Sylvia Lee S et al. (2000) East Coast Worm Meeting "Chemosensory modulation of the daf-2/insulin signaling pathway"

Ruvkun GB, Sylvia Lee S

[East Coast Worm Meeting, 2000]

The daf-2 insulin-like, daf-7 TGFb-like, and daf-11 guanylate cyclase signaling pathways regulate dauer formation, whereas daf-2 signaling also controls longevity. Although environmental cues are critical in regulating dauer formation, not much is known about how such environmental signals are integrated into the downstream signaling components. Ciliated sensory neurons play important roles in sensing the environment and epistasis studies have placed cilia mutants upstream of daf-2 and daf-7, but downstream of daf-11, in controlling dauer arrest. Recently, mutations affecting the sensory cilia have also been shown to extend adult lifespan (Apfeld & Kenyon, 1999), a phenotype likely mediated through the daf-2 signaling pathway. Interestingly, double mutants harboring defects in both daf-2 signaling pathway and sensory cilia function exhibit synthetic lethality, possibly due to enhanced daf-2 defects in the mutant animals. We reasoned that genetic screens which identify suppressors of this synthetic lethality would likely further our understanding of the signaling pathway between sensory cilia and the DAF-2 insulin-like receptor and identify outputs from chemosensory neurons which modulate the daf-2 signaling. In a screen of 10,000 EMS-mutagenized daf-2;cilia mutant F2s, we identified ~20 suppressors which exhibited reduced lethality. Progress on the characterization of these mutants will be reported.

Lee, S. et al. (2017) International Worm Meeting "The Novel Utility of the Central Pattern Generator within C. elegans."

Arisaka, K., Lee, S., Hydari, S., Zeng, C.

[International Worm Meeting, 2017]

Central pattern generators, or CPGs, are intrinsic neural rhythms generated in the absence of sensory information. The CPG underlies several diverse and essential behaviors, functionally streamlined and localized for their non-divorceable utility. Traditionally, the CPG has been understood unjustly in the confines of rhythmicity and motor processes. Beginning and framing the C. elegans as a foundational model for the incredible versatility of the basic CPG, we delve into the CPGs unconventional essentiality in sensorimotor integration, substantiation of the physical principles of causality and locality, and its novelty in the context of understanding more complex behaviors, such as human saccadic eye movement. We then discuss the global trends and emergent properties of such rhythms across several organisms that characterize the manifestation of a meaningful patterned neural activity, premised again, in the lens of the highly elucidative neural system of the C. elegans. Penultimately, we demarcate the morphological evolution of the neural information code and neuromodulatory vehicles that biological pressures have enforced on the increasingly complex design of organisms, starring the C. elegans, once again, as the basis and transitional prism for the evolution of this information code from an amplitude based phenomena (graded potential code) to a frequency based phenomena (action potential code). Ultimately, the thematic motif of this article demonstrates the immense utility in understanding the overlooked 302-neuron nervous system of the C. elegans towards unveiling powerful truths in the evolutionary ladder and trending homologies in more complex organisms- of particular interest here, pattern generated behaviors. Our analyses incepts and extends from a compilation of personal lab discoveries and extrinsic literary interpretations of the idiosyncrasies of the C. elegans nervous system, and has been protracted homologies across various Animalia and processes, including the aforementioned saccades in human oculomotor codes.

Lee, S. et al. (2019) International Worm Meeting "A novel repeat-containing protein supports male and female reproduction in C.elegans."

Ahnn, J., Hahn, Y., Durnaoglu, S., Lee, S., Jung, H.

[International Worm Meeting, 2019]

Here, we report that CNP-2 is a novel protein that contains repeats, which are frequently found in proteins displaying flexibility. GFP driven by cnp-2 promoter is highly expressed in the spermatheca, the spermathecal-uterus valve, and male tail, which are elastic. cnp-2 is critical in reproductive processes in both hermaphrodites and male. KSP repeat domain rescued defective phenotypes of cnp-2 lf mutants, although it was distantly related and shows low sequence similarity and identity to CNP-2. Expression of KSP repeats from both worm obscurin and human NF-M reversed most defects in cnp-2 worm mutants in a repeat-number-dependent manner; a greater number of KSP repeats showed more efficient effects. Altogether, the data suggest that cnp-2 is a KRSTPE-rich repeat protein that shares common features with the KSP repeats of NF-M, including regulatory molecular extension to support inter-molecular interactions, and contractile and elastic cellular structures.

Lee, S. et al. (2011) International Worm Meeting "Secreted VAPB/ALS8 major sperm protein domains regulate mitochondrial fusion and fission machinery localization in body wall muscle."

Miller, M., Lee, S.

[International Worm Meeting, 2011]

Mitochondria are dynamic organelles that are critical for energy homeostasis and cell survival. Abnormalities in mitochondrial morphology and function are associated with diverse neurodegenerative diseases, but the mechanisms are not understood. C. elegans VPR-1 and human VAPB/ALS8 are homologous proteins with an N-terminal major sperm protein domain (MSP) and C-terminal transmembrane domain. A missense mutation (P56S) in the VAPB/ALS8 MSP domain is associated with amyotrophic lateral sclerosis and late-onset spinal muscular atrophy, two neuropathies characterized by progressive muscle atrophy and motor neuron degeneration. Our lab has shown that the VPR-1 MSP domain is secreted by neurons and functions as a hormone that signals through Roundabout and Lar-like growth cone guidance receptors to modulate mitochondrial localization, division (fission), and function in body wall muscle (see abstract by Han et al.). MSPs signal to mitochondria by regulating actin-related protein 2/3 complex (Arp2/3) activity, stabilizing mitochondria at actin-rich I-bands. Here I show that VPR-1 regulates the localization of ARX-2, a component of the actin-related protein 2/3 complex, as well as multiple proteins that control mitochondrial fusion and fission in body wall muscle. In wild-type hermaphrodites, ARX-2 primarily localizes to the dense bodies, where it functions to promote mitochondrial elongation along I-bands. However, in vpr-1 null mutants, ARX-2 mislocalizes to the muscle belly along with mitochondria. Together with data presented by Han et al., my results support the model that secreted MSPs promote Arp2/3 complex localization at the dense bodies. In vpr-1 mutants, mislocalized Arp2/3 causes ectopic actin filaments in the muscle belly that displace mitochondria from the I-bands and modulate mitochondrial fission and fusion sites. These abnormal events promote mitochondrial mobility, elongation, and branching, and inhibit energy metabolism. My results reveal a critical role for the Arp2/3 complex in modulating the mitochondrial fission/fusion balance. I will also present preliminary data suggesting that the survival of motor neuron 1 protein SMN-1 functions to transport RNAs to the dense bodies that are critical for Arp2/3 activity and mitochondrial morphology.

Lee S et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Inhibition of Mitochondrial Respiration Extends C. elegans' Lifespan via Reactive Oxygen Species that Activate HIF-1"

Kenyon C, Lee S, Hwang A B

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Inhibition of mitochondrial respiration can increase the lifespans of yeast, C. elegans, Drosophila and mice. However, the molecular mechanisms by which impaired respiration promotes longevity are poorly understood. One environmental condition that reduces rates of respiration is hypoxia (low oxygen). Thus it is possible that mechanisms that sense oxygen levels play a role in the longevity response to reduced respiration rates. While performing a genome-wide RNAi screen to identify genes that affect the activity of the hypoxia inducible factor HIF-1, we found that knockdown of many genes involved in respiration can activate HIF-1. This increased HIF-1 activity seems to play an essential role in the longevity caused by reduced respiration, since we found that the long lifespan of respiration-defective clk-1 and isp-1 mutants was significantly decreased by hif-1 mutations. We also showed that the longevity conferred by down-regulation of vhl-1 (ubiquitin ligase) or egl-9 (prolyl hydroxylase), which normally stabilizes HIF-1 protein, was not increased by mutations that inhibit respiration. These data suggest that the respiration mutations and the defects in the vhl-1 and egl-9 genes act in the same pathway. As a potential mechanism by which conditions that reduce respiration increase HIF-1 activity, we hypothesized that reactive oxygen species (ROS) levels rise in mutants with defective respiration, and that this ROS, in turn, may activate HIF-1. We showed that the respiratory clk-1 and isp-1 mutants displayed elevated levels of ROS. Moreover, we found that chemically generated ROS, produced by using paraquat, can up-regulate the HIF-1 activity. Finally, we demonstrated that treatment of low level (e.g. 250 muM) of paraquat can increase the lifespan of C. elegans in a hif-1-dependent manner. Taken together our data show that longevity caused by inhibition of respiration activates HIF-1 and that this process is mediated by ROS. In contrast to the free radical theory of aging, which suggests that ROS generated from mitochondria are one of the main determinants of aging, our findings indicate that ROS generated by mitochondrial dysfunction can actually be beneficial to aging.

Nicole F Liachko et al. (2005) International Worm Meeting "Characterization of putative DAF-16 target genes"

Siu Sylvia Lee, Nicole F Liachko

[International Worm Meeting, 2005]

The forkhead transcription factor DAF-16 is a key mediator of longevity, metabolism, and development. An informatic search using the known DAF-16 DNA recognition sequence to predict putative DAF-16 target genes has been performed (1). To further characterize the predicted DAF-16 target genes, we are optimizing a chromatin immunoprecipitation (ChIP) procedure using larvae and adult worms to demonstrate the presence of DAF-16 at the promoter of the target genes. In addition, we are using a promoter GFP fusion strategy to examine the expression of a subset of the DAF-16 target genes in worms. This approach allows study of when and where the target genes are expressed, as well as study of the dynamic regulation of the expression of the target genes in reponse to environmental stresses. Preliminary results show cell-type and developmental stage specific expression of the genes of interest. (1) S.S. Lee, S. Kennedy, A.C. Tolonen, G. Ruvkin, Science 300, 644 (2003).

Pinan-Lucarre, Berangere et al. (2009) International Worm Meeting "Transgenic C. elegans expressing the fungal prion protein HET-s as a model for the study of amyloid infectivity and toxicity."

Qiao, Yujie, Saupe, Sven, Pinan-Lucarre, Berangere, Driscoll, Monica

[International Worm Meeting, 2009]

Amyloids are protein fibers rich in beta sheet structure that are associated with numerous neurodegenerative diseases, including Alzheimer disease. Amyloids have two major biological properties. First, they can be infectious--meaning that they can spread the altered amyloid conformation to the same protein or even among proteins of distinct primary sequence (the latter phenomenon is known as cross-polymerization). Infectious amyloids are called prions. Second, amyloids can be toxic, whether they are infectious or not. Both properties have tremendous fundamental and biomedical impact. [Het-s] is a prion of the fungus Podospora anserina involved in a defense cell suicide mechanism named heterokaryon incompatibility, which disables mixing of different strains by somatic fusion, an ubiquitous feature in filamentous fungi. The HET-s protein exists in a soluble state or in an aggregated, prion state named [Het-s]. The [Het-s] prion is not toxic by itself in P. anserina or in yeast. However cell death is rapidly triggered by the lethal interaction between 2 alleles of the het-s locus: het-S (het-"large S") and het-s (het-"small s") when the HET-s protein adopts the prion form. This prion has been extensively characterized and it is the only prion for which tridimensional structure is available to date. The critical point is that in the [Het-s] system, transgenic expression of het-s allows propagating a non-toxic prion while co-expression of het-s and het-S generates toxicity. We constructed strains expressing het-s or the prion domain only het-s(218-289) (the 218-289 fragment of the protein) as GFP fusions under the control of the ubiquitous promoter dpy-30. The fluorescence of pdpy-30het-s::gfp appears to be mainly diffuse in the cytoplasm while it shows strong aggregation in pdpy-30het-s(218-289)::gfp strains. We constructed strains expressing het-s(218-289)::gfp in the touch neurons using mec-4 promoter and observed that the fusion protein is mostly aggregated into a large fiber spanning the cell body and the proximal part of the axon. These aggregates remain to be shown as infectious [Het-s] amyloids. In future experiments, we will aim to generate neuronal toxicity through the co-expression of het-s(218-289) and het-S. This C. elegans model for prion studies, filling a gap between simple fungal systems and highly complex mammalian systems, might allow a better understanding of the molecular and cellular basis of amyloid infectivity and toxicity.

Dana L. Miller et al. (2007) International Worm Meeting "Adaptation to hydrogen sulfide increases thermotolerance and lifespan."

Mark B. Roth, Dana L. Miller

[International Worm Meeting, 2007]

Hydrogen sulfide (H₂S), which is naturally produced in animal cells, has been shown to effect physiological changes that improve the capacity of mammals to survive environmental changes. We have investigated the physiological response of C. elegans to H₂S to begin to elucidate the molecular mechanisms of H₂S action. We show that nematodes exposed to H₂S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. However, we observed that animals exposed to H₂S had increased thermotolerance and lifespan and survived subsequent exposure to otherwise lethal concentrations of H₂S. Increased thermotolerance and lifespan is not observed in the sir-2.1(ok434) deletion mutant exposed to H₂S. However, mutants in the insulin signaling pathway (both daf-2 and daf-16), animals with mitochondrial dysfunction (isp-1 and clk-1) and a genetic model of caloric restriction (eat-2) all exhibit H₂S-induced increased thermotolerance. These data suggest that H₂S activates a pathway including SIR-2.1 that is separate from dietary restriction and insulin signaling that results in increased lifespan. Moreover, these studies suggest that SIR-2.1 activity may translate environmental change into physiological alterations that improve survival. It is interesting to consider the possibility that the mechanisms by which H₂S increases thermotolerance and lifespan in nematodes are conserved, and that studies using C. elegans may help explain beneficial effects observed in mammals exposed to H₂S.

Oppenheimer AJ et al. (1995) International C. elegans Meeting "G-PROTEIN SIGNALING IN C. ELEGANS"

Kaplan JM, Oppenheimer AJ

[International C. elegans Meeting, 1995]

We are interested in determining how G-proteins modulate the activity of ion channels in the C. elegans nervous system. To generate behavioral phenotypes dependent on G-protein signaling, we targeted expression of mammalian G-proteins to a set of neurons including those controlling foraging and locomotion (RMD, AVA, AVB, AVD, and PVC). Ectopic expression of several G-proteins under the control of the not-3 promoter results in behavioral defects. Expression of constitutively active rat G-alpha-i1 causes uncoordinated forward and backward locomotion. Expression of wild-type rat G-alpha-s impairs backward locomotion, while constitutively active rat G-alpha-s causes defective backward locomotion and lethargy. In order to identify components of the G-alpha-s signaling pathway, we have initiated a screen for mutations that suppress the behavioral effects of activated G-alpha-s expression. In a screen of approximately 7000 haploid genomes, we have identified 11 independent mutations that improve backward locomotion. Several of these mutations result in a hyperactive phenotype in the absence of activated G-alpha-s expression. Mapping and complementation tests will determine whether the suppressors are allelic to other hyperactive mutants isolated by D. Elkes and J. Kaplan. By cloning the suppressor genes, we hope to identify ion channels regulated by G-alpha-s as well as other components of the G-alpha-s signaling pathway. We also plan to determine whether the behavioral effects of G-alpha-s expression are mediated by known second messenger systems. We are first testing the role of cAMP-dependent protein kinase (PKA), because it is activated by G-alpha-s in other systems and has been shown to phosphorylate ion channels. If G-alpha-s signals through PKA, increasing the level of PKA activity should mimic the effect of activated G-alpha-s expression, and inhibiting PKA should suppress the effect of activated G- alpha-s expression.

Chaudhary, S. et al. (2019) International Worm Meeting "A strategy for neuron identification in whole-brain videos."

Lee, S., Patel, D., Chaudhary, S., Lu, H.

[International Worm Meeting, 2019]

A current bottleneck in generating meaningful interpretation of the neuron activity dynamics extracted from C. elegans whole-brain videos is determining the identity of neurons. Previous strategies to determine neuron identity include, manual comparison of images with an atlas [1] and polychromatic barcoding of neurons [2]. The former strategy is labor intensive, prone to human bias and error, and requires expert supervision. The latter strategy requires custom designed microscope with several excitation-emission channels that may not be available to all researchers. Here we present an alternative strategy based on neuronal landmarks for assisting researchers in unbiased identification of neurons. We developed a framework [3] that uses known geometrical relationships among positions of neurons and automatically generates a probabilistic identity for each neuron in whole brain stacks. We tested the effect of neuron position variability on prediction accuracy by perturbing ideal data (atlas data) to mimic real images. Next, we established that either spatially distributed landmarks or landmarks in lateral ganglion will provide optimal prediction accuracy. Guided by our analysis, we generated whole-brain imaging strains with CyOFP-labeled neuronal landmarks in the head, which are easily identified in each worm. They establish a coordinate system with which neurons are identified by matching to the atlas. We also validated the automatic prediction accuracy on many real worm data sets. Further, using CyOFP enables 3-channel (GCaMP, RFP and landmarks) imaging with only 2 emission-excitation filter sets that are commonly available, thus freeing a channel for other purposes such as optogenetic manipulations. We use this strategy to compare neurons' responses to chemical stimulation in different individuals, and show how experimental context can be included in the prediction framework as additional constraint. We expect that our algorithm will help realize the full potential of whole-brain imaging techniques by enabling complex experiments and analyses using methods that require neuron identity. References [1] Kato S, et al. Cell. 2015;163: 656-669. doi:10.1016/j.cell.2015.09.034 [2] Personal communication (Ev Yemini and Oliver Hobert) [3] Lafferty J, et al. ICML '01 Proc Eighteenth Int Conf Mach Learn. 2001;8: 282-289. doi:10.1038/nprot.2006.61