scholarly journals IRI-1, a LIN-15B Homologue, Interacts with Inositol-1,4,5-Triphosphate Receptors and Regulates Gonadogenesis, Defecation, and Pharyngeal Pumping in Caenorhabditis elegans

2004 ◽  
Vol 15 (7) ◽  
pp. 3073-3082 ◽  
Author(s):  
Denise S. Walker ◽  
Sung Ly ◽  
Nicholas J.D. Gower ◽  
Howard A. Baylis
Keyword(s):  
Caenorhabditis Elegans ◽  
Single Gene ◽  
Loss Of Function ◽  
Yeast Two Hybrid ◽  
Uncharacterized Protein ◽  
Pumping Rate ◽  
Wide Range ◽  
Pharyngeal Pumping ◽  
And Control ◽  
Two Hybrid

Inositol-1,4,5-triphosphate receptors (IP3Rs) are ligand-gated Ca2+ channels that control Ca2+ release from intracellular stores. They are central to a wide range of cellular responses. IP3Rs in Caenorhabditis elegans are encoded by a single gene, itr-1, and are widely expressed. Signaling through IP3 and IP3Rs is important in ovulation, control of the defecation cycle, modulation of pharyngeal pumping rate, and embryogenesis. To further elucidate the molecular basis of the diversity of IP3R function, we used a yeast two-hybrid screen to search for proteins that interact with ITR-1. We identified an interaction between ITR-1 and IRI-1, a previously uncharacterized protein with homology to LIN-15B. Iri-1 is widely expressed, and its expression overlaps significantly with that of itr-1. In agreement with this observation, iri-1 functions in known itr-1-mediated processes, namely, upregulation of pharyngeal pumping in response to food and control of the defecation cycle. Knockdown of iri-1 in an itr-1 loss-of-function mutant potentiates some of these effects and sheds light on the signaling pathways that control pharyngeal pumping rate. Knockdown of iri-1 expression also results in a sterile, evl phenotype, as a consequence of failures in early Z1/Z4 lineage divisions, such that gonadogenesis is severely disrupted.

scholarly journals Two LIM Domain Proteins and UNC-96 Link UNC-97/PINCH to Myosin Thick Filaments in Caenorhabditis elegans Muscle

2007 ◽  
Vol 18 (11) ◽  
pp. 4317-4326 ◽  
Author(s):  
Hiroshi Qadota ◽  
Kristina B. Mercer ◽  
Rachel K. Miller ◽  
Kozo Kaibuchi ◽  
Guy M. Benian
Keyword(s):  
Caenorhabditis Elegans ◽  
Binding Assays ◽  
Lim Domain ◽  
Yeast Two Hybrid ◽  
Lim Domains ◽  
Thick Filaments ◽  
Vitro Binding ◽  
Two Hybrid ◽  
Hybrid Screening

By yeast two-hybrid screening, we found three novel interactors (UNC-95, LIM-8, and LIM-9) for UNC-97/PINCH in Caenorhabditis elegans. All three proteins contain LIM domains that are required for binding. Among the three interactors, LIM-8 and LIM-9 also bind to UNC-96, a component of sarcomeric M-lines. UNC-96 and LIM-8 also bind to the C-terminal portion of a myosin heavy chain (MHC), MHC A, which resides in the middle of thick filaments in the proximity of M-lines. All interactions identified by yeast two-hybrid assays were confirmed by in vitro binding assays using purified proteins. All three novel UNC-97 interactors are expressed in body wall muscle and by antibodies localize to M-lines. Either a decreased or an increased dosage of UNC-96 results in disorganization of thick filaments. Our previous studies showed that UNC-98, a C2H2 Zn finger protein, acts as a linkage between UNC-97, an integrin-associated protein, and MHC A in myosin thick filaments. In this study, we demonstrate another mechanism by which this linkage occurs: from UNC-97 through LIM-8 or LIM-9/UNC-96 to myosin.

scholarly journals Caenorhabditis elegans SMG-2 Selectively Marks mRNAs Containing Premature Translation Termination Codons

2007 ◽  
Vol 27 (16) ◽  
pp. 5630-5638 ◽  
Author(s):  
Lisa Johns ◽  
Andrew Grimson ◽  
Sherry L. Kuchma ◽  
Carrie Loushin Newman ◽  
Philip Anderson
Keyword(s):  
Caenorhabditis Elegans ◽  
Mammalian Cells ◽  
Translation Termination ◽  
Yeast Two Hybrid ◽  
Eukaryotic Mrnas ◽  
Nonsense Mediated Mrna Decay ◽  
Endogenous Genes ◽  
Alternatively Spliced ◽  
Two Hybrid ◽  
Premature Translation Termination

ABSTRACT Eukaryotic mRNAs containing premature translation termination codons (PTCs) are rapidly degraded by a process termed “nonsense-mediated mRNA decay” (NMD). We examined protein-protein and protein-RNA interactions among Caenorhabditis elegans proteins required for NMD. SMG-2, SMG-3, and SMG-4 are orthologs of yeast (Saccharomyces cerevisiae) and mammalian Upf1, Upf2, and Upf3, respectively. A combination of immunoprecipitation and yeast two-hybrid experiments indicated that SMG-2 interacts with SMG-3, SMG-3 interacts with SMG-4, and SMG-2 interacts indirectly with SMG-4 via shared interactions with SMG-3. Such interactions are similar to those observed in yeast and mammalian cells. SMG-2-SMG-3-SMG-4 interactions require neither SMG-2 phosphorylation, which is abolished in smg-1 mutants, nor SMG-2 dephosphorylation, which is reduced or eliminated in smg-5 mutants. SMG-2 preferentially associates with PTC-containing mRNAs. We monitored the association of SMG-2, SMG-3, and SMG-4 with mRNAs of five endogenous genes whose mRNAs are alternatively spliced to either contain or not contain PTCs. SMG-2 associates with both PTC-free and PTC-containing mRNPs, but it strongly and preferentially associates with (“marks”) those containing PTCs. SMG-2 marking of PTC-mRNPs is enhanced by SMG-3 and SMG-4, but SMG-3 and SMG-4 are not detectably associated with the same mRNPs. Neither SMG-2 phosphorylation nor dephosphorylation is required for selective association of SMG-2 with PTC-containing mRNPs, indicating that SMG-2 is phosphorylated only after premature terminations have been discriminated from normal terminations. We discuss these observations with regard to the functions of SMG-2 and its phosphorylation during NMD.

Interacting genes required for pharyngeal excitation by motor neuron MC in Caenorhabditis elegans.

Genetics ◽  
1995 ◽  
Vol 141 (4) ◽  
pp. 1365-1382 ◽  
Author(s):  
D M Raizen ◽  
R Y Lee ◽  
L Avery
Keyword(s):  
Caenorhabditis Elegans ◽  
Genetic Interactions ◽  
Neuron Type ◽  
Loss Of Function ◽  
Ach Release ◽  
Pharyngeal Muscle ◽  
Recessive Mutations ◽  
Allele Specific ◽  
Pharyngeal Pumping ◽  
Pumping Rates

Abstract We studied the control of pharyngeal excitation in Caenorhabditis elegans. By laser ablating subsets of the pharyngeal nervous system, we found that the MC neuron type is necessary and probably sufficient for rapid pharyngeal pumping. Electropharyngeograms showed that MC transmits excitatory postsynaptic potentials, suggesting that MC acts as a neurogenic pacemaker for pharyngeal pumping. Mutations in genes required for acetylcholine (ACh) release and an antagonist of the nicotinic ACh receptor (nAChR) reduced pumping rates, suggesting that a nAChR is required for MC transmission. To identify genes required for MC neurotransmission, we screened for mutations that cause slow pumping but no other defects. Mutations in two genes, eat-2 and eat-18, eliminated MC neurotransmission. A gain-of-function eat-18 mutation, ad820sd, and a putative loss-of-function eat-18 mutation, ad1110, both reduced the excitation of pharyngeal muscle in response to the nAChR agonists nicotine and carbachol, suggesting that eat-18 is required for the function of a pharyngeal nAChR. Fourteen recessive mutations in eat-2 fell into five complementation classes. We found allele-specific genetic interactions between eat-2 and eat-18 that correlated with complementation classes of eat-2. We propose that eat-18 and eat-2 function in a multisubunit protein complex involved in the function of a pharyngeal nAChR.

scholarly journals Mammalian Homologue of the Caenorhabditis elegans UNC-76 Protein Involved in Axonal Outgrowth Is a Protein Kinase C ζ–interacting Protein

1999 ◽  
Vol 144 (3) ◽  
pp. 403-411 ◽  
Author(s):  
Shun'ichi Kuroda ◽  
Noritaka Nakagawa ◽  
Chiharu Tokunaga ◽  
Kenji Tatematsu ◽  
Katsuyuki Tanizawa
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Caenorhabditis Elegans ◽  
Protein Kinase ◽  
Rat Brain ◽  
Axonal Outgrowth ◽  
Yeast Two Hybrid ◽  
Interacting Protein ◽  
Kinase C ◽  
Two Hybrid

By the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C ζ (PKCζ) as a bait, we have cloned a gene coding for a novel PKCζ-interacting protein homologous to the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth and fasciculation. The protein designated FEZ1 (fasciculation and elongation protein zeta-1) consisting of 393 amino acid residues shows a high Asp/Glu content and contains several regions predicted to form amphipathic helices. Northern blot analysis has revealed that FEZ1 mRNA is abundantly expressed in adult rat brain and throughout the developmental stages of mouse embryo. By the yeast two-hybrid assay with various deletion mutants of PKC, FEZ1 was shown to interact with the NH2-terminal variable region (V1) of PKCζ and weakly with that of PKCε. In the COS-7 cells coexpressing FEZ1 and PKCζ, FEZ1 was present mainly in the plasma membrane, associating with PKCζ and being phosphorylated. These results indicate that FEZ1 is a novel substrate of PKCζ. When the constitutively active mutant of PKCζ was used, FEZ1 was found in the cytoplasm of COS-7 cells. Upon treatment of the cells with a PKC inhibitor, staurosporin, FEZ1 was translocated from the cytoplasm to the plasma membrane, suggesting that the cytoplasmic translocation of FEZ1 is directly regulated by the PKCζ activity. Although expression of FEZ1 alone had no effect on PC12 cells, coexpression of FEZ1 and constitutively active PKCζ stimulated the neuronal differentiation of PC12 cells. Combined with the recent finding that a human FEZ1 protein is able to complement the function of UNC-76 necessary for normal axonal bundling and elongation within axon bundles in the nematode, these results suggest that FEZ1 plays a crucial role in the axon guidance machinery in mammals by interacting with PKCζ.

The mammalian homologue of the Caenorhabditis elegans polarity protein PAR-6 is a binding partner for the Rho GTPases Cdc42 and Rac1

2000 ◽  
Vol 113 (18) ◽  
pp. 3267-3275 ◽  
Author(s):  
A. Johansson ◽  
M. Driessens ◽  
P. Aspenstrom
Keyword(s):  
Caenorhabditis Elegans ◽  
Cell Polarity ◽  
Hybrid System ◽  
Mdck Cells ◽  
Pdz Domain ◽  
Yeast Two Hybrid ◽  
C Elegans ◽  
Yeast Two Hybrid System ◽  
Mammalian Homologue ◽  
Two Hybrid

A mammalian homologue of the PDZ domain containing Caenorhabditis elegans protein PAR-6 was found in a yeast two-hybrid system screen as binding to the Rho family member Cdc42. PAR-6 contains a PDZ domain and in C. elegans it has been shown to be crucial for the asymmetric cleavage and establishment of cell polarity during the first cell divisions in the growing embryo. Mammalian PAR-6 interacted with Cdc42 and Rac1 both in the yeast two-hybrid system and in in vitro binding assays. Co-immunoprecipitation experiments, employing transiently transfected Cos-1 cells, further confirmed that Cdc42 and Rac1 are physiological binding partners for PAR-6. We found that, in epithelial Madin-Darby canine kidney cells (MDCK), endogenous PAR-6 was present in the tight junctions, as judged from its co-localisation with the tight junction protein ZO-1, however, PAR-6 was also detected in the cell nucleus. Stimulation of MDCK cells with scatter factor/hepatocyte growth factor induced a loss of PAR-6 from the areas of cell-cell contacts in conformity with their progressive breakdown. In C. elegans PAR-6 co-localises with PAR-3 and has been suggested to form a direct complex. In agreement with earlier studies, mammalian PAR-3 was found to be present in tight junctions of MDCK cells but, in contrast to PAR-6, the protein could not be detected in the nucleus. Furthermore, co-immunoprecipitation experiments, employing Cos-1 cells, demonstrated that mammalian PAR-6 and PAR-3 formed a direct complex. These findings, together with the reported roles of PAR-6 and PAR-3 in C. elegans, suggest that Cdc42 and Rac1 and PAR-6/PAR-3 are involved in the establishment of cell polarity in epithelial cells.

scholarly journals FIGNL1 associates with KIF1Bβ and BICD1 to restrict dynein transport velocity during axon navigation

2019 ◽  
Vol 218 (10) ◽  
pp. 3290-3306 ◽  
Author(s):  
Melody Atkins ◽  
Laïla Gasmi ◽  
Valérie Bercier ◽  
Céline Revenu ◽  
Filippo Del Bene ◽  
...  
Keyword(s):  
Molecular Motor ◽  
Motor Axon ◽  
Regulatory Mechanisms ◽  
Axon Pathfinding ◽  
Loss Of Function ◽  
Yeast Two Hybrid ◽  
Neuronal Connectivity ◽  
Transport Velocity ◽  
Bidirectional Transport ◽  
Two Hybrid

Neuronal connectivity relies on molecular motor-based axonal transport of diverse cargoes. Yet the precise players and regulatory mechanisms orchestrating such trafficking events remain largely unknown. We here report the ATPase Fignl1 as a novel regulator of bidirectional transport during axon navigation. Using a yeast two-hybrid screen and coimmunoprecipitation assays, we showed that Fignl1 binds the kinesin Kif1bβ and the dynein/dynactin adaptor Bicaudal D-1 (Bicd1) in a molecular complex including the dynactin subunit dynactin 1. Fignl1 colocalized with Kif1bβ and showed bidirectional mobility in zebrafish axons. Notably, Kif1bβ and Fignl1 loss of function similarly altered zebrafish motor axon pathfinding and increased dynein-based transport velocity of Rab3 vesicles in these navigating axons, pinpointing Fignl1/Kif1bβ as a dynein speed limiter complex. Accordingly, disrupting dynein/dynactin activity or Bicd1/Fignl1 interaction induced motor axon pathfinding defects characteristic of Fignl1 gain or loss of function, respectively. Finally, pharmacological inhibition of dynein activity partially rescued the axon pathfinding defects of Fignl1-depleted larvae. Together, our results identify Fignl1 as a key dynein regulator required for motor circuit wiring.

scholarly journals UNC-98 and UNC-96 Interact with Paramyosin to Promote Its Incorporation into Thick Filaments of Caenorhabditis elegans

2008 ◽  
Vol 19 (4) ◽  
pp. 1529-1539 ◽  
Author(s):  
Rachel K. Miller ◽  
Hiroshi Qadota ◽  
Kristina B. Mercer ◽  
Kim M. Gernert ◽  
Guy M. Benian
Keyword(s):  
Caenorhabditis Elegans ◽  
Light Microscopy ◽  
Polarized Light ◽  
Wild Type ◽  
Binding Region ◽  
Yeast Two Hybrid ◽  
Thick Filaments ◽  
Separate Region ◽  
Two Hybrid ◽  
Null Mutants

Mutations in unc-96 or -98 cause reduced motility and a characteristic defect in muscle structure: by polarized light microscopy birefringent needles are found at the ends of muscle cells. Anti-paramyosin stains the needles in unc-96 and -98 mutant muscle. However there is no difference in the overall level of paramyosin in wild-type, unc-96, and -98 animals. Anti-UNC-98 and anti-paramyosin colocalize in the paramyosin accumulations of missense alleles of unc-15 (encodes paramyosin). Anti-UNC-96 and anti-UNC-98 have diffuse localization within muscles of unc-15 null mutants. By immunoblot, in the absence of paramyosin, UNC-98 is diminished, whereas in paramyosin missense mutants, UNC-98 is increased. unc-98 and -15 or unc-96 and -15 interact genetically either as double heterozygotes or as double homozygotes. By yeast two-hybrid assay and ELISAs using purified proteins, UNC-98 interacts with paramyosin residues 31-693, whereas UNC-96 interacts with a separate region of paramyosin, residues 699-798. The importance of surface charge of this 99 residue region for UNC-96 binding was shown. Paramyosin lacking the C-terminal UNC-96 binding region fails to localize throughout A-bands. We propose a model in which UNC-98 and -96 may act as chaperones to promote the incorporation of paramyosin into thick filaments.

scholarly journals The SH3 domain of UNC-89 (obscurin) interacts with paramyosin, a coiled-coil protein, in Caenorhabditis elegans muscle

2016 ◽  
Vol 27 (10) ◽  
pp. 1606-1620 ◽  
Author(s):  
Hiroshi Qadota ◽  
Olga Mayans ◽  
Yohei Matsunaga ◽  
Jonathan L. McMurry ◽  
Kristy J. Wilson ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Consensus Sequence ◽  
Coiled Coil ◽  
Thick Filament ◽  
Sh3 Domain ◽  
Structural Features ◽  
Loss Of Function ◽  
Heavy Chains ◽  
Binding Partners ◽  
Two Hybrid

UNC-89 is a giant polypeptide located at the sarcomeric M-line of Caenorhabditis elegans muscle. The human homologue is obscurin. To understand how UNC-89 is localized and functions, we have been identifying its binding partners. Screening a yeast two-hybrid library revealed that UNC-89 interacts with paramyosin. Paramyosin is an invertebrate-specific coiled-coil dimer protein that is homologous to the rod portion of myosin heavy chains and resides in thick filament cores. Minimally, this interaction requires UNC-89’s SH3 domain and residues 294–376 of paramyosin and has a KD of ∼1.1 μM. In unc-89 loss-of-function mutants that lack the SH3 domain, paramyosin is found in accumulations. When the SH3 domain is overexpressed, paramyosin is mislocalized. SH3 domains usually interact with a proline-rich consensus sequence, but the region of paramyosin that interacts with UNC-89’s SH3 is α-helical and lacks prolines. Homology modeling of UNC-89’s SH3 suggests structural features that might be responsible for this interaction. The SH3-binding region of paramyosin contains a “skip residue,” which is likely to locally unwind the coiled-coil and perhaps contributes to the binding specificity.

scholarly journals Enhanced Healthspan in Caenorhabditis elegans Treated With Extracts From the Traditional Chinese Medicine Plants Cuscuta chinensis Lam. and Eucommia ulmoides Oliv.

2021 ◽  
Vol 12 ◽  
Author(s):  
Shimaa M. A. Sayed ◽  
Karsten Siems ◽  
Christian Schmitz-Linneweber ◽  
Walter Luyten ◽  
Nadine Saul
Keyword(s):  
Caenorhabditis Elegans ◽  
Chinese Medicine ◽  
Traditional Chinese Medicine ◽  
Eucommia Ulmoides ◽  
Pumping Rate ◽  
C Elegans ◽  
Beneficial Effects ◽  
Physiological Fitness ◽  
Pharyngeal Pumping ◽  
Cuscuta Chinensis

To uncover potential anti-aging capacities of Traditional Chinese Medicine (TCM), the nematode Caenorhabditis elegans was used to investigate the effects of Eucommia ulmoides and Cuscuta chinensis extracts, selected by screening seven TCM extracts, on different healthspan parameters. Nematodes exposed to E. ulmoides and C. chinensis extracts, starting at the young adult stage, exhibited prolonged lifespan and increased survival after heat stress as well as upon exposure to the pathogenic bacterium Photorhabdus luminescens, whereby the survival benefits were monitored after stress initiation at different adult stages. However, only C. chinensis had the ability to enhance physical fitness: the swimming behavior and the pharyngeal pumping rate of C. elegans were improved at day 7 and especially at day 12 of adulthood. Finally, monitoring the red fluorescence of aged worms revealed that only C. chinensis extracts caused suppression of intestinal autofluorescence, a known marker of aging. The results underline the different modes of action of the tested plants extracts. E. ulmoides improved specifically the physiological fitness by increasing the survival probability of C. elegans after stress, while C. chinensis seems to be an overall healthspan enhancer, reflected in the suppressed autofluorescence, with beneficial effects on physical as well as physiological fitness. The C. chinensis effects may be hormetic: this is supported by increased gene expression of hsp-16.1 and by trend, also of hsp-12.6.

scholarly journals Some Phenotypic Characteristics of Nematode Caenorhabditis elegans Strains with Defective Functions of the Sestrin (cSesn) gene

2018 ◽  
Vol 11 (2) ◽  
pp. 759-767 ◽  
Author(s):  
A. O. Zeltukhin ◽  
G. V. Ilyinskaya ◽  
A. V. Budanov ◽  
P. M. Chumakov
Keyword(s):  
Caenorhabditis Elegans ◽  
Redox Homeostasis ◽  
Functional Decline ◽  
Extended Period ◽  
Locomotory Activity ◽  
Pumping Rate ◽  
Phenotypic Differences ◽  
C Elegans ◽  
Age Related ◽  
Pharyngeal Pumping

In mammals a small family of genes called Sestrins play important roles in the maintenance of metabolic and redox homeostasis, suggesting that the genes may positively affect the lifespan and counteract the age-related functional decline. The nematode genome contains a single cSesn gene that makes the Caenorhabditis elegans an excellent model for studying functions of the sestrin family. We describe phenotypic differences of worms that have compromised expression of cSesn gene. By comparing three different cSesn-deficient modes with the wild-type C. elegans strain we show that the abrogation of cSesn expression results in an increased body size, an extended period of body growth, a reduces brood size and number of offspring per a single worm, an accelerated decline in muscular functions revealed as a rapid decrease in the pharyngeal pumping rate and in the overall locomotory activity. The results are consistent with the potential roles of cSesn in counteracting the process of aging in C. elegans.

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