Chih-Wei W Chen et al. (2010) East Asia Worm Meeting "Mechanisms of UNC-104/dynein complex interactions in the nervous system of C. elegans"

Chih-Wei W Chen, Ying-Cheng Yan, Oliver I Wagner

[East Asia Worm Meeting, 2010]

Molecular motors as kinesins and dynein/dynactin complex are major proteins for cargo transport along axonal microtubules in neuronal cells. Recent evidence has shown that defects in molecular motors may lead to severe neurodegenerative diseases. In addition, the accumulation of cargo (as, e.g., tau and neurofilaments) is a hallmark of neuron degeneration. However, only little evidence gives rise on how molecular motors are regulated. The frequently observed directional changes of motors and synaptic vesicles, however, might be explained by cooperative interactions between kinesins and dynein or by the so called tug-of-war model. We use C. elegans to observe and analyze the motility of the major synaptic vesicle transporter UNC-104/KIF1A. Here, we set up crosses of worms expressing a UNC-104::GFP rescue construct in worms carrying either mutations in the dynein or dynactin gene. We further observe the movement of synaptic vesicles by expressing SNB-1::GFP (synaptobrevin-1) in dynein/dynactin mutant worms. Interestingly, both, motor and cargo seemed to have reduced speeds in C. elegans mutants. We also analyze the distribution pattern of UNC-104 and SNB-1 along the neurons and found that both motor and vesicle tend to accumulate along the axons. We thus hypothesize a direct and functional interaction between UNC-104 and dynein/dynactin. Indeed, yeast-two hybrid assays revealed strong interactions between UNC-104 and the dynein Tctex-1 light chain, roadblock light chain, and p27 domains. Future experiments include co-immunoprecipitation assays to investigate UNC-104/dynein in vitro interactions as well as RNAi to investigate the specific role of genes encoding dynein and dynactin subcomplexes.

Tonks NK (2009) Cell "Pseudophosphatases: grab and hold on."

Tonks NK

[Cell, 2009]

Catalytically inactive pseudophosphatases are able to signal in the absence of enzymatic activity. Analyzing the oocyte-to-zygote transition in the worm, Cheng et al. (2009) and Parry et al. (2009) now show how two pseudophosphatases, EGG-4 and EGG-5, can regulate signaling by the DYRK family kinase MBK-2.

Pop, C et al. (2005) Cell "The nematode death machine in 3D."

Pop, C, Salvesen, GS

[Cell, 2005]

Regulated apoptosis is part of the development of the nematode Caenorhabditis elegans. In a recent paper in Nature, Yan et al. (2005) describe the in vitro reconstitution of the core components of the worm apoptotic pathway. Based on a structural analysis of the complex between the death activator CED-4 and the antiapoptotic protein CED-9, the authors explain the regulation of activity of CED-4. Intriguingly, CED-4 comprises a AAA+ type ATPase domain yet does not seem to need ATP hydrolysis for activity.

Malinow, R.A. et al. (2019) International Worm Meeting "Functional dissection of C. elegans bZip-protein CEBP-1."

Kim, K.W., Koorman, T., Boxem, M., Ying, P., Malinow, R.A., Jin, Y.

[International Worm Meeting, 2019]

C. elegans CEBP-1 is a C/EBP bZIP transcription factor and essential for axon regeneration. Previous studies have focused on the role of bZIP domain at the C-terminus of CEBP-1 in axon regeneration (Yan et al., 2009 Cell). However, CEBP-1 also has a long N-terminal fragment that contains no known protein motifs and how it is involved in CEBP-1 function remains unknown. Here, we report novel structural motifs in the N-terminus of CEBP-1 for axon regeneration and nuclear import. Using two types of forward genetic screens, we have identified a unique domain in the N-terminus that is critical for CEBP-1's in vivo functions. This N' domain also has a predicted propensity to form alpha helices. Furthermore, using yeast two-hybrid screening with N-terminal CEBP-1 fragments as baits, we have identified IMA-3/Importin-? as a CEBP-1 binding partner. In transgenic expression studies, we characterized the subcellular localization of CEBP-1 fragments and identified three nuclear localization signals (NLS) in CEBP-1, one which interacts with IMA-3. Lastly, we showed that ima-3 is required for axon regeneration. Taken together, our findings uncover new protein structural elements for CEBP-1, and further reinforce the importance of Importin-mediated nuclear import system for promoting axon regeneration. Yan, D., Wu, Z., Chisholm, A.D., and Jin, Y. (2009). The DLK-1 kinase promotes mRNA stability and local translation in C. elegans synapses and axon regeneration. Cell 138, 1005-1018.

Calvo AC et al. (2008) FASEB J "Anabolic function of phenylalanine hydroxylase in Caenorhabditis elegans."

Loer CM, Calvo AC, Ying M, Martinez A, Pey AL

[FASEB J, 2008]

In humans, liver phenylalanine hydroxylase (PAH) has an established catabolic function, and mutations in PAH cause phenylketonuria, a genetic disease characterized by neurological damage, if not treated. To obtain novel evolutionary insights and information on molecular mechanisms operating in phenylketonuria, we investigated PAH in the nematode Caenorhabditis elegans (cePAH), where the enzyme is coded by the pah-1 gene, expressed in the hypodermis. CePAH presents similar molecular and kinetic properties to human PAH [S0.5(L-Phe) approximately 150 microM; Km for tetrahydrobiopterin (BH4) approximately 35 microM and comparable Vmax], but cePAH is devoid of positive cooperativity for L-Phe, an important regulatory mechanism of mammalian PAH that protects the nervous system from excess L-Phe. Pah-1 knockout worms show no obvious neurological defects, but in combination with a second cuticle synthesis mutation, they display serious cuticle abnormalities. We found that pah-1 knockouts lack a yellow-orange pigment in the cuticle, identified as melanin by spectroscopic techniques, and which is detected in C. elegans for the first time. Pah-1 mutants show stimulation of superoxide dismutase activity, suggesting that cuticle melanin functions as oxygen radical scavenger. Our results uncover both an important anabolic function of PAH and the change in regulation of the enzyme along evolution.-Calvo, A. C., Pey, A. L., Ying, M., Loer, C. M., Martinez, A. Anabolic function of phenylalanine hydroxylase in Caenorhabditis elegans.

G Kao et al. (1999) International C. elegans Meeting "Characterization of Laminin beta and gamma Genes."

G Kao, C Huang, WG Wadsworth

[International C. elegans Meeting, 1999]

We have characterized laminin b (lam-1) and laminin g (lam-2) genes. The sequencing project has uncovered genes for one isoform each of the two subunits and two isoforms of laminin a , epi-1 and lam-3. (See abstract by Cheng et al.). This indicates that two types of trimers likely are form in basement membranes : a A bg and a B bg . A hypomorphic mutation (rh219) exists in lam-1, whose phenotypes we have described in previous meetings (IWM 1997). Both lam-1(RNAi) and lam-2(RNAi) animals arrest development during morphogenesis. Significantly, this phenotype is identical to the arrest seen in double knockout of epi-1 and lam-3, and is more severe than either knockout alone. We examined the expression patterns of lam-1 and lam-2 using promoter:GFP fusions. Both genes are expressed in body wall muscles, pharynx, vulval muscles, the rectal muscle group and the distal tip cells. The expression patterns of b and g subunit genes overlap with and are more widespread than either epi-1 or lam-3 alone. Taken together these results suggest that lam-1 and lam-2 are found in all basement membranes and indicate that trimers a A bg and a B bg are formed.

Cho, Jihye et al. (2021) International Worm Meeting "Identify the function of mechanosensitive channel PEZO-1 in C. elegans males"

Cho, Jihye, Kim, Kyuhyung

[International Worm Meeting, 2021]

The conversion of mechanical force to biological signals is crucial for the survival of animals. Mechanosensitive ion channels play a direct role in sensing physical stimuli and are evolutionarily conserved from bacteria to mammals. In mammals, PIEZO channels have been identified as ion channels that directly detect mechanical stimuli (Coste et al., 2010, Gnanasambandam et al., 2015, Syeda et al., 2015). C. elegans genome has a single PIEZO gene, pezo-1, which encodes 14 isoforms (Bai X et al., 2020). However, the function of PEZO-1 in C. elegans has not been fully understood yet. To investigate its function, we first grouped 14 isoforms into short or long isoforms depending on the mRNA length and observed their expression patterns. We found that the promoter region of short isoforms is expressed in several tail neurons, including the 6th ray neurons of C. elegans males. Nine pairs of male ray neurons appear to have functional specialization and have been implicated to be involved in mating behavior (Zhang H, Yue X, Cheng H, et al., 2018). Interestingly, sensory endings of ray 6 neurons, but not other ray neurons, are not exposed to the external environment (Sulston and Horvitz, 1977), suggesting a distinct role in mechanotransduction during C. elegans mating. We are currently examining the contribution of PEZO-1 to mating behavior in males.

Towlson EK et al. (2018) Philos Trans R Soc Lond B Biol Sci "Caenorhabditis elegans and the network control framework-FAQs."

Schafer WR, Walker DS, Towlson EK, Yan G, Vertes PE, Barabasi AL, Chew YL

[Philos Trans R Soc Lond B Biol Sci, 2018]

Control is essential to the functioning of any neural system. Indeed, under healthy conditions the brain must be able to continuously maintain a tight functional control between the system's inputs and outputs. One may therefore hypothesize that the brain's wiring is predetermined by the need to maintain control across multiple scales, maintaining the stability of key internal variables, and producing behaviour in response to environmental cues. Recent advances in network control have offered a powerful mathematical framework to explore the structure-function relationship in complex biological, social and technological networks, and are beginning to yield important and precise insights on neuronal systems. The network control paradigm promises a predictive, quantitative framework to unite the distinct datasets necessary to fully describe a nervous system, and provide mechanistic explanations for the observed structure and function relationships. Here, we provide a thorough review of the network control framework as applied to <i>Caenorhabditis elegans</i> (Yan <i>et al.</i> 2017 <i>Nature</i><b>550</b>, 519-523. (doi:10.1038/nature24056)), in the style of Frequently Asked Questions. We present the theoretical, computational and experimental aspects of network control, and discuss its current capabilities and limitations, together with the next likely advances and improvements. We further present the Python code to enable exploration of control principles in a manner specific to this prototypical organism.This article is part of a discussion meeting issue 'Connectome to behaviour: modelling <i>C. elegans</i> at cellular resolution'.

Rand, Jim et al. (2009) International Worm Meeting "Neuroligin deficient mutants of C. elegans are hypersensitive to oxidative stress and some heavy metals."

Heatherly, Jessica, Hunter, Jerrod, Rand, Jim, McManus, John, Duke, Angie, Mullen, Greg

[International Worm Meeting, 2009]

Neuroligins are postsynaptic cell adhesion molecules that bind specifically to a set of presynaptic membrane proteins called neurexins. Mutations in the human neuroligin genes NLGN3 and NLGN4 are associated with a subset of cases of autism spectrum disorders (Jamain et al., 2003; Laumonnier et al., 2004; Yan et al., 2005). C. elegans has a single neuroligin gene (nlg-1), and approximately one-sixth of C. elegans neurons, including some sensory neurons, interneurons, and a subset of cholinergic motor neurons, express a neuroligin transcriptional reporter (see abstract by Hunter et al.). Null nlg-1 mutants are viable, with a grossly normal nervous system, and although they are not deficient in any major motor functions, they are defective in a subset of sensory behaviors and sensory processing (see abstract by Heatherly et al.). In addition, nlg-1 mutants are hypersensitive to oxidative stress (i.e., paraquat treatment); this is a completely unexpected phenotype for a synaptic protein mutant. Like many other stress-sensitive mutants, nlg-1 mutants have a reduced lifespan and an increased level of oxidatively damaged proteins. nlg-1 mutants are also hypersensitive to inorganic (HgCl2) and organic (thimerosal) mercury compounds and copper compounds, but not to cadmium (CdCl2). There is a body of literature documenting in individuals with autism the presence of biomarkers associated with oxidative stress, and it is striking that a mutation which, in humans, is associated with autism produces a similar oxidative stress phenotype in nematodes. (Supported by a grant from Autism Speaks).

Riga, Amalia et al. (2019) International Worm Meeting "The role of basolateral polarity regulators in epithelial tissue homeostasis."

Boxem, Mike, Pires, Helena, Riga, Amalia

[International Worm Meeting, 2019]

Polarization of epithelial cells into apical and basolateral domains is essential for the functioning of epithelia as selectively permeable barriers. The basolateral Scribble group proteins (Scrib, Lgl, Dlg) were originally identified as tumor suppressors in D. melanogaster and were later shown to play key roles in the establishment and maintenance of polarity in a broad range of cell types. In addition to promoting basolateral identity, Scribble group proteins regulate diverse processes including tissue growth, differentiation and directed cell migration, and therefore are major regulators of tissue development and homeostasis. In C. elegans, loss of LET-413/Scrib and DLG-1/Dlg results in defects in epithelial polarization and junction formation during embryonic development, resulting in embryonic lethality. However, their importance in larval epithelia is less well understood. To study the role of LET-413 in larval epithelia, we generated an allele that allows inducible protein degradation using the auxin-inducible system recently developed for C. elegans (1). We find that ubiquitous degradation of LET-413 in larval tissues results in a developmental arrest. Tissue-specific degradation of LET-413 in the larval intestine does not cause defects in development. In contrast, degradation of LET-413 in the seam cells and hypodermis mimics the developmental arrest seen upon ubiquitous LET-413 degradation. Live-cell imaging of seam cells shows a severe effect on outgrowth and reattachment following the L1 asymmetric division. In addition, we observed that loss of LET-413 results in punctate and discontinuous localization pattern of DLG-1, similar to previous observations in C. elegans embryos. We are currently following the effects of LET-413 inactivation on cell recognition and migration pathways to understand the outgrowth defects of the seam cells. (1) Zhang L, Ward JD, Cheng Z, Dernburg AF. Development. 2015 Dec 15;142(24):4374-84.