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Kida, Katarzyna, Blacque, Oliver E., Williams, Corey L., Leroux, Michel R., Yoder, Bradley K., Li, Chunmei, Jensen, Victor L.
[
International Worm Meeting,
2011]
C. elegans perceives its environment mainly by way of sensory neurons which have cilia at the distal ends of dendrites, much like mammals can smell with the use of olfactory cilia or can see with photoreceptor cilia. In studying several human disorders involving cilia dysfunction (ciliopathies), we have uncovered a novel molecular pathway necessary for ciliogenesis. The disorders in question-nephronophthisis (NPHP), Meckel syndrome (MKS), Joubert syndrome (JBTS), Senior-Loken syndrome (SLSN), and Leber congenital amaurosis (LCA)-present with overlapping ailments, such as retinopathy, kidney disease, liver fibrosis and brain malformations. They also show considerable allelism between at least twelve causative genes, suggesting a common molecular aetiology that remains unexplained. We demonstrate using C. elegans that the recently-identified MKS-6 and MKS-2 proteins, together with MKS-1, MKSR-1, MKSR-2, MKS-5, NPHP-1 and NPHP-4, collectively function at the base of cilia, in a region termed transition zone (TZ), to orchestrate cilium formation. Specifically, the proteins act as two distinct modules, which we term MKS and NPHP, to facilitate basal body-transition zone anchoring to the membrane; disruption of the TZ proteins results in defects in prominent ciliary TZ and axoneme formation defects, and thus, chemosensory anomalies. This first pathway is independent of a second pathway specifically required for the formation and function of the ciliary organelles, involving intraflagellar transport (IFT) and Bardet-Biedl syndrome (BBS) proteins. Our genetic and cell biology analyses reveal a hierarchical organisation of the TZ proteins, with MKS-5 as the central anchor, followed by B9 domain-containing proteins (MKS-1, MKSR-1, MKSR-2). Together, our findings expand the interaction network of ciliopathy-associated proteins and suggest a two-stage ciliogenic pathway that first involves transition zone proteins, followed by an intraflagellar transport (IFT)-dependent formation of the remaining axoneme.
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Bialas, Nathan, Inglis, Peter, Kida, Katarzyna, Leroux, Michel, Li, Chunmei, Blacque, Oliver
[
International Worm Meeting,
2009]
Cilia are membrane-ensheathed organelles with motile and/or sensory functions that are important for a wide array of physiological and developmental processes. Many of the proteins destined for localisation to cilia are trafficked on post-Golgi vesicles that must fuse at the base. The proteins may then be captured by a non-vesicular intraflagellar transport (IFT) machinery that docks at transitional fibers and is mobilised within the organelle by virtue of kinesin motor(s). The transitional fibers, along with a neighbouring region (transition zone) possessing Y-shaped crosslinks, join the microtubule axoneme to the membrane and as such form a ''ciliary gate'' that restricts vesicle entry and perhaps also acts as a molecular gatekeeper akin to the nuclear pore complex. However, the components that form or maintain such a ciliary gate are unknown. Candidates include approximately 12 different proteins, all of which are implicated in ciliopathies collectively characterised by retinal degeneration, kidney disease and other ailments; these proteins localise at or near the transition zone and perform essentially unknown roles related to cilia function. Here, we demonstrate using C. elegans that a functional interaction between a novel C2 domain-containing protein (MKS-6), recently implicated in the ciliopathy Meckel syndrome, and the Nephrocystin protein NPH-4, is required for the structural integrity of this ciliary gate. The loss of these two proteins causes a disruption in the ultrastructure of the transition zone region, characterised by an expanded membrane, absence of Y-links and the accumulation of membranous-type material. Our findings expand the interaction network of proteins present within the ciliary transition zone region, and point to the importance of ciliopathy-associated proteins in maintaining a functional ciliary gate.
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[
C.elegans Neuronal Development Meeting,
2008]
Meckel syndrome (MKS) is a developmental disorder associated with central nervous system malformations, cystic kidney disease, liver fibrosis and polydactyly. An identifying feature of MKS1, one of three MKS-associated proteins identified to date, is the presence of a B9 domain of unknown function. Our comprehensive phylogenetic analyses reveal that this domain occurs exclusively within a family of three proteins distributed widely in ciliated organisms. We show that all three C. elegans B9 domain-containing proteins, MKS-1 and MKS1 related proteins 1 and 2 (MKSR-1, MKSR-2), localise to transition zones (akin to basal bodies) at the base of sensory cilia. Their subcellular localisation is largely co-dependent, consistent with a functional relationship between the three proteins. Importantly, this localisation is evolutionarily conserved, since the three human orthologues also localise to basal bodies. Single, double and triple C. elegans mks/mksr mutants do not display overt ciliary structure defects based on fluorescencent cilia markers and electron microscopy observations. Moreover, live imaging of GFP-tagged intraflagellar transport (IFT) proteins reveal no anomalies in the ciliary transport system that is responsible for the transport of ciliary cargo, and hence, ciliogenesis. Lastly, no chemosensory defects are observed in the mutants. We demonstrate, however, genetic interactions between all double mks/mksr mutant combinations, which manifest as an increase in lifespan. Such a longevity phenotype is often associated with clear defects in ciliary structures, for example those observed in IFT gene mutants. We also show that the lifespan changes are mediated through anomalies in the DAF-2-DAF-16-dependent insulin signaling pathway. These results suggest that the B9 domain-containing proteins play important roles in supporting the insulin-signaling pathway at the base of cilia, perhaps by regulating the entry of particular signaling molecules. Overall, our findings therefore demonstrate functional interactions between members of a novel protein family, and provide new insights into the molecular etiology of a pleiotropic human disorder.
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[
J Pharmacol Exp Ther,
2015]
NaCT (SLC13A5) is a Na(+)-coupled transporter for Krebs cycle intermediates and is expressed predominantly in the liver. Human NaCT is relatively specific for citrate compared with other Krebs cycle intermediates. The transport activity of human NaCT is stimulated by Li(+), whereas that of rat NaCT is inhibited by Li(+). We studied the influence of Li(+) on NaCTs cloned from eight different species. Li(+) stimulated the activity of only NaCTs from primates (human, chimpanzee, and monkey); by contrast, NaCTs from nonprimate species (mouse, rat, dog, and zebrafish) were inhibited by Li(+). Caenorhabditis elegans NaCT was not affected by Li(+). With human NaCT, the Li(+)-induced increase in transport activity was associated with the conversion of the transporter from a low-affinity/high-capacity type to a high-affinity/low-capacity type. H(+) was able to substitute for Li(+) in eliciting the stimulatory effect. The amino acid Phe500 in human NaCT was critical for Li(+)/H(+)-induced stimulation. Mutation of this amino acid to tryptophan (F500W) markedly increased the basal transport activity of human NaCT in the absence of Li(+), but the ability of Li(+) to stimulate the transporter was almost completely lost with this mutant. Substitution of Phe500 with tryptophan in human NaCT converted the transporter from a low-affinity/high-capacity type to a high-affinity/low-capacity type, an effect similar to that of Li(+) on the wild-type NaCT. These studies show that Li(+)-induced activation of NaCT is specific for the transporter in primates and that the region surrounding Phe500 in primate NaCTs is important for the Li(+) effect.
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[
International C. elegans Meeting,
1997]
Lithium (Li) has long been known to have teratogenic effects on the development of many organisms, including sea urchin, Xenopus and Dictyostelium. In Li-treated Xenopus embryos, ventral blastmeres are respecified to develop into dorsal structures, leading to dorsalized embryos lacking ventral mesodermal tissues. In Dictyostelium, Li alters the fate of prespore cells to become prestalk cells instead. Besides teratogenic effects, Li is also known to be a most effective treatment of manic-depressive illness.!@Although several models have been proposed to explain Li action, the molecular mechanism has remained unclear. Recently, GSK (glycogen synthase kinase)-3b was proposed to be a target of Li, suggesting that Li affects the wnt signaling pathway. To understand the mechanism of Li action, I first examined the effect of Li on C. elegans embryogenesis. I inoculated N2 animals at the late L4 stage onto NG plates containing 20 mM LiCl, incubated them at 20 oC. The number of eggs produced by treated animals was reduced to about half of the untreated control. Although cell division seemed to proceed, no embryos hatched on Li plates. Treated-embryos developed to produce gut granules, but did not execute normal morphogenesis at later embryonic stages. To identify genes involved in the action of Li, I have begun to screen for Li-resistant mutants that propagated on Li-containing medium. Several mutants were isolated, and one of them was mapped on the left side of
unc-42 on chromosome V. On Li plates, the hatching rate of mutant eggs cross-fertilized by wild-type males was essentially the same as that for self-fertilized mutant eggs. On the contrary, no wild-type eggs cross-fertilized by mutant males hatched on Li-containing plates. So, this mutation appeared to be maternal. Further genetic analyses of the mutants and the observation on the cellular phenotype of Li-treated embryos are underway.
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[
Worm Breeder's Gazette,
1996]
Lithium (Li) has long been known to have teratogenic effects on the development of many organisms, including sea urchin, Xenopus and Dictyostelium. In Li-treated Xenopus embryos, ventral blastmeres are respecified to develop into dorsal structures, leading to dorsalized embryos lacking ventral mesodermal tissues. In Dictyostelium, Li alters the fate of prespore cells to become prestalk cells instead. Besides teratogenic effects, Li is also known to bea most effective treatment of manic-depressive illness. Although several models have been proposed to explain Li action, the molecular mechansm remains unclear. The most widely accepted model is the inositol depletion hypothesis, in which Li is thought to affect inositol phosphate turnover by inhibiting inositol monophosphatase, thus resulting in the depletion of endogenous inositol. To understand the mechanism of Li action, I first examined the effect of Li on C. elegans embryogenesis. I inoculated N2 animals at the late L4 stage onto NG plates containing 20 mM LiCl, incubated them at 20 C and observed the laid embryos. The number of eggs produced by treated animals was reduced to about half of the untreated control. Although cell division seemed to proceed, no embryos hatched on Li plates. Treated-embryos developed to produce gut granules, but did not execute normal morphogenesis at later embryonic stages. To identify genes involved in the action of Li, I have begun to screen for Li-resistant mutants, which propagated on Li-containing medium. So far, I obtained one mutant. The mutation was tentatively assigned to chromosome V. Preliminary genetic analysis showed that the mutation showed maternal effect. On Li plates, the hatching rate of mutant eggs cross-fertilized by wild-type males was essentially the same as that for self-fertilized mutant eggs. On the contrary, no wild-type eggs cross-fertilized by mutant males hatched on Li-containing plates. I am now trying to isolate other mutants and also to identify early defects of embryogenesis caused by lithium treatment. I would like to thank J. Miwa for encouragement and discussions.
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[
J Appl Toxicol,
2015]
Lithium (Li) has been widely used to treat bipolar disorder, and industrial use of Li has been increasing; thus, environmental pollution and ecological impacts of Li have become a concern. This study was conducted to clarify the potential biological effects of LiCl and Li(2)CO(3) on a nematode, Caenorhabditis elegans as a model system for evaluating soil contaminated with Li. Exposure of C. elegans to LiCl and Li(2)CO(3) decreased growth/maturation and reproduction. The lowest observed effect concentrations for growth, maturation and reproduction were 1250, 313 and 10 000m, respectively, for LiCl and 750, 750 and 3000m, respectively, for Li(2)CO(3). We also investigated the physiological function of LiCl and LiCO(3) in C. elegans using DNA microarray analysis as an eco-toxicogenomic approach. Among approximately 300 unique genes, including metabolic genes, the exposure to 78m LiCl downregulated the expression of 36 cytochrome P450, 16 ABC transporter, 10 glutathione S-transferase, 16 lipid metabolism and two vitellogenin genes. On the other hand, exposure to 375m Li(2)CO(3) downregulated the expression of 11 cytochrome P450, 13 ABC transporter, 13 lipid metabolism and one vitellogenin genes. No gene was upregulated by LiCl or Li(2)CO(3). These results suggest that LiCl and Li(2)CO(3) potentially affect the biological and physiological function in C. elegans associated with alteration of the gene expression such as metabolic genes. Our data also provide experimental support for the utility of toxicogenomics by integrating gene expression profiling into a toxicological study of an environmentally important organism such as C. elegans.
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[
J Biol Chem,
2008]
Lithium (Li+) has been used to treat mood affect disorders, including bipolar, for decades (1;2). This drug is neuroprotective and has several identified molecular targets. However, it has a narrow therapeutic range and the underlying mechanism(s) of its therapeutic action is not understood. Here we describe a pharmacogenetic study of Li+ in the nematode Caenorhabditis elegans. Exposure to Li+ at clinically relevant concentrations throughout adulthood increases survival during normal aging (up to 46% median increase). Longevity is extended via a novel mechanism with altered expression of genes encoding nucleosome-associated functions. Li+ treatment results in reduced expression of the worm ortholog of LSD-1 (T08D10.2), a histone demethylase; knockdown by RNA interference (RNAi) of T08D10.2 is sufficient to extend longevity (~25% median increase), suggesting Li+ regulates survival by modulating histone methylation and chromatin structure.
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[
International C. elegans Meeting,
2001]
Little is known about how ions permeate the C. elegans gut and cuticle. How various ions influence development and behavior is also not understood. One ion with considerable impact in human biology is Li + , which modulates bipolar disorder by an unknown mechanism. In particular, the targets of lithium that cause side effects remain to be identified. We are using C. elegans to genetically identify targets of lithium and to elaborate on mechanisms of transport, which may have an impact on human health. Li + has dosage-dependent effects on C. elegans embryos. When L4 animals mature on NGM plates with Li + added to the final concentrations of 10mM-20mM, they produce embryos that are unable to hatch. We demonstrated that this failure to hatch is due to defects in cytokinesis that result in multi-nucleated embryos and symmetrically partitioned cells. Li + also has a dosage-dependent effect on larval development. When adult hermaphrodites are permitted to lay embryos on 10mM-20mM Li + NGM plates, the resulting offspring experience a developmental delay proportional to the concentration of Li + . The offspring become progressively paralyzed as they reach adulthood. We demonstrated earlier that there is a delay in the entry into the S phase of the cell cycle larval stages. To learn more about the biology of Li + sensitivity, we screened for Li + resistant mutants. We developed three screens that took advantage of the embryonic arrest and the larval delay caused by Li + . We isolated one mutant
bz71 , in a screen of 44,000 haploid genomes, that is resistant to both the larval and embryonic blocks of 16mM Li + .
bz71 is dominant. Using classical mapping techniques, we positioned it on LGIII between
unc-32 and
dpy-18 . We are currently using SNP strategy to obtain a higher resolution map and we hope to report on the identification and cloning of this locus.
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[
Worm Breeder's Gazette,
1992]
Characterization of the axonal guidance and outgrowth gene
unc-33 W. Li, R. K. Herman and J. E. Shaw Department of Genetics and Cell biology, University of Minnesota, St Paul, MN 55108