Srinivasan S (2015) Annu Rev Physiol "Regulation of body fat in Caenorhabditis elegans."

Srinivasan S

[Annu Rev Physiol, 2015]

Over the past decade, studies conducted in Caenorhabditis elegans have helped to uncover the ancient and complex origins of body fat regulation. This review highlights the powerful combination of genetics, pharmacology, and biochemistry used to study energy balance and the regulation of cellular fat metabolism in C. elegans. The complete wiring diagram of the C. elegans nervous system has been exploited to understand how the sensory nervous system regulates body fat and how food perception is coupled with the production of energy via fat metabolism. As a model organism, C. elegans also offers a unique opportunity to discover neuroendocrine factors that mediate direct communication between the nervous system and the metabolic tissues. The coming years are expected to reveal a wealth of information on the neuroendocrine control of body fat in C. elegans.

Supriya Srinivasan et al. (2007) International Worm Meeting "The Role of Serotonin in Fat Regulation."

Kaveh Ashrafi, Supriya Srinivasan, Leila Sadegh

[International Worm Meeting, 2007]

The neurotransmitter serotonin is an important sensor of metabolic status and food availability in many species. In C. elegans, the exogenous administration of serotonin has a profound, dose-dependent reduction in fat content and interestingly, a parallel increase in food intake. The pharmacological data are corroborated by genetic evidence from the tph-1(mg280)mutant which lacks the ability to make serotonin. Animals deficient in serotonin production have increased fat and reduced food intake. To understand the relationship between the serotonergic regulation of feeding rate and fat content, we have used a combination of forward genetics, RNAi-mediated gene inactivation and pharmacology to identify molecular components that underlie these processes. Interestingly, we have identified genes that fall into 3 classes: i) those required for reduced fat content of serotonin-treated animals without affecting feeding rate, ii) those required for increased feeding rate of serotonin-treated animals without concomitant effects on fat accumulation and iii) genes that suppress both the fat content and the feeding rate of serotonin-treated animals. Tissue localization experiments show that genes regulating fat content are expressed either in the nervous system or in the intestinal fat-storing cells, while genes regulating feeding rate are expressed predominantly in the nervous system of animals. We have also found that serotonin reduces fat content by regulating fatty acid oxidation pathways rather than fatty acid synthesis pathways. Together, these results suggest that serotonin coordinately modulates behavior and metabolism to regulate fat content. Currently, we are focused on deciphering molecular mechanisms that couple neuronal serotonergic signaling to intestinal fat oxidation pathways.

Jagan Srinivasan et al. (2007) International Worm Meeting "Evolution of a sensory response network."

Jagan Srinivasan, Paul W. Sternberg

[International Worm Meeting, 2007]

We are trying to understand how neural circuits evolve to mediate ethologically relevant behaviors. While model systems help elucidate how sensory information from diverse environmental conditions is represented and processed in the neural, remarkably little is known about the evolution of neural circuits, and how selective forces shape the final architecture of neural circuits and processing circuits (Dumont & Richardson, 1986, Science). Nematodes are an ancient phylum comprising millions of species from diverse habitats. They have molecularly diverse nervous systems which in principle can help them sense several types of environmental stimuli. To trace the evolutionary history of such a sensory repertoire, we tested three different avoidance behaviors; osmotic avoidance, response to nose touch, and volatile chemical repellence in six diverse species of free-living nematodes, a) Caenorhabditis elegans (N2), b) Caenorhabditis briggsae (AF16), c) Caenorhabditis sp. 3 (PS1010), d) Pristionchus pacificus (PS312) e) Cruznema tripartitum (PS1351) and f) Panagrellus redivivus (PS2298). We found that all species tested exhibit the three avoidance behaviors. However, we also find that sensory sensitivity to the different stimuli differs among the tested nematode species. In C. elegans , response to these stimuli are mediated by the ‘polymodal ASH neurons (Kaplan and Horvitz PNAS, 1993, Hart et al. Nature, 1995, Hilliard et al Curr Biol. 2002). We identified the pairs of putative ASH neurons in different nematode species by their anatomical positions and ablated them. Ablation of the ASH neurons in these species resulted in an inability to avoid these stimuli. By further ablation experiments, we find that there is increase or decrease in the set of sensory neurons mediating osmosensation and mechanosensation. In P. pacificus , osmosensation involves the ADL neuron. In Caenorhabditis sp. 3 , mechanosensation is solely mediated by the ASH neuron as compared to 3 neurons in C. elegans . The overall conservation of ASH mediated behaviors suggests that polymodality is an ancestral feature and is evolutionarily stable, but can evolve by alterations in the level of sensitivity and the relative contributions of sensory neurons.

Srinivasan J et al. (1985) Worm Breeder's Gazette "isolation of mec mutants in TR679."

Chalfie M, Srinivasan J, Goldstein R, Mindich J

[Worm Breeder's Gazette, 1985]

We have isolated nineteen mec mutants from the mut(r459) strain TR679 (kindly provided by Phil Anderson). Mutants were picked at 20 C from plates grown from strips from dauered plates. When a mutant was found, all plates from the same dauered plate were discarded. Sets of plates that yielded no mutants were used as sources of additional dauers for the next rounds of screening. Thus, each mutant was presumed to be an independent isolate (but see below). As seen in the accompanying table, the distribution and relative frequency of these spontaneous mutations were different from those of mutations arising from EMS mutagenesis. [See Figure 1] In addition, no spontaneous mutations have been identified yet in mec-5 or mec-7, genes that are readily mutated by EMS (freq. = 3.3 X 10+E-4) and 2.3 X 10+E-4) respectively; many of the mec-5 alleles are ts, but the frequency of EMS mutation at 15 C is still 0.9 X 10+E-4). Three of the spontaneous mec mutations, mec-1(u295), 5), and mec-4 (u348) reverted spontaneously. A fourth mutation, mec-3(u300), that appears to have a copy of Tc1 inserted in the gene (see Way and Chalfie in this 'newsletter) did not revert (150,000 chromosomes examined). We are examining the Tc1 patterns in these mutants. A caution: We cannot be certain that all of the spontaneous mec mutations are independent. If a mec mutation arose on a chromosome carrying a lethal mutation, it may remain cryptic in the population. Because of the subculturing used in our screening, this could mean that the same mec mutation could populate a number of presumably independent plates. Thus, at this time we do not know whether all of the mec mutations are independent. In these experiments, we did two mass screenings of TR679, each from a different stock from Wisconsin. All nine mec-9 mutations came from the first screening, yet a screen by complementation for a cryptic mec-9 allele was unsuccessful. We feel, however, that at least two mutations in mec-1 and mec-4 and three of the mec-3 mutations are independent. It seems that a better way to determine the frequency of spontaneous mutations would be by precomplementation screening.

Srinivasan J et al. (2008) BMC Biol "Evolution of a polymodal sensory response network."

Durak O, Srinivasan J, Sternberg PW

[BMC Biol, 2008]

ABSTRACT: BACKGROUND: Avoidance of noxious stimuli is essential for the survival of an animal in its natural habitat. Some avoidance responses require polymodal sensory neurons, which sense a range of diverse stimuli, whereas other stimuli require a unimodal sensory neuron, which senses a single stimulus. Polymodality might have evolved to help animals quickly detect and respond to diverse noxious stimuli. Nematodes inhabit diverse habitats and most nematode nervous systems are composed of a small number of neurons, despite a wide assortment in nematode sizes. Given this observation, we speculated that cellular contribution to stereotyped avoidance behaviors would also be conserved between nematode species. The ASH neuron mediates avoidance of three classes of noxious stimuli in Caenorhabditis elegans. Two species of parasitic nematodes also utilize the ASH neuron to avoid certain stimuli. We wanted to extend our knowledge of avoidance behaviors by comparing multiple stimuli in a set of free-living nematode species. RESULTS: We used comparative behavioral analysis and laser microsurgery to examine three avoidance behaviors in six diverse species of free-living nematodes. We found that all species tested exhibit avoidance of chemo-, mechano- and osmosensory stimuli. In C. elegans, the bilaterally symmetric polymodal ASH neurons detect all three classes of repellant. We identified the putative ASH neurons in different nematode species by their anatomical positions and showed that in all six species ablation of the ASH neurons resulted in an inability to avoid noxious stimuli. However, in the nematode Pristionchus pacificus, the ADL neuron in addition to the ASH neuron contributed to osmosensation. In the species Caenorhabditis sp. 3, only the ASH neuron was required to mediate nose touch avoidance instead of three neurons in C. elegans. These data suggest that different species can increase or decrease the contribution of additional, non-ASH sensory neurons mediating osmosensation and mechanosensation. CONCLUSIONS: The overall conservation of ASH mediated polymodal nociception suggests that it is an ancestral evolutionarily stable feature of sensation. However, the finding that contribution from non-ASH sensory neurons mediates polymodal nociception in some nematode species suggests that even in conserved sensory behaviors, the cellular response network is dynamic over evolutionary time, perhaps shaped by adaptation of each species to its environment.

DG Srinivasan et al. (1999) International C. elegans Meeting "Creating a C. elegans cell line"

DG Srinivasan, S van den Heuvel

[International C. elegans Meeting, 1999]

Powerful genetic and cell biological approaches can be used to study gene function in C. elegans ; however, biochemical characterization of such functions is troublesome. To facilitate biochemical studies, we are attempting to establish a C. elegans cell line. As potential sources, we are using embryonic, germline and somatic tissues. Embryonic cells are used for their proliferative capacity. Moreover, culture conditions for embryonic cells have been established previously. We treat embryos with chitinase-chymotrypsin to obtain suspensions of single cells and small cell clumps. The cells are plated in a Schneider's-based medium yet are viable for only 1-3 days, and continuous divisions are rare. We are combining these methods with random mutagenesis to create cells with improved proliferative potential. Expression of rnr::gfp , an S-phase reporter (R. Roy and V. Ambros), will serve as a marker for viability and proliferation. The second approach utilizes germline tissue in combination with well characterized mutations: gld-2(dx32) gld-1(q485) double mutants contain a tumorous, mitotic germ line, epi-1(rh191ts) mutants grown at 20deg C form cellularized germ cells, and cells in ced-3(n717) animals do not undergo apoptosis. Combination of these mutations may allow us to culture an autonomously proliferating germ cell line. Germ cells, dissected from these mutant gonads, will be monitored for continuous proliferation. As a final approach, we are attempting to culture adherent somatic cells. Mammalian culture methods often select for adherent cells following digestion of tissue samples. Using a similar strategy, mixed stage worms are dissected, briefly trypsinized and plated in a Schneider's-based medium. Cell masses that tend to grow out from the cut sites and free cells are examined for proliferation and adherence. This technique will be combined with random mutagenesis and putatively oncogenic mutations to create cells with proliferative advantages. These techniques may allow us to obtain a cell line from at least of one of these tissue sources. Because of its proliferative capacity and totipotent nature, we consider the germ line the optimal source for a tissue culture cell line. Successful establishment of a cell line will aid us in probing the molecular nature of genetic and cellular interactions.

Srinivasan DG et al. (2000) East Coast Worm Meeting "Biochemical purification of mitotic LIN-5 protein complexes"

van den Heuvel S, Srinivasan DG

[East Coast Worm Meeting, 2000]

We use C. elegans as a genetic model for studying the events of cell division. Phenotypic characterizations of lin-5 mutants indicated an essential role for lin-5 in chromosome and spindle movements during mitosis. LIN-5 localizes to spindle microtubules, centrosomes and the cell cortex. Together, these results suggest LIN-5 is involved in spindle force generation (Lorson et al, 2000). To reveal the molecular mechanisms that underlie the mitotic functions of lin-5, we have initiated biochemical analyses of LIN-5 and its associated proteins. Many mitotic proteins are regulated by mitotic kinase activities. Western blots of LIN-5 from embryonic lysates reveal a protein with an apparent molecular weight of 100 kD and a minor, more slowly migrating form. LIN-5 contains several putative Ser/Thr phosphorylation sites, three of which conform to the consensus phosphorylation sites of the mitotic cyclin-dependent kinase, Cdc2/Cdk1. Experiments to determine if LIN-5 is phosphorylated are currently underway. Identification and study of LIN-5-associated proteins may reveal the molecular function of lin-5. To determine the size of LIN-5 protein complexes, taxol-treated lysates were fractionated by gel filtration. This analysis showed that LIN-5 is part of a large protein complex greater than 1 megadalton in size. This LIN-5 protein complex will be affinity purified using antibodies recognizing endogenous or epitope-tagged LIN-5, and interacting proteins will be identified. In addition, two classes of candidate LIN-5 interacting proteins are being tested for coimmunoprecipitation from embryonic lysates: interactors identified in two-hybrid screens and proteins involved in spindle force generation. Genes identified as LIN-5 interacting proteins will be tested genetically for their role in mitosis. Analysis of the functions of LIN-5 and its associated proteins should contribute to our understanding of the processes of chromosome segregation.

Jagan Srinivasan et al. (2001) International C. elegans Meeting "Towards a physical and genetic map of Pristionchus pacificus"

Waltraud Sinz, Ralf J Sommer, Jagan Srinivasan

[International C. elegans Meeting, 2001]

We are studying the evolution of cell fate specification, using vulva development as a model system and compare C. elegans with Pristionchus pacificus . We performed various genetic screens to isolate a number of mutants defective in vulva formation, many of which result in phenotypes unknown in C.elegans . To facilitate the molecular characterization of these mutants, we have initiated a physical and genetic map project of Pristionchus pacificus . The Pristionchus genome is approximately 100 MB in size. We have constructed a BAC library (with the help of Keygene N.V., Netherlands) with approximately 14 fold coverage of the Pristionchus genome and have performed BAC end sequencing (in collaboration with the Genome Sequencing Center, MPI Tuebingen) for half of the clones. We are using the sequence information from the BAC ends to create a polymorphism map using the strain Pristionchus pacificus var. Washington. It has been observed that the Washington strain shows a high degree of polymorphism with respect to Pristionchus pacificus var. California. The AFLP analysis indicated 55% of the bands (368 of the 630 bands) to be polymorphic between the two populations (Srinivasan et al , 2001). More than 1,000 BAC ends were tested for polymorphisms using the Single Stranded Conformation Polymorphism (SSCP) technique. 200 of the polymorphic markers were used to construct a genetic linkage map. The two parental strains from California and Washington were crossed and genetic marker segregation was followed in 48 random F2 offspring for each of the 200 polymorphic markers thereby generating a genetic linkage map. We also have initiated to construct a restriction fingerprint map of Pristionchus . Combining the polymorphism data, the genetic linkage map and the restriction fingerprint map will facilitate the cloning of mutants in Pristionchus pacificus . References: 1. Srinivasan. J et al . (2001) Evol. and Devel. (in Press)

Srinivasan B et al. (2015) Nat Chem Biol "Extracellular 4'-phosphopantetheine is a source for intracellular coenzyme A synthesis."

Schaap O, Baratashvili M, Colombelli C, Tiranti V, Hayflick S, Nollen EA, Grzeschik NA, Podgorsek A, van der Zwaag M, Kanon B, Lambrechts RA, Kosec G, Reijngoud DJ, Sibon OC, Srinivasan B, Petkovic H

[Nat Chem Biol, 2015]

The metabolic cofactor coenzyme A (CoA) gained renewed attention because of its roles in neurodegeneration, protein acetylation, autophagy and signal transduction. The long-standing dogma is that eukaryotic cells obtain CoA exclusively via the uptake of extracellular precursors, especially vitamin B5, which is intracellularly converted through five conserved enzymatic reactions into CoA. This study demonstrates an alternative mechanism that allows cells and organisms to adjust intracellular CoA levels by using exogenous CoA. Here CoA was hydrolyzed extracellularly by ectonucleotide pyrophosphatases to 4'-phosphopantetheine, a biologically stable molecule able to translocate through membranes via passive diffusion. Inside the cell, 4'-phosphopantetheine was enzymatically converted back to CoA by the bifunctional enzyme CoA synthase. Phenotypes induced by intracellular CoA deprivation were reversed when exogenous CoA was provided. Our findings answer long-standing questions in fundamental cell biology and have major implications for the understanding of CoA-related diseases and therapies.

Srinivasan J et al. (2008) Nature "A blend of small molecules regulates both mating and development in Caenorhabditis elegans."

Kaplan F, Schroeder FC, Malik RU, Sternberg PW, Zachariah C, Ajredini R, Teal PE, Srinivasan J, Alborn HT, Edison AS

[Nature, 2008]

In many organisms, population-density sensing and sexual attraction rely on small-molecule-based signalling systems. In the nematode Caenorhabditis elegans, population density is monitored through specific glycosides of the dideoxysugar ascarylose (the ''ascarosides'') that promote entry into an alternative larval stage, the non-feeding and highly persistent dauer stage. In addition, adult C. elegans males are attracted to hermaphrodites by a previously unidentified small-molecule signal. Here we show, by means of combinatorial activity-guided fractionation of the C. elegans metabolome, that the mating signal consists of a synergistic blend of three dauer-inducing ascarosides, which we call ascr#2, ascr#3 and ascr#4. This blend of ascarosides acts as a potent male attractant at very low concentrations, whereas at the higher concentrations required for dauer formation the compounds no longer attract males and instead deter hermaphrodites. The ascarosides ascr#2 and ascr#3 carry different, but overlapping, information, as ascr#3 is more potent as a male attractant than ascr#2, whereas ascr#2 is slightly more potent than ascr#3 in promoting dauer formation. We demonstrate that ascr#2, ascr#3 and ascr#4 are strongly synergistic, and that two types of neuron, the amphid single-ciliated sensory neuron type K (ASK) and the male-specific cephalic companion neuron (CEM), are required for male attraction by ascr#3. On the basis of these results, male attraction and dauer formation in C. elegans appear as alternative behavioural responses to a common set of signalling molecules. The ascaroside signalling system thus connects reproductive and developmental pathways and represents a unique example of structure- and concentration-dependent differential activity of signalling molecules.