50K Papers found (0.03 seconds)

Bland, Tom, Hirani, Nisha, Goehring, Nathan W., Ng, KangBo

[

MicroPubl Biol,

2022]

Engineered analog sensitive kinases provide a highly effective method for acute, controllable, and highly selective inhibition of kinase activity. Here we describe the design and characterization of an analog sensitive allele of the polarity kinase, PKC-3. This allele supports normal function as measured by its ability to exclude PAR-2 from the anterior membrane of zygotes, and is rapidly and reversibly inhibited in a dose-dependent manner by the ATP analog 1NA-PP1. This allele provides a new tool to explore the role of PKC-3 in diverse contexts within C. elegans , particularly those in which acute and reversible control of PKC-3 kinase activity may be desired.

Hollon NG et al. (2020) Neuron "Neural Implementation of Behavioral Hierarchy."

Jin X, Hollon NG

[

Neuron,

2020]

A vast array of animal behavior-from locomotion to human speech-is thought to consist of different hierarchical levels, but its neural implementation remains poorly understood. In this issue of Neuron, Kaplan etal. (2020) identify neuronal circuit dynamics responsible for multiple levels and timescales of hierarchical locomotion control in Caenorhabditis elegans.

Ng SS et al. (2009) J Biol Chem "Phosphatidylinositol 3-kinase signaling does not activate the wnt cascade."

Mahmoudi T, Bejaoui I, Schutte M, Ng SS, Clevers H, de Lau W, Korswagen HC, Danenberg E

[

J Biol Chem,

2009]

Mutational activation of the phosphatidylinositol 3-kinase (PI3K) pathway occurs in a wide variety of tumors, whereas activating Wnt pathway mutants are predominantly found in colon cancer. Because GSK3 is a key component of both pathways, it is widely assumed that active PI3K signaling feeds positively into the Wnt pathway by protein kinase B (PKB)-mediatefd inhibition of GSK3. In addition, PKB has been proposed to modulate the canonical Wnt signaling through direct stabilization and nuclear localization of beta-catenin. Here, we show that compartmentalization by Axin of GSK3 prohibits cross-talk between the PI3K and Wnt pathways and that Wnt-mediated transcriptional activity is not modulated by activation of the PI3K/PKB pathway.

Ng HWY et al. (2021) Dis Model Mech "Genetically altered animal models for ATP1A3-related disorders."

Clapcote SJ, Ng HWY, Ogbeta JA

[

Dis Model Mech,

2021]

Within the past 20 years, particularly with the advent of exome sequencing technologies, autosomal dominant and de novo mutations in the gene encoding the neurone-specific alpha3 subunit of the Na+,K+-ATPase (NKA alpha3) pump, ATP1A3, have been identified as the cause of a phenotypic continuum of rare neurological disorders. These allelic disorders of ATP1A3 include (in approximate order of severity/disability and onset in childhood development): polymicrogyria; alternating hemiplegia of childhood; cerebellar ataxia, areflexia, pes cavus, optic atrophy and sensorineural hearing loss syndrome; relapsing encephalopathy with cerebellar ataxia; and rapid-onset dystonia-parkinsonism. Some patients present intermediate, atypical or combined phenotypes. As these disorders are currently difficult to treat, there is an unmet need for more effective therapies. The molecular mechanisms through which mutations in ATP1A3 result in a broad range of neurological symptoms are poorly understood. However, in vivo comparative studies using genetically altered model organisms can provide insight into the biological consequences of the disease-causing mutations in NKA alpha3. Herein, we review the existing mouse, zebrafish, Drosophila and Caenorhabditis elegans models used to study ATP1A3-related disorders, and discuss their potential contribution towards the understanding of disease mechanisms and development of novel therapeutics.

Ng LF et al. (2019) Bio Protoc "Measurement of Respiration Rate in Live Caenorhabditis elegans."

Ng LF, Gruber J

[

Bio Protoc,

2019]

Mitochondrial function and dysfunction are at the core of aging and involved in many age-dependent diseases. Rate of oxygen consumption is a measure of mitochondrial function and energy production rate. The nematode Caenorhabditis elegans (C. elegans) offers an opportunity to study "living" mitochondria without the need for mitochondrial extraction, purification and associated artifacts. Oxygen consumption rate (OCR) is traditionally measured using single-chamber Clark electrodes with or without the addition of metabolic modulators. More recently, multi-well oxygen electrodes with automated injection system have been developed to enable rapid measurement of OCR under different conditions. Here, we describe a detailed protocol that we have adapted from existing protocols to measure coupled and uncoupled mitochondrial respiration (with and without metabolic modulators) in live respiring nematodes using a Seahorse XFe96 extracellular flux analyzer. We present details on our protocol, including preparation of nematode culture, use of metabolic modulators, execution of Seahorse XF assay as well as post-experimental data analysis. As a reference, we provide results of a series of experiments in which the metabolic activity of N2 wild-type nematodes was compared to N2 nematode treated with paraquat, a compound that generates reactive oxygen species (ROS), thus causing oxidative damage and mitochondrial dysfunction. These data illustrate the kind of insights that can be obtained even using a low number of nematodes (10 animals only per well).

Egilmez NG et al. (1993) Worm Breeder's Gazette "Quantitation of Tc1 Elements in Various Strains of C. elegans"

Egilmez NG, Shmookler Reis RJ

[

Worm Breeder's Gazette,

1993]

We have employed the transposable element Tc1 as a polymorphic marker in order to define the evolutionary relationships between strains of C elegans. We first determined the number of Tc1 elements in strains N2 ,PA2 ,C12 aand Ga1 ,reported to be low-copy number strains and RC301 ,TR403 ,DH424 ,RW7000 and BO, described as high-copy variants (1). N2 is commonly referred to as having 30 Tc1s in its genome, while the values reported for BO vary between 300 and 500. No specific values have been reported for the other strains mentioned above. We utilized two different approaches to determine the Tc1 copy numbers. Initially, genomic DNAs from each strain were digested with EcoRI and separated on large, slow-running 0.8% agarose gels (23cm, 20V, 96 hours). Southern blots of these gels were then probed with a 1.6 kb cloned Tc1 element (2). Tc1 elements of the low-copy number strains could be resolved sufficiently well on these blots to allow direct enumeration from the autoradiographs. The results obtained from these blots for the low-copy strains are shown in Table 1. Tc1 copy numbers varied between 26 and 30 and the overall patterns were similar with minor differences between strains. RC301 ,contrary to previous reports (1), appeared to be a low-copy strain, containing 26 Tc1 bands. Re-analysis of fresh RC301 stocks, re-ordered from the Caenorhabditis Genetics Center and kindly provided by J. Hodgkin (MRC, Cambridge, England), confirmed our initial results. The band patterns of GA1 and N2 were identical. Other low-copy strains were distinct from N2 and from each other, in their Tc1 band patterns, but retained 21-22 bands with mobilities indistinguishable from N2 bands. C12 aand PA2 were clearly different from N2 but were nearly identical to each other differing at only one band. RC301 appeared to be the most distant relative of N2 based on the number and pattern of its Tc1 elements. DNA dot-blots were utilized to analyze the Tc1 copy numbers of the high-copy strains. Genomic DNAs from N2 ,RC301 ,TR403 ,DH424 ,RW7000 and BO were blotted at four different dilutions and were probed with the cloned Tc1 element. The values for DNA loads were corrected by probing the same blot with a cloned single-copy gene fragment (2). The blots were analyzed by direct phosphor-imaging of -emissions on a Phosphor Imager Computing Densitometer (Molecular Dynamics). Intensities of phosphor-images were integrated directly using ImageQuant software and the results are summarized in Figure 1. All values were within the linear range of the phosphor- screen extending over a 105 range of signal intensities thus ensuring reliable quantitation. Using RC301 as a reference standard, at 26 Tc1s ,the Tc1 copy numbers for the strains TR403 ,DH424 ,RW7000 and BO were estimated to be 205, 260, 410 and 494 respectively, based on least-squares linear regression. The slope for RW7000 ,a separately maintained stock of Bergerac-BO, has a large standard error and is not significantly different from that of BO. Nevertheless, the difference could be genuine, since different levels of Tc1 -transpositionmay have accumulated in these two stocks of an active mutator strain. In all other cases, the lines fit the data well and thus the values determined should be highly accurate. References: (1) Cassada, R. WBG 9(3):29, 1986; Hodgkin, J. WBG 10(2):140-141, 1988; Hodgkin, J. WBG 11(5):60, 1991. (2) Egilmez, N.K. & R.J. Shmookler Reis, manuscript in preparation.

Q Ch Ng et al. (2004) European Worm Meeting "LABELLING DENSE CORE VESICLE IN C. ELEGANS."

Q Ch Ng, J Kaplan

[

European Worm Meeting,

2004]

How do nervous systems process information? In individual neurons, information is transmitted and processed along two separate channels represented by two classes of morphologically distinct secretory vesicles: the synaptic vesicles (SVs) and the dense core vesicles (DCVs). SVs and DCVs deliver different neurotransmitters and are mobilized by different patterns of neuronal activity. They also differ in their origins, cellular localization, calcium sensitivity for release and the release rate of their contents. These differences necessitate differences in the biochemistry and cell biology of DCV and SV release. Neurons must distinguish between different patterns of neuronal activity before mobilizing the appropriate class of vesicles to ensure that the correct information is transmitted. Currently, little is known about the cellular mechanisms that separate SV and DCV release. To study how this kind of specificity is encoded during exocytosis and thus how neurons process and transmit information, I seek to develop in vivo assays in C. elegans to detect DCV release and to identify genes required for this process that separate it from SV release. DCVs contain neuropeptides and the enzymes involved in their processing and maturation. To visualize DCVs in vivo, I have tagged some of these proteins with Venus, YFP variant that is capable of fluorescing in the acidic compartment of DCVs when GFP is normally quenched. Venus tagged EGL-21 (a carboxypeptidase E that processes neuropeptides) is localized to discrete puncta and are retained in the cell body in unc-104 mutants, consistent with localization to vesicles. Some of these Venus::EGL-21 puncta move. Venus::EGL-21 molecules accumulate in the coelomocytes (scavenger cells that inhabit the pseudocoelomic cavity of C. elegans) suggesting that they are secreted into the pseudocoeloem and taken up by these cells. Similar results have also been obtained with Venus fusions to neuropeptides. These Venus fusions will be used to develop optical assays for detecting DCV release in vivo.

Ng LF et al. (2019) Front Genet "Mitochondrial DNA Damage Does Not Determine C. elegans Lifespan."

Ng LF, Gruber J, van Breugel M, Ng LT, Halliwell B

[

Front Genet,

2019]

The mitochondrial free radical theory of aging (mFRTA) proposes that accumulation of oxidative damage to macromolecules in mitochondria is a causative mechanism for aging. Accumulation of mitochondrial DNA (mtDNA) damage may be of particular interest in this context. While there is evidence for age-dependent accumulation of mtDNA damage, there have been only a limited number of investigations into mtDNA damage as a determinant of longevity. This lack of quantitative data regarding mtDNA damage is predominantly due to a lack of reliable assays to measure mtDNA damage. Here, we report adaptation of a quantitative real-time polymerase chain reaction (qRT-PCR) assay for the detection of sequence-specific mtDNA damage in C. elegans and apply this method to investigate the role of mtDNA damage in the aging of nematodes. We compare damage levels in old and young animals and also between wild-type animals and long-lived mutant strains or strains with modifications in ROS detoxification or production rates. We confirm an age-dependent increase in mtDNA damage levels in C. elegans but found that there is no simple relationship between mtDNA damage and lifespan. MtDNA damage levels were high in some mutants with long lifespan (and vice versa). We next investigated mtDNA damage, lifespan and healthspan effects in nematode subjected to exogenously elevated damage (UV- or -radiation induced). We, again, observed a complex relationship between damage and lifespan in such animals. Despite causing a significant elevation in mtDNA damage, -radiation did not shorten the lifespan of nematodes at any of the doses tested. When mtDNA damage levels were elevated significantly using UV-radiation, nematodes did suffer from shorter lifespan at the higher end of exposure tested. However, surprisingly, we also found hormetic lifespan and healthspan benefits in nematodes treated with intermediate doses of UV-radiation, despite the fact that mtDNA damage in these animals was also significantly elevated. Our results suggest that within a wide physiological range, the level of mtDNA damage does not control lifespan in C. elegans.

Sharon Yp Ng et al. (2005) International Worm Meeting "A novel role for the innexin UNC-7 in chemical synapse formation"

Ed Yeh, Sharon Yp Ng, Mei ZHEN

[

International Worm Meeting,

2005]

UNC-7 is a member of the innexin family that is required for formation of neuron specific gap junctions. We isolated a new unc-7 allele, hp121, in a forward genetic screen using the active zone marker SYD-2::GFP specifically expressed in the GABAergic neurons to identify regulators of active zone formation (for details see abstract by T. Kawano). unc-7(hp121) shows a reduced number of SYD-2::GFP puncta throughout development, but shows no obvious defect in the localization of the synaptic vesicle marker SNB-1::GFP. We found that three of the previously identified unc-7 alleles display a similar defect. This suggests that unc-7 does not affect the overall presynaptic structure; rather, unc-7 may specifically regulate the incorporation of active zone markers into the presynaptic termini. Consistent with this notion, we determined that the synaptic temperature sensitive period of unc-7 coincides with the time of synaptogenesis in C.elegans. Interestingly, unc-7 shows a genetic epistatic interaction with the presynaptic serine/threonine kinase SAD-1 (Crump et al, 2001). sad-1 loss of function alleles suppress both the behavioral defects and SYD-2::GFP defects of unc-7 mutants, while overexpression of SAD-1 enhances the weaker unc-7(hs9) allele. This suggests that unc-7 negatively regulates SAD-1 to promote synapse formation. Furthermore, we expressed a functional UNC-7::GFP fusion protein in the GABAergic neurons which localizes in a punctate pattern along both the dorsal and ventral cords similar to the pattern of en passant chemical synapses, suggesting that UNC-7 localizes to synapses. It is widely accepted that gap junctions localize adjacent to neuron cell bodies to indirectly mediate formation/development of chemical synapses through electrical coupling of neurons. However, the subcellular localization of GABAergic specific UNC-7::GFP argues against this traditional view. We propose that innexin UNC-7 may function as a hemichannel or even as a monomer at the presynaptic termini to negatively regulate the activity of SAD-1 kinase, thereby allowing the incorporation of active zone proteins and formation of chemical synapses. We will present more evidence to support our model.

Forger NG et al. (2010) Curr Opin Neurobiol "Cell death and sexual differentiation of behavior: worms, flies, and mammals."

Forger NG, de Vries GJ

[

Curr Opin Neurobiol,

2010]

Sex differences in the nervous system are found throughout the animal kingdom. Here, we discuss three prominent genetic models: nematodes, fruit flies, and mice. In all three, differential cell death is central to sexual differentiation and shared molecular mechanisms have been identified. Our knowledge of the precise function of neural sex differences lags behind. One fruitful approach to the 'function' question is to contrast sexual differentiation in standard laboratory animals with differentiation in species exhibiting unique social and reproductive organizations. Advanced genetic strategies are also addressing this question in worms and flies, and may soon be applicable to vertebrates.

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