitor of the CRM1/exportin-alpha pathway. Intriguingly, β-catenin colocalizes with LZTS2 in the cytoplasm of cells in the absence of LMB but in the nuclei of cells in the presence of LMB. Increasing the LZTS2 protein in cells reduces the level of nuclear β-catenin in SW480 cells. Taken together, these data demonstrate that LZTS2 is a β-catenin-interacting protein that can modulate β-catenin signaling and localization.

scholarly journals Calreticulin Is a Receptor for Nuclear Export

2001 ◽  
Vol 152 (1) ◽  
pp. 127-140 ◽  
Author(s):  
James M. Holaska ◽  
Ben E. Black ◽  
Dona C. Love ◽  
John A. Hanover ◽  
John Leszyk ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Hormone Receptors ◽  
Fluorescent Protein ◽  
Kinase Inhibitor ◽  
Steroid Hormone ◽  
Nuclear Export Signal ◽  
Steroid Hormone Receptors ◽  
Permeabilized Cell ◽  
Green Fluorescent

In previous work, we used a permeabilized cell assay that reconstitutes nuclear export of protein kinase inhibitor (PKI) to show that cytosol contains an export activity that is distinct from Crm1 (Holaska, J.M., and B.M. Paschal. 1995. Proc. Natl. Acad. Sci. USA. 95: 14739–14744). Here, we describe the purification and characterization of the activity as calreticulin (CRT), a protein previously ascribed to functions in the lumen of the ER. We show that cells contain both ER and cytosolic pools of CRT. The mechanism of CRT-dependent export of PKI requires a functional nuclear export signal (NES) in PKI and involves formation of an export complex that contains RanGTP. Previous studies linking CRT to downregulation of steroid hormone receptor function led us to examine its potential role in nuclear export of the glucocorticoid receptor (GR). We found that CRT mediates nuclear export of GR in permeabilized cell, microinjection, and transfection assays. GR export is insensitive to the Crm1 inhibitor leptomycin B in vivo, and it does not rely on a leucine-rich NES. Rather, GR export is facilitated by its DNA-binding domain, which is shown to function as an NES when transplanted to a green fluorescent protein reporter. CRT defines a new export pathway that may regulate the transcriptional activity of steroid hormone receptors.

scholarly journals Nanoluciferase-Based Method for Detecting Gene Expression in Caenorhabditis elegans

Genetics ◽  
2019 ◽  
Vol 213 (4) ◽  
pp. 1197-1207 ◽  
Author(s):  
Ivana Sfarcic ◽  
Theresa Bui ◽  
Erin C. Daniels ◽  
Emily R. Troemel
Keyword(s):  
Gene Expression ◽  
Caenorhabditis Elegans ◽  
Promoter Activity ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Fluorescent Reporters ◽  
C Elegans ◽  
Link Type ◽  
Highly Sensitive ◽  
Green Fluorescent

Genetic reporters such as the green fluorescent protein (GFP) can facilitate measurement of promoter activity and gene expression. However, animal autofluorescence limits the sensitivity of GFP and other fluorescent reporters in whole-animal settings like in the nematode Caenorhabditis elegans. Here, we present a highly sensitive Nanoluciferase (NanoLuc)-based method in a multiwell format to detect constitutive and inducible gene expression in C. elegans. We optimize detection of bioluminescent signals from NanoLuc in C. elegans and show that it can be detected at 400,000-fold over background in a population of 100 animals expressing intestinal NanoLuc driven by the vha-6 promoter. We can reliably detect signal in single vha-6p::Nanoluc-expressing worms from all developmental stages. Furthermore, we can detect signal from a 1/100 dilution of lysate from a single vha-6p::Nanoluc-expressing adult and from a single vha-6p::Nanoluc-expressing adult “hidden” in a pool of 5000 N2 wild-type animals. We also optimize various steps of this protocol, which involves a lysis step that can be performed in minutes. As a proof-of-concept, we used NanoLuc to monitor the promoter activity of the pals-5 stress/immune reporter and were able to measure 300- and 50-fold increased NanoLuc activity after proteasome blockade and infection with microsporidia, respectively. Altogether, these results indicate that NanoLuc provides a highly sensitive genetic reporter for rapidly monitoring whole-animal gene expression in C. elegans.

scholarly journals Receptor-mediated Endocytosis in the Caenorhabditis elegans Oocyte

1999 ◽  
Vol 10 (12) ◽  
pp. 4311-4326 ◽  
Author(s):  
Barth Grant ◽  
David Hirsh
Keyword(s):  
Caenorhabditis Elegans ◽  
Fluorescent Protein ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Receptor Mediated Endocytosis ◽  
C Elegans ◽  
Density Lipoprotein Receptor ◽  
New Gene ◽  
Green Fluorescent

The Caenorhabditis elegans oocyte is a highly amenable system for forward and reverse genetic analysis of receptor-mediated endocytosis. We describe the use of transgenic strains expressing a vitellogenin::green fluorescent protein (YP170::GFP) fusion to monitor yolk endocytosis by theC. elegans oocyte in vivo. This YP170::GFP reporter was used to assay the functions of C. eleganspredicted proteins homologous to vertebrate endocytosis factors using RNA-mediated interference. We show that the basic components and pathways of endocytic trafficking are conserved between C. elegans and vertebrates, and that this system can be used to test the endocytic functions of any new gene. We also used the YP170::GFP assay to identify rme(receptor-mediated endocytosis) mutants. We describe a new member of the low-density lipoprotein receptor superfamily, RME-2, identified in our screens for endocytosis defective mutants. We show that RME-2 is the C. elegans yolk receptor.

Identification of a functional nuclear export signal in the green fluorescent protein asFP499

2006 ◽  
Vol 342 (4) ◽  
pp. 1178-1182 ◽  
Author(s):  
Huseyin Mustafa ◽  
Bernd Straßer ◽  
Sabine Rauth ◽  
Robert A. Irving ◽  
Kim L. Wark
Keyword(s):  
Green Fluorescent Protein ◽  
Nuclear Export ◽  
Fluorescent Protein ◽  
Nuclear Export Signal ◽  
Green Fluorescent ◽  
Export Signal

scholarly journals CRM1 Mediates the Export of ADAR1 through a Nuclear Export Signal within the Z-DNA Binding Domain

2001 ◽  
Vol 21 (22) ◽  
pp. 7862-7871 ◽  
Author(s):  
Hanne Poulsen ◽  
Jakob Nilsson ◽  
Christian K. Damgaard ◽  
Jan Egebjerg ◽  
Jørgen Kjems
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Protein A ◽  
Nuclear Export Signal ◽  
Nuclear Accumulation ◽  
Adenosine Deaminases ◽  
Editing Activity ◽  
Green Fluorescent ◽  
Export Signal

ABSTRACT RNA editing of specific residues by adenosine deamination is a nuclear process catalyzed by adenosine deaminases acting on RNA (ADAR). Different promoters in the ADAR1 gene give rise to two forms of the protein: a constitutive promoter expresses a transcript encoding (c)ADAR1, and an interferon-induced promoter expresses a transcript encoding an N-terminally extended form, (i)ADAR1. Here we show that (c)ADAR1 is primarily nuclear whereas (i)ADAR1 encompasses a functional nuclear export signal in the N-terminal part and is a nucleocytoplasmic shuttle protein. Mutation of the nuclear export signal or treatment with the CRM1-specific drug leptomycin B induces nuclear accumulation of (i)ADAR1 fused to the green fluorescent protein and increases the nuclear editing activity. In concurrence, CRM1 and RanGTP interact specifically with the (i)ADAR1 nuclear export signal to form a tripartite export complex in vitro. Furthermore, our data imply that nuclear import of (i)ADAR1 is mediated by at least two nuclear localization sequences. These results suggest that the nuclear editing activity of (i)ADAR1 is modulated by nuclear export.

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