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[
West Coast Worm Meeting,
2004]
Advances in understanding multi-cellular organisms are encumbered because of inherent problems with cell and/or tissue heterogeneity. Particularly challenging, with regard to studies of tissue-specific gene expression, is the fact that cells and tissues of interest often cannot be isolated away from unwanted cells and tissues. The inability to precisely isolate tissue types can lead to incorrect assignment of gene expression, and to misinterpretation of promoter and gene function. This difficulty slows advances in understanding multi-cellular organisms, such as C. elegans, despite extensive genomic sequence data. To address this problem, SL-addition trans-splicing is engineered to act as a tool for tissue-specific profiling of genes in C. elegans. Synthetic sequences are inserted adjacent to or within the spliced leader mini-exon. Alternatively, the spliced leader mini-exon is mutated. In keeping with the nomenclature exon and intron, the synthetic RNA sequences that are "tagged on" to the 5'-end of genes in a SL-addition trans-splicing reaction are designated "Tagon™ sequences", and the genes that donate them as "Tagon™-SLRNA" genes. After Tagon™-SLRNAs are spliced onto pre-mRNAs the Tagon™ sequence can be used to purify expressed genes by simple oligonucleotide-mediated hybridization. These isolated mRNAs can be used to generate cDNA libraries, or directly labeled with fluorescent and/or radioactive tags for use as a 'probe' in microarray studies. Alternatively, Tagon™ trans-spliced mRNAs can be preferentially cloned by priming second-strand cDNA synthesis in a RACE (rapid amplification of cDNA ends) reaction, using a designed oligonucleotide corresponding to the synthetic Tagon™sequence. In order to enable the recovery of expressed genes subsets from defined tissues and/or cells, a Tagon™-SLRNA gene is cloned downstream of a known cell type specific or tissue-specific structural gene promoter. SL-RNA genes have an internal promoter element that must be obliterated to achieve tissue-specific expression of the Tagon™-SLRNA gene construct. Tagon™ profiling can be multiplexed. Because a Tagon™ sequence provides a unique nucleic acid "tag" for eventual recovery, Tagon™-SLRNA genes each encoding a unique Tagon™ sequence can be cloned and expressed under the control of different promoters. An individual strain can be created containing multiple different Promoter::Tagon™-SLRNA constructs (PromoterX::Tagon™1-SLRNA, PromoterY::Tagon™2-SLRNA, etc.). This technique enables the recovery of different populations of trans-spliced tissue-specific mRNAs from the same animal, and the creation of precisely defined cDNA libraries from C. elegans and other organisms previously refractory to this type of analysis.
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[
International Worm Meeting,
2013]
Asymmetric cell division (ACD) is a process that generates cell diversity. Both intrinsic and extrinsic mechanisms can distribute developmental potential asymmetrically to generate daughter cells of different fates and position the cleavage furrow asymmetrically to generate cells of different sizes. Wnts are evolutionarily conserved secreted glycoproteins that are utilized throughout development and play a role in ACD.1 During C. elegans development, Wnts regulate asymmetric divisions by controlling the distributions of the b-catenin SYS-1 and the LEF/TCF POP-1.2 They can also regulate the orientation of the spindle.3 In other organisms, Wnts can also regulate cytoskeletal dynamics via the Planar Cell Polarity (PCP) pathway, which allows polarization of neighboring cells along an axis orthogonal to the apical-basal axis within an epithelial sheet.4 The ACDs of the Q.a and Q.p neuroblasts to give rise to a larger daughter that lives and a smaller cell destined to die. We find that two Frizzled homologs LIN-17 and MOM-5 together are necessary for both apoptotic fates, for the asymmetric distribution of POP-1 in the Q.a and Q.p daughter cells, and for the asymmetric position of the Q.a and Q.p furrows that produce daughter cells of different sizes. Reduction of Wnt signaling, however, fails to generate the same robust disturbance of POP-1 distribution and does not affect the furrow localization. Instead, reduction of Wnt signaling can result in a reversal of POP-1 asymmetry, a phenotype that is enhanced by loss of the Van Gogh homolog VANG-1, a component of the PCP pathway. 1.Munro and Bowerman. CSH Perspectives. 2009. 2.Mizumoto and Sawa. Trends in Cell Biology. 2007. 3.Walston and Hardin. Seminars in Cell and Developmental Biology. 2006. 4.Segalen and Bellaiche. Seminars in Cell and Developmental Biology. 2009.
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[
J Mol Biol,
2018]
Titin-like kinases are muscle-specific kinases that regulate mechanical sensing in the sarcomere. Twitchin kinase (TwcK) is the best-characterized member of this family, both structurally and enzymatically. TwcK activity is auto-inhibited by a dual intrasteric mechanism, in which N- and C-terminal tail extensions wrap around the kinase domain, blocking the hinge region, the ATP binding pocket and the peptide substrate binding groove. Physiologically, kinase activation is thought to occur by a stretch-induced displacement of the inhibitory tails from the kinase domain. Here, we now show that TwcK inhibits its catalysis even in the absence of regulatory tails, by undergoing auto-phosphorylation at mechanistically important elements of the kinase fold. Using mass spectrometry, site-directed mutagenesis and catalytic assays on recombinant samples, we identify residues T212, T301, T316 and T401 as primary auto-phosphorylation sites in TwcK in vitro. Taken together, our results suggest that residue T316, located in the peptide substrate binding P+1 loop, is the dominantly regulatory site in TwcK. Based on these findings, we conclude that TwcK is regulated through a triple-inhibitory mechanism consisting of phosphorylation and intrasteric blockage, which is responsive not only to mechanical cues but also to biochemical modulation. This implies that mechanically stretched conformations of TwcK do not necessarily correspond to catalytically active states, as previously postulated. This further suggests a phosphorylation-dependent desensitization of the TwcK-mediated mechanoresponse of the sarcomere in vivo.
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[
J Toxicol Environ Health A,
2000]
Caenorhabditis elegans has proven useful in toxicity testing of known toxicants, but its potential for assessing the toxicity of new pharmaceuticals is relatively unexplored. In this study the procedures used in aquatic testing of toxicants were modified to permit testing of small amounts (<40 mg) of gadolinium-based magnetic resonance imaging (MRI) compounds. Five blinded compounds were tested. The toxicity of these compounds determined using C. elegans was compared to existing mammalian test system data (minimum lethal dose [MLD] values for mice). Four of five compounds tested had the same relative sensitivity with C. elegans as with the mouse test system. Testing with C. elegans is efficient and could markedly reduce the cost of screening potentially useful compounds.
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[
J Exp Neurosci,
2018]
<i>Caenorhabditis elegans</i> is a powerful model to study the neural and biochemical basis of behavior. It combines a small, completely mapped nervous system, powerful genetic tools, and a transparent cuticle, allowing Ca<sup>++</sup> imaging without the need for dissection. However, these approaches remain one step removed from direct pharmacological and physiological characterization of individual neurons. Much can still be learned by "getting under the hood" or breaching the cuticle and directly studying the neurons. For example, we recently combined electrophysiology, Ca<sup>++</sup> imaging, and pharmacological analysis on partially dissected ASH nociceptors showing that serotonin (5-HT) potentiates depolarization by inhibiting Ca<sup>++</sup> influx. This study challenges the tacit assumption that Ca<sup>++</sup> transient amplitudes and depolarization strength are positively correlated and has validated a new paradigm for interpreting Ca<sup>++</sup> signals. Bypassing the cuticle was critical for the success of these experiments, not only for performing electrical recordings but also for the acute and reversible application of drugs. By contrast, drug soaking or mutating genes can produce long-term effects and compensatory changes, potentially confounding interpretations significantly. Therefore, direct studies of the physiological response of individual neurons should remain a critical objective, to provide key molecular insights complementing global Ca<sup>++</sup> imaging neural network studies.
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[
J Neurosci,
2018]
Neuromodulators such as serotonin (5-HT) alter neuronal excitability and synaptic strengths, and define different behavioral states. Neuromodulator-dependent changes in neuronal activity patterns are frequently measured using calcium reporters, since calcium imaging can easily be performed on intact functioning nervous systems. With only 302 neurons, the nematode Caenorhabditis elegans provides a relatively simple, yet powerful, system to understand neuromodulation at the level of individual neurons. C. elegans hermaphrodites are repelled by 1-octanol, and the initiation of these aversive responses are potentiated by 5-HT. 5-HT acts on the ASH polymodal nociceptors that sense the 1-octanol stimulus. Surprisingly, 5-HT suppresses ASH Ca++ transients while simultaneously potentiating 1-octanol-dependent ASH depolarization. Here we further explore this seemingly inverse relationship. Our results show first, that 5-HT acts downstream of depolarization, through Gq-mediated signaling and calcineurin, to inhibit L-type voltage-gated Ca++ channels; second, that the 1-octanol-evoked Ca++ transients in ASHs inhibit depolarization; and third, that the Ca++-activated K+ channel, SLO-1, acts downstream of 5-HT, and is a critical regulator of ASH response dynamics. These findings define a Ca++-dependent inhibitory feedback loop that can be modulated by 5-HT to increase neuronal excitability and regulate behavior, and highlight the possibility that neuromodulator-induced changes in the amplitudes of Ca++ transients do not necessarily predict corresponding changes in depolarization.SIGNIFICANCE STATEMENT:Neuromodulators such as 5-HT modify behavior by regulating excitability and synaptic efficiency in neurons. Neuromodulation is often studied using Ca++ imaging, whereby neuromodulator-dependent changes in neuronal activity levels can be detected in intact, functioning circuits. Here we show that 5-HT reduces the amplitude of depolarization-dependent Ca++ transients in a C. elegans nociceptive neuron, through Gq signaling and calcineurin, but that Ca++ itself inhibits depolarization, likely through Ca++-activated K+ channels. The net effect of 5-HT, therefore, is to increase neuronal excitability through disinhibition. These results establish a novel 5-HT signal transduction pathway, and demonstrate that neuromodulators can change Ca++ signals and depolarization amplitudes in opposite directions, simultaneously, within a single neuron.
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[
J Vis Exp,
2011]
Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans(1). The main difficulty of this assay is the perceived cost ($25-100K) and technical expertise required for implementing a laser ablation system(2,3). However, solid-state pulse lasers of modest costs (<$10K) can provide robust performance for laser ablation in transparent preparations where target axons are "close" to the tissue surface. Construction and alignment of a system can be accomplished in a day. The optical path provided by light from the focused condenser to the ablation laser provides a convenient alignment guide. An intermediate module with all optics removed can be dedicated to the ablation laser and assures that no optical elements need be moved during a laser ablation session. A dichroic in the intermediate module allows simultaneous imaging and laser ablation. Centering the laser beam to the outgoing beam from the focused microscope condenser lens guides the initial alignment of the system. A variety of lenses are used to condition and expand the laser beam to fill the back aperture of the chosen objective lens. Final alignment and testing is performed with a front surface mirrored glass slide target. Laser power is adjusted to give a minimum size ablation spot (<1 um). The ablation spot is centered with fine adjustments of the last kinematically mounted mirror to cross hairs fixed in the imaging window. Laser power for axotomy will be approximately 10X higher than needed for the minimum ablation spot on the target slide (this may vary with the target you use). Worms can be immobilized for laser axotomy and time-lapse imaging by mounting on agarose pads (or in microfluidic chambers(4)). Agarose pads are easily made with 10% agarose in balanced saline melted in a microwave. A drop of molten agarose is placed on a glass slide and flattened with another glass slide into a pad approximately 200 um thick (a single layer of time tape on adjacent slides is used as a spacer). A "Sharpie" cap is used to cut out a uniformed diameter circular pad of 13 mm. Anesthetic (1 ul Muscimol 20mM) and Microspheres (Chris Fang-Yen personal communication) (1 ul 2.65% Polystyrene 0.1 um in water) are added to the center of the pad followed by 3-5 worms oriented so they are lying on their left sides. A glass coverslip is applied and then Vaseline is used to seal the coverslip and prevent evaporation of the sample.
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[
J Am Soc Nephrol,
2010]
Cilia dysfunction contributes to renal cyst formation in multiple human syndromes including nephronophthisis (NPHP), Meckel-Gruber syndrome (MKS), Joubert syndrome (JBTS), and Bardet-Beidl syndrome (BBS). Although genetically heterogeneous, these diseases share several loci that affect cilia and/or basal body proteins, but the functions and interactions of these gene products are incompletely understood. Here, we report that the ciliated sensory neurons (CSNs) of C. elegans express the putative transmembrane protein MKS-3, which localized to the distal end of their dendrites and to the cilium base but not to the cilium itself. Localization of MKS-3 and other known MKS and NPHP proteins partially overlapped. By analyzing
mks-3 mutants, we found that ciliogenesis did not require MKS-3; instead, cilia elongated and cilia-mediated chemoreception was abnormal. Genetic analysis indicated that
mks-3 functions in a pathway with other mks genes. Furthermore,
mks-1 and
mks-3 genetically interacted with a separate pathway (involving
nphp-1 and
nphp-4) to influence proper positioning, orientation, and formation of cilia. Combined disruption of nphp and mks pathways had cell nonautonomous effects on C. elegans sensilla. Taken together, these data demonstrate the importance of mutational load on the presentation and severity of ciliopathies and expand the understanding of the interactions between ciliopathy genes.
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[
J Cell Biol,
1994]
By taking advantage of a lethal phenotype characteristic of Caenorhabditis elegans embryos that fail to move, we have identified 13 genes required for muscle assembly and function and discovered a new lethal class of alleles for three previously known muscle-affecting genes. By staining mutant embryos for myosin and actin we have recognized five distinct classes of genes: mutations in four genes disrupt the assembly of thick and thin filaments into the myofilament lattice as well as the polarized location of these components to the sarcolemma. Mutations in another three genes also disrupt thick and thin filament assembly, but allow proper polarization of lattice components based on the myosin heavy chain isoform that we analyzed. Another two classes of genes are defined by mutations with principal effects on thick or thin filament assembly into the lattice, but not both. The final class includes three genes in which mutations cause relatively minor defects in lattice assembly. Failure of certain mutants to stain with antibodies to specific muscle cell antigens suggest that two genes associated with severe disruptions of myofilament lattice assembly may code for components of the basement membrane and the sarcolemma that are concentrated where dense bodies (Z-line analogs) and M-lines attach to the cell membrane. Similar evidence suggests that one of the genes associated with mild effects on lattice assembly may code for tropomyosin. Many of the newly identified genes are likely to play critical roles in muscle development and function.
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[
Int J Mol Sci,
2011]
In this article we propose a systematic development method for rational drug design while reviewing paradigms in industry, emerging techniques and technologies in the field. Although the process of drug development today has been accelerated by emergence of computational methodologies, it is a herculean challenge requiring exorbitant resources; and often fails to yield clinically viable results. The current paradigm of target based drug design is often misguided and tends to yield compounds that have poor absorption, distribution, metabolism, and excretion, toxicology (ADMET) properties. Therefore, an in vivo organism based approach allowing for a multidisciplinary inquiry into potent and selective molecules is an excellent place to begin rational drug design. We will review how organisms like the zebrafish and Caenorhabditis elegans can not only be starting points, but can be used at various steps of the drug development process from target identification to pre-clinical trial models. This systems biology based approach paired with the power of computational biology; genetics and developmental biology provide a methodological framework to avoid the pitfalls of traditional target based drug design.