Lucinda Carnell et al. (2001) International C. elegans Meeting "Measuring neurotransmitter levels in isolated C.elegans vesicles by HPLC."

Jennifer Hsu, Steven McIntire, Lucinda Carnell

[International C. elegans Meeting, 2001]

We are in the process of developing an assay to measure transmitter content in synaptic vesicles from C.elegans in order to study the function of various vesicular neurotransmitter transporters. We circumvented the problem of obtaining large amounts of tissue sample by measuring transmitter levels using HPLC. Starting with about half a gram of worms, we can by differential centrifugation isolate a crude vesicle fraction. We can demonstrate enrichment of synaptic vesicles in this fraction by the detection of full length unc-47 ::GFP (the GABA vesicular transporter) using Western blot analysis. The presence of unc-47 ::GFP in this fraction suggests it contains synaptic vesicles because this construct is localized to synaptic vesicles and can rescue the unc-47 mutant phenotype (McIntire et al. (1997), Nature 389: p 870-879). We measured amino acid neurotransmitter levels in these fractions by HPLC which is sensitive enough to allow small concentrations (ng/ml) to be reliably quantified. Although we used this procedure to primarily examine the neurotransmitter GABA, this method could also be used to measure quantities of other neurotransmitters such as dopamine, serotonin, octopamine and acetylcholine. In vesicle preparations from N2 animals we demonstrated that GABA is contained in this fraction. As a control for the purity of the synaptic vesicle preparation, we measured GABA levels in unc-47 mutants which should contain no GABA in their vesicles and observed a 3-4 fold reduction compared to N2 animals. We also examined GABA levels in synaptic vesicles of unc-46 mutant animals. unc-46 encodes a novel protein and unc-46 mutants have a similar phenotype to unc-47 animals (K. Schuske and E. Jorgensen, personal communication). Since overexpression of UNC-47 is capable of rescuing the unc-46 mutant phenotype, UNC-46 may function together with UNC-47 to regulate vesicular transport of GABA (K. Schuske and E. Jorgensen, personal communication). We found GABA levels were also 3-4 fold lower in these mutants further supporting UNC-46's role in regulating transport of GABA into synaptic vesicles. This technique can also be used to quantitate neurotransmitter levels in whole worm extracts. For example, unc-25 animals, which are missing the enzyme glutamic acid decarboxylase, have significantly decreased GABA levels in their extracts compared to N2 animals. We currently are focusing our attention on obtaining a purer vesicle preparation to reduce the levels of GABA we see in the unc-47 mutants. Once established, this technique can be used to characterize and identify genes involved in the function and regulation of vesicular transporters in C.elegans .

Lucinda Carnell et al. (2002) West Coast Worm Meeting "Genetic Analysis of the Behavioral Effects of Amphetamine in C. elegans."

Shannon Behrman, Jennifer Hsu, Steven L McIntire, Lucinda Carnell

[West Coast Worm Meeting, 2002]

Amphetamine as well as other psychostimulants such as cocaine and methylphenidate have a high potential for abuse. Their primary mechanism of action is the inhibition of the clearance of dopamine from the synapse via the dopamine plasma membrane transporter (DAT). The CeDAT has been cloned and shown in vitro to bind both amphetamine and cocaine (LD Jayanthi et al., Mol Pharm 54:601-9, 1998). There is also evidence that even a single exposure to psychostimulants can produce long term potentiation on the mesolimbic dopamine system (Ungless et al., Nature 411:583-587, 2001) which is part of the reward pathway activated by drugs of abuse. These synaptic changes may be important for the subsequent addiction to these types of drugs. We are interested in characterizing further the mechanisms that contribute to the effects of amphetamine modulation on dopaminergic pathways. To examine the effects of amphetamine on synaptic transmission in C. elegans we pretreated worms with amphetamine in liquid culture prior to transferring animals to agar plates for behavioral analysis. We found that amphetamine pretreatment resulted in suppression of locomotion, egg laying, and pharyngeal pumping in a dose dependent manner. These behavioral effects are similar to those produced by exposing animals to exogenous dopamine, suggesting amphetamine is acting on dopaminergic pathways. We then initiated a genetic screen to identify mutants that showed reduced sensitivity to the effects of amphetamine on locomotion. We have identified eight candidate mutants and are in the process of mapping and cloning the genes responsible for their resistance. Most of these mutants are visibly wild type with the exception of two of the mutants which appeared hyperactive for locomotion. This hyperactivity was confirmed by assaying for aldicarb sensitivity. The other mutants were found to be either wild-type or resistant to the effect of aldicarb on movement. One of these mutants also displayed reduced sensitivity to the effects of amphetamine on pharyngeal pumping and two other mutants displayed reduced sensitivity to the effects of amphetamine on egg laying. We are characterizing the mutants from our screen for defects in dopamine mediated behaviors. Preliminary results indicate that two of these mutants are defective in the dopamine dependent basal slowing response on bacteria (Sawin et al., Neuron 26:619-631, 2000).

Kai-Chia Yeh et al. (2004) East Asia Worm Meeting "Identification of a kinase that functions in the engulfment of cell-corpses in C. elegans"

Yi-Chun Wu, Tsung-Yuan Hsu, Kai-Chia Yeh

[East Asia Worm Meeting, 2004]

Programmed cell death (PCD) plays important functions during the development of multicellular organisms. After cells undergo PCD, cell corpses are recognized and degraded by engulfing cells. In C.elegans, two redundant genetic pathways act during cell-corpse engulfment : psr-1 , ced-2 , ced-5 , ced-10 and ced-12 in one and ced-1 , ced-6 and ced-7 in the other. To identify more genes that function during cell-corpse engulfment, an RNA-based screen was performed. In the screen we identified a kinase, whose inactivation by RNA interference (please see abstract Tsung-Yang Hsu et.al) increased cell corpses at comma, 1.5-fold and 2-fold stages. We generated polyclonal antibodies against bacterial expressed GST-kinase fusion protein. The antibodies recognized bacterial expressed GST-kinase protein and His-tagged kinase protein expressed in the baculovirus system on western blot. By immunostaining, this kinase appeared to be localized on (or near) the cell membrane in all cells during early and mid-staged embryogenesis. We are currently establishing a kinase assay, and will test if previously identified proteins, which functions in cell-corpse engulfment pathway, are the potential substrates of this kinase.

Jennifer Pilipiuk et al. (2006) European Worm Meeting "Analysis of Epithelial Genes during Postembryonic Development of C. elegans"

Olaf Bossinger, Jennifer Pilipiuk, Gisela Helbig

[European Worm Meeting, 2006]

Jennifer Pilipiuk, Gisela Helbig and Olaf Bossinger. We wish to understand how epithelial polarity and tissue integrity is maintained. Here, we consider the role of DLG-1 (Discs large), LET-413 (Scribble), ERM-1 (Ezrin-Radixin-Moesin), and the catenin-cadherin complex (HMP-1HMP-2HMR-1) during postembryonic development of C. elegans. In the course of embryogenesis these genes play an important role in the establishment and maintenance of epithelial polarity, the formation of the lumen, and cell-cell adhesion. In contrast, during larval and adult development only newly established epithelia (e.g. the spermatheka or the vulva) show severe defects after bacterial RNAi against DLG-1, LET-413 and ERM-1, while the depletion of the catenin-cadherin complex seems not to cause visible defects. Nevertheless, how polarity of already established epithelia (e.g. the intestine or the hypodermis) is maintained during postembryonic development remains elusive. In the case of DLG-1, our results suggest that a small amount of protein is sufficient, but cannot fall below a certain threshold without causing defects. Intestinal and hypodermal polarity during larval and adult development of C. elegans might also depend on other, so far unidentified proteins. A detailed analysis of DLG-1, LET-413 and ERM-1 phenotypes during postembryonic epithelial development in C. elegans will be presented and discussed.

Giurumescu, Claudiu et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Collective movements of substrate neurons during epidermal enclosure"

Chisholm, Andrew, Giurumescu, Claudiu

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

In the embryonic development of the C. elegans nervous system, neuronal precursors undergo several collective movements, including closure of the ventral cleft following gastrulation. We are interested in how these collective movements are controlled and their relationship to other embryonic morphogenetic processes such as epidermal enclosure. To visualize these and other cell movements we have used computer-aided tracking of histone-GFP labeled nuclei. We have developed new software that has allowed semi-automated tracking of >600 nuclei (>520 live nuclei + cell deaths). Details of our methods and algorithms will be presented separately at this meeting (Giurumescu, Hsu and Chisholm, New tools for semi-automated tracking of nuclei in embryos). We find that during epidermal enclosure neurons undergo widespread collective movements. Each phase in epidermal enclosure is accompanied by movements of underlying neurons. The movement of the four epidermal leading cells is paralleled by midline migrations of the RMDD, SMB and anterior DB neurons. Closure of the ventral pocket is accompanied by convergence of the precursors of the ventral cord motor neurons, which appear to displace more medial precursors anteriorly and posteriorly to form the retrovesicular and preanal ganglia. Finally, enclosure of the anterior head involves anteriorly converging movements of a large number of anterior neurons. The widespread and dynamic nature of the neuronal migrations during enclosure raises the question of whether neurons are being passively pushed by epidermal cell movements, or whether the neuronal movements could actively contribute to enclosure. We are using a combination of genetics and laser killing to understand the origin of the forces in these neuronal movements.

Pei-ken Hsu et al. (2004) West Coast Worm Meeting "Structural and Functional Analysis of PSR-1 in C. elegans"

Ming-chia Lee, Yi-Chun Wu, Pei-ken Hsu

[West Coast Worm Meeting, 2004]

In the development of multicellular organisms, the proper removal of cell corpses generated by programmed cell death prevents the possible dispersal of harmful cellular contents from dying cells. A recent study has shown that psr-1 , the C. elegans homolog of human PSR (phosphatidylserine receptor), is important for cell-corpse engulfment (Wang et al., 2003). PSR-1 is proposed to function as a receptor and act upstream of the pathway mediated by CED-2, CED-5, CED-10, and CED-12 signaling molecules, possibly through physical interactions with CED-5 and CED-12 to control the engulfment process. In order to understand how PSR-1 interacts with CED-5 and CED-12, we mapped the binding region in PSR-1 for CED-5 and CED-12 interactions. We found that the amino acids 135-257 are required but not sufficient for CED-5 binding and the amino acids 212-257 of PSR-1 are required and sufficient for CED-12 binding. In addition, PSR-1 is predicted to have one tyrosine phosphorylation site. To investigate its function in cell-corpse engulfment we changed the tyrosine to phenylalanine in PSR-1 and found that the mutant PSR-1 failed to rescue the cell-corpse engulfment defect in psr-1 mutants. Therefore, the tyrosine residue and possibly its phosphorylation is important for PSR-1 function. To explore the expression pattern of PSR-1, we generated polyclonal antibodies against GST-PSR-1 fusion protein. We are currently investigating the subcellular localization of PSR-1. Reference: Wang, X., Wu, Y., Fadok, V.A, Lee, M., Gengyo-Ando. K., Cheng, L., Ledwich, D., Hsu, P., Chen, J., Chou, B., Henson, P., Mitani, S., Xue, D. Science 302, 1563-1566 (2003).

Ala Berdichevsky et al. (2003) International Worm Meeting "A Genetic Screen for New Genes Involved in Aging"

Ala Berdichevsky, Leonard Guarente, Bob Horvitz

[International Worm Meeting, 2003]

Over the last decade, several lines of experiments using C. elegans and other organisms have resulted in progress towards understanding molecular mechanisms of aging. Mutations in genes involved in the insulin-like pathway or in mitochondrial function significantly affect C. elegans lifespan. Research in the field has largely focused on the study of genes that normally function to reduce longevity, that is, genes that mutate to extend lifespan. This focus is primarily a consequence of the difficulties in distinguishing accelerated aging from sickness in short-lived mutants. Recently, Garigan et al.1 and Herndon et al.2 investigated a number of old-age characteristics of C. elegans, such as muscle deterioration, cuticle thickening, and accumulation of autofluorescence. Such observations may help to distinguish between aged and sick worms and therefore facilitate the study of the aging process in C. elegans. The accumulation of a fluorescent pigment in the gut of old worms has long been known to be a marker of aging. The amount of pigment is consistent among wild-type animals of the same age and gradually increases throughout adulthood. We measured the spectrum of the autofluorescence and quantitated the amount of the pigment. At all ages tested, daf-2(e1370) animals accumulated less fluorescence than did wild-type animals, whereas daf-16(mgDf50) animals accumulated more fluorescence than did wild-type worms. These findings correlate with the longer lifespan and delayed aging of daf-2 mutants and the shorter lifespan of daf-16 mutants. To identify genes that when disrupted cause premature aging, we are performing a screen for mutants that show early accumulation of gut autofluorescence. Mutants isolated from this screen will be tested for other aging-related characteristics. 1. Garigan, Hsu, Fraser, Kamath, Ahringer and Kenyon, Genetics 161: 1101-1112 (2002); 2. Herndon, Schmeissner, Dudaronek, Brown, Listner, Sakano, Paupard, Hall and Driscoll, Nature 419: 808-814 (2002).

Ala Berdichevsky et al. (2004) East Coast Worm Meeting "A Genetic Screen for New Genes Involved in Aging"

Leonard Guarente, Ala Berdichevsky, Bob Horvitz

[East Coast Worm Meeting, 2004]

Over the last decade, studies of C. elegans and other organisms have resulted in progress towards understanding molecular mechanisms of aging. Mutations in genes involved in the insulin-like pathway or in mitochondrial function significantly affect C. elegans lifespan. Research in the field has largely focused on genes that normally function to reduce longevity, that is, genes that when mutated extend lifespan. This focus is primarily a consequence of the difficulties in distinguishing accelerated aging from sickness in short-lived mutants. Recently, Garigan et al. (1) and Herndon et al. (2) described characteristics of aging C. elegans , including decline in locomotion, muscle deterioration, and accumulation of autofluorescence in the intestine of aging animals. Such observations help distinguish aged from sick worms and therefore facilitate the study of the aging process in C. elegans . We are interested in identifying and studying genes that function to prevent or delay the aging of C. elegans . Loss-of-function mutations of such genes should result in accelerated aging, similar to human progeria. We are performing a genetic screen for mutants that age prematurely, using accumulation of intestinal autofluorescence as a marker. The accumulation of a lipofuscin-like fluorescent pigment in the gut of old worms has long been known to be a marker of aging. The amount of pigment is consistent among wild-type animals of the same age and gradually increases throughout adulthood. We measured the spectrum of the autofluorescence of old worms and found that emission peaks at 420 nm upon excitation at 350 nm. We are using a filter set with similar excitation and emission values to visualize lipofuscin and to screen for mutants showing early accumulation of this pigment. So far we have screened worms corresponding to 20,000 mutagenized haploid genomes and identified 12 isolates that exhibit premature accumulation of gut autofluorescence. Eight of these isolates have significantly shorter lifespans than the wild type. We are currently mapping the mutations and characterizing the mutants with respect to other aging-related characteristics, such as behavioral decline and tissue deterioration. 1. Garigan, Hsu, Fraser, Kamath, Ahringer and Kenyon, Genetics 161 : 1101-1112 (2002). 2. Herndon, Schmeissner, Dudaronek, Brown, Listner, Sakano, Paupard, Hall and Driscoll, Nature 419 : 808-814 (2002).

Jennifer Schisa et al. (2003) International Worm Meeting "Investigation of chemotaxis mutants in an undergraduate genetics laboratory"

Jennifer Schisa, Katie M Morrison

[International Worm Meeting, 2003]

A three-week series of C. elegans-based laboratories has been implemented in an introductory college genetics course (JS) that is based upon those used successfully in a high school program (KMM) and others (Jennifer Vowels, Debbie Birnby, Wendy Schackwitz, Jim Thomas). These labs are performed near the end of the semester, after students have been introduced to basic molecular biology concepts and at the same time that the lecture discusses the use of genetics to study behavior. The first session is a computer-based laboratory to introduce students to C. elegans and the vast array of information available at the C. elegans WWW server, Wormbase, and NCBI. Students are guided through questions and prompts to learn how to find different types of information such as: the literature published regarding chemotaxis in C. elegans, the names of researchers working on this problem, and the proteins that have been implicated in regulating chemotaxis. The second lab introduces students to the worm via three activities: 1) distinguishing males, adult hermaphrodites, and L4 worms; 2) identifying basic anatomical parts of the worm; and 3) sorting unc, dpy, and rol worms from a plate containing all three mutants. The first and third activity give students the opportunity to learn how to pick worms correctly, a skill they will need in the third lab. At the end of the second lab, students are assigned three unknowns (that they will identify in the third lab via chemotaxis assays) and pick worms to fresh plates in preparation for lab 3. The unknowns include two osm mutants, 1 che mutant, 1 che mutant marked with dpy, and wild-type. The third lab includes an attractance assay (based on Bargmann et al., 1993) and an avoidance assay, each performed with the three unknowns and wild-type controls. Students calculate avoidance and attractance indexes and perform statistical analyses to determine the identity of their unknowns. This series of laboratories could easily be expanded to incorporate student-directed projects, investigating other possible effectors of C. elegans behavior. Bargmann, C.I., Hartweig, E., and Horvitz, H.R. 1993. Odorant-selective genes and neurons mediate olfaction in C. elegans. Cell 74: 515-527.

Xiaochen Wang et al. (2005) International Worm Meeting "Identification and Characterization of the genetic enhancers of the psr-1 mutant"

Monica Darland, Ding Xue, Xiaochen Wang, Chonglin Yang, Adam Seluzicki

[International Worm Meeting, 2005]

Phagocytosis of apoptotic cells is an integral part of the cell death program and an important event in tissue remodeling, suppression of inflammation, as well as regulation of immune responses. During apoptosis, phosphatidylserine (PS), which normally is restricted to the inner leaflet of plasma membrane, is exposed on the surface of apoptotic cells and likely serves as a dying cell signal for engulfment. How PS is externalized, recognized, and used to trigger cell corpse engulfment is largely unclear. Our previous study suggests that psr-1, the C.elegans homologue of phosphatidylserine receptor (PSR), is important for cell corpse engulfment in vivo (1). PSR-1 binds PS preferentially, functions upstream of CED-2, CED-5, CED-12, and CED-10, and may transduce the PS engulfment signal through direct interactions with CED-5 and CED-12. However, PSR-1 is unlikely to be the only engulfment receptor in the ced-2, ced-5, ced-10, and ced-12 signaling pathway, since the psr-1 mutant displays a significantly weaker engulfment defect than that of ced-2, ced-5, ced-12, and ced-10 mutants. Identification of other engulfment receptors that also function through this pathway will help us understand how apoptotic cells are recognized and removed. In order to identify engulfment receptors that may function redundantly with psr-1, we performed a psr-1 enhancer screen to search for mutations that enhance the engulfment defect of the psr-1 mutant. In a screen of 3600 C.elegans haploid genomes, we isolated three candidate mutants: sm211, sm212, and sm237. Preliminary genetic analysis indicates that sm212 affects a known gene, ced-8. We are in the process of mapping the other two mutations, sm211 and sm237. We will also carry out a large scale screen to isolate more mutants. Characterization of the psr-1 enhancers may identify new players that act with psr-1 to mediate cell corpse engulfment. 1. Wang, X.C., Wu, Y.C., Fadok, V., Lee, M.C., Gengyo-Ando, K., Cheng, L.C., Ledwich, D., Hsu, P.K., Chen, J.Y., Chou, B.K., Henson, P., Mitani, S., and Xue, D. (2003). Cell Corpse Engulfment Mediated by C. elegans Phosphatidylserine Receptor Through CED-5 and CED-12. Science 302, 1563-1566.