Ding, S.S. et al. (2019) International Worm Meeting "Shared behavioral mechanisms underlie C. elegans aggregation and swarming."

Endres, R.G., Javer, A.E., Schumacher, L.J., Ding, S.S., Brown, A.E.X., Sarkisyan, K.S.

[International Worm Meeting, 2019]

In complex biological systems, simple individual-level behavioral rules can give rise to emergent group-level behavior. While collective behavior has been well studied in cells and larger organisms, the mesoscopic scale is less understood, as it is unclear which sensory inputs and physical processes matter a priori. Here, we investigate collective feeding in the roundworm C. elegans at this intermediate scale, using quantitative phenotyping and agent-based modeling to identify behavioral rules underlying both aggregation and swarming-a dynamic phenotype only observed at longer timescales. Using fluorescence multi-worm tracking, we quantify aggregation in terms of individual dynamics and population-level statistics. Then we use agent-based simulations and approximate Bayesian inference to identify three key behavioral rules for aggregation: cluster-edge reversals, a density-dependent switch between crawling speeds, and taxis towards neighboring worms. Our simulations suggest that swarming is simply driven by local food depletion but otherwise employs the same behavioral mechanisms as the initial aggregation. We currently extend this work by studying swarming at very high worm numbers, as well as by visualising and quantifying feeding using a bioluminescent bacterial system.

Allyson Whittaker et al. (2001) International C. elegans Meeting "Control of the backing and turning steps of male mating behavior of C. elegans."

Allyson Whittaker, Gary Schindelman, Shahla Gharib, Paul Sternberg

[International C. elegans Meeting, 2001]

Behavior in its simplest form consists of an organism sensing the environment and executing a motor output based on those signals. The next level is coordination of distinct behaviors, that is, processing a number of signals from the environment and triggering the appropriate response. C. elegans male mating behavior provides an excellent model for studying how behavior is coordinated and also for furthering our understanding of sensory perception. Mating behavior in C. elegans consists of a series of sub-behaviors with the initiation of one step correlating with the repression of the previous step. Understanding coordination of these behaviors will require furthering our knowledge of the molecular and neural pathways through which the different sub-steps are controlled. We are currently focusing on two of the earliest steps in mating behavior, backing and turning. Efforts are being made to address how these steps are triggered and executed and how one step is repressed when the next step is initiated. Previous research has provided information about the sensory, inter and motorneurons as well as the neurotransmitters required for the execution of these steps (1,2,3). We are currently doing a genetic screen to isolate new mutations that affect male mating behavior. The design of the screen will also allow us to identify mutations in the hermaphrodite which disrupt mating behavior enabling us to determine the hermaphrodite signals which trigger male responses. Further characterization of previously isolated mutations affecting backing and turning will also be described. 1. Liu, K.S. and Sternberg, P.W., Sensory regulation of male mating behavior in Caenorhabditis elegans . Neuron 14, 79-89 (1995). 2. Liu, K.S., Male mating behavior in Caenorhabditis elegans Ph.D. thesis, California Institute of Technology, Pasadena, California U.S.A. (1995). 3. Loer, C.M. and Kenyon, C.J. Serotonin-deficient mutants and male mating-behavior in the nematode Caenorhabditis elegans . J. Neurosci. 13, 5407-5417.

Hirotsu T et al. (1997) International C. elegans Meeting "ISOLATION AND CHARACTERIZATION OF MALE MATING-DEFECTIVE MUTANTS"

Hirotsu T, Yamamoto M, Iino Y

[International C. elegans Meeting, 1997]

Male mating behavior is the most complex behavior known in C. elegans. We are interested in the mechanism whereby this male-specific and innate behavior is genetically programmed. Since 1983 (J. Hodgkin, Genetics 103, 43), many male mating-defective mutants have been isolated in several labs. A majority of these mutants have morphological defects in the copulatory organs in the tail (called mab mutants). We have started another round of mutant screening aiming at isolation of new mating-defective mutants, especially those that are morphologically normal. We mutagenized the plg-1; him-5 strain and 39 mutants that fail to deposit the copulatory plug, which is deposited over the hermaphrodite vulva after sperm transfer by the plg-1 males, at a normal rate were isolated. Male mating behavior is composed of "response to contact", "turning", "vulva location", "spicule insertion" and "sperm transfer" (K.S. Liu and P.W. Sternberg, Neuron 14, 79, 1995). Fourteen of the mutants had defects in response to contact, 23 in turning, and 5 in vulva location. Some of them had defects in multiple steps. Twenty of the mutants had morphological defects in the tail. These morphological defects included fusion and deletion of rays and shortening of rays. Six of the mutants occasionally showed projection of the spicules, suggesting defects in the spicule insertion/retraction step. Further analysis with DiO staining revealed defects in the sensory neurons in some of the morphologically normal mutants. Two of the mutants totally failed to adsorb DiO into the head amphid neurons, suggesting defects like "cilium-structure mutants". Some other mutants showed staining of supernumerary cells around amphid cell bodies. Further analysis of the mutants is under way.

Garca LR et al. (2000) West Coast Worm Meeting "Execution and regulation of male C. elegans spicule muscle contractions during mating"

Garca LR, Sternberg PW

[West Coast Worm Meeting, 2000]

HHMI & California Institute of Technology, Division of Biology, Pasadena, CA 91125 U.S.A. As the C. elegans male attempts to penetrate the hermaphrodite vulva with his spicules, the protractor muscles that are attached to these copulatory structures contract and relax rapidly such that the spicules prod the vulva slit at a frequency of 7.2 1.3 Hz. Phasic protractor muscle contractions persist until the vulva slit is partially penetrated. Once the vulva is breached, the protractor muscles contract tonically and the spicules extend completely into the hermaphrodite. The spicule muscles stay contracted for 75 20 seconds, sufficient time for sperm to transfer into the hermaphrodite. The male protractor muscles are innervated by the SPC motor neurons (2), and removal of these cells results in male impotency (1). The SPC neurons are essential for tonic, but not phasic spicule muscle contractions; the spicules of SPC-ablated males can not fully extend into the hermaphrodite, but can prod the vulva at a frequency of 5.1 1.1 Hz. The ACh agonists levamisole, nicotine, arecoline, and oxotremorine can stimulate the protractor muscles to contract; and the SPC motor neurons support the expression of the unc-17-encoded VAChT. Therefore ACh most likely regulates the contractile state of the spicule muscles. The ACh agonists differentially require egl-19 and unc-68-encoded calcium channels to mediate the behavioral output; thus suggesting that calcium mobilization from these channels might have non-overlapping roles during copulation. During mating the spicules of egl-19 males can prod the vulva at a frequency of 5.0 1.5 Hz, but they can not breach the vulva lips. In contrast, the spicules of unc-68 males can insert into the hermaphrodite, but prior to penetration, the spicules prod the vulva at a frequency of 0.48 1.4 Hz. Therefore, the UNC-68 channel is utilized in phasic muscle contractions, and the EGL-19 channel is required for tonic muscle contractions. 1. Liu, K.S., and Sternberg, P.W. (1995).Neuron 14, 79-89. 2. Sulston, J.E., Albertson, D.G., and Thomson, J.N. (1980). Dev. Biol. 78, 542-576.

McKim KS et al. (1991) International C. elegans Meeting "GENETIC ANALYSIS OF SPONTANEOUS DUPLICATION LOSS AND BREAKAGE."

Rose AM, McKim KS

[International C. elegans Meeting, 1991]

Duplications spontaneously break at a frequency which is related to their mitotic stability. We have analyzed the structure and mitotic stability of 32 duplications resulting from spontaneous breakage events. Most of these duplications were derived from pre-existing chromosome I duplications, although three arose from spontaneous breakage of an X-chromosome into which a large piece of chromosome I ( hDp14) had inserted. Where it has been possible to observe a cluster of spontaneous shortening events, only a single exceptional duplication was produced by any one worm. Thus, most of the spontaneous duplications may have formed during meiosis. The structures of the spontaneous duplications were determined by genetic complementation. In general there were no hot spots for breakage. Derivatives from four of the duplications had complex structures. Two of these four progenitors were of spontaneous origin. The structures were most simply resolved by proposing the progenitor duplications were ring chromosomes with a superimposed inversion. Similar analysis of most of the other duplications were consistent with a linear structure. Most of the proposed ring chromosomes were mitotically unstable, suggesting ring structures increase the frequency of chromosome loss. Unlike the other duplications, the spontaneous breakage events of hDp14 were clustered; they all appeared to occur close to or at the site of the chromosome I insertion. Thus, the insertion appeared to create a type of fragile site. Most of the spontaneous duplications remained free. Three, however, had become linked to other autosomes. Like free duplications of chromosome 1, these linked duplications (Dp(I;A)) tended to segregate from the X- chromosome in males. Interestingly, only a subset of males carrying these linked duplications demonstrated Dp(I;A) - X segregation patterns. Half of the males demonstrated random segregation of the Dp( I;A) and the X-chromosome. This was not observed with the free duplications. It is possible that in each individual male, premeiotic events determine if the Dp(I;A) and X-chromosome segregate at meiosis. The mitotic stabilities of the spontaneous free duplications were variable, indicating that they acquired different structures during their formation. Supported by an MRC studentship to K.S. McKim and an NSERC grant to A. M. Rose.

Erik M Peden et al. (2003) International Worm Meeting "Characterization of Genes Required for the Distinct Substeps of Male Mating Behavior."

Maureen M Barr, Erik M Peden

[International Worm Meeting, 2003]

We are genetically dissecting Caenorhabditis elegans male mating behavior to identify and characterize genes that underlie neural sensory transduction. Because TRP ion channels are widespread sensory receptors and the C. elegans pkd2 TRP gene functions in male sensory neurons, we wanted to determine if other TRP family members are required for male mating behavior. Males from existing mutant TRP allele strains were scored in mating efficiency tests and mating behavior tests for execution of the substeps of male mating: response to hermaphrodite contact; backing; turning; location of vulva (Lov); spicule insertion; and sperm transfer (Liu and Sternberg, 1995). Of 8 TRP mutants tested, only osm-9 and ocr-2 males exhibited a specific mating defect. Interestingly, males from both strains show an identical sperm transfer defect and severity of the phenotype is not affected in osm-9; ocr-2 double mutants, suggesting that these genes function in the same pathway. Moreoever, OSM-:9::GFP and OCR-2::GFP fusion proteins express in and localize to the cilia of spicule sensory neurons that regulate sperm transfer. osm-9 and ocr-2 have previously been demonstrated to function coordinately in C. elegans olfaction and nose-touch responses (Tobin et al., 2002), and our data implicates a similar model of coordinate sensory transduction function in sperm transfer behavor. Further experiments will address spicule neuron signal trasnduction pathways relating to TRP channel activity. To identify new candidate neurosensory genes, we are mapping and cloning a mutation, the sy511 allele, which was originally isolated in the Sternberg Lab in a screen for plugging and Lov defective mutants. sy511 males are response and Lov defective, and no other behavioral phenotypes are seen in males or hermaphrodites. Development and differentiation of sensory neurons in sy511 males appear normal. We hypothesize that this gene encodes a sensory transducer molecule specific to male mating behavior neurons. Genetic (3 factor cross) and physical (SNP) mapping place sy511 within a small interval on Linkage III, and cosmid injections are underway to rescue the sy511 mating behavior defects. 1. Liu, K.S. and Sternberg, PW. Neuron 1995, 14: 79. 2. Tobin, D. et al. Neuron 2002, 35: 307-318.

Ayako Hasegawa et al. (2003) International Worm Meeting "C.elegans homologue of yeast Mdm38 and mammalian Letm1 is essential for mitochondrial morphology"

Ayako Hasegawa, Alexander M van der Bliek

[International Worm Meeting, 2003]

Mitochondria are dynamic structures that divide and fuse throughout the life of a cell. Several proteins have been identified to affect mitochondrial outer membrane division and fusion. Our lab discovered that dynamin related protein called DRP-1 plays a key role in mitochondrial outer membrane division (1). The detailed analysis of the dominant-negative phenotype of DRP-1, which causes a block in outer membrane division, indicates that inner membrane division occurs independent of outer membrane division. 3D reconstruction of mitochondrial inner membrane and cristae by electron tomography also indicates that fusion occurs within mitochondria. Cristae often fuse to each other or to inner membrane (2). These data suggest that there are separate mechanisms of division and fusion for inner membrane aside from those for outer membrane, and a mechanism that coordinates different membrane remodelling. Our lab has been looking for novel proteins that are involved in regulation of mitochondrial morphology. C.elegans body wall muscle cells are used because they are large and flat, and their mitochondria are fairly well organized. Mitochondrially targeted fluorescence protein marker under muscle-specific promoter was used to visualize the mitochondria with minimum background. We are analyzing the function of a C.elegans homologue of the yeast Mdm38 protein, which has previously been implicated in mitochondrial morphology (3). This protein is also homologous to the mammalian protein Letm1, which is deleted in some patients with Wolf Hirschhorn Syndrome, a disease of as yet unknown etiology. We find that this protein is localized to mitochondria and affect mitochondrial morphology. Strong MDM38 loss-of-function causes abnormally enlarged and distended mitochondria, while mild loss-of-function causes enlarged and indented mitochondria, resembling oxidation-phophorylation complex loss-of-function phenotypes. Over expression of MDM38 appears to cause loss of mitochondrial potential and shriveling of matrix and/or swelling of outer membrane. We suspect that MDM38 homologous protein affects cristae remodeling, however the functionality of this protein is yet to be discovered. 1. Labrousse, A.M., Zappaterra, M.D., Rube, D.A., and van der Bliek, A.M. Molecular, Cell 2: 815-826. 1999. 2. Frey, T.G., Mannella, C.A. Trends Biochem Sci 25(7): 319-324. 2000. 3. Dimmer, K.S., Fritz, S., Fuchs, F., Messerschmitt, M., Weinbach, N., Neupert, W., Westermann, B. Mol Biol Cell 13(3): 847-853. 2002.

Koelle MR et al. (1996) East Coast Worm Meeting "BIOCHEMICAL ANALYSIS OF EGL-10 AND ITS INTERACTIONS WITH THE G PROTEIN GOA-1"

Horvitz HR, Koelle MR

[East Coast Worm Meeting, 1996]

The frequency of egg-laying behavior by C. elegans is regulated by environmental conditions: in the presence of abundant food eggs are laid about every 30 minutes, but in the absence of food egg laying rarely occurs. To understand how the activities of the egg-laying neurons and/or muscles are modulated to achieve this regulation, we are studying mutations that disrupt the regulation of egg laying. Two of the genes identified by these mutations have been cloned: egl-10 positively regulates egg laying and encodes a homolog of sst2, a gene that controls G protein signaling in yeast (1); goa-1 negatively regulates egg laying and encodes a G protein alpha subunit homolog (2,3). These results suggest that the control of egg laying involves a G protein signaling pathway that may mediate the action of neurotransmitters on the egg-laying neurons and/or muscles to modulate the activities of these cells. egl-10 is of particular interest, since it is a member of a newly discovered family of homologous genes that we have named RGS genes (RGS, regulator of G protein signaling). This family includes egl-10, sst2, at least three additional C. elegans genes, and at least 15 mammalian genes that we identified by their similarity to egl-10. Genetic studies in worms and yeast suggest that the EGL-10 and Sst2p proteins have similar functions: each appears to act upstream of a G protein to inhibit signaling. We hypothesize that the other members of the family of RGS proteins might function to regulate many different G protein signaling pathways. We would like to understand the biochemical mechanism of action of the RGS proteins. One class of molecular models consistent with the genetic studies in worms and yeast involves a direct interaction between RGS proteins and G proteins. This class of models is supported by the recent identification of an interaction between an RGS protein and a G protein alpha subunit in a yeast two-hybrid system (4). RGS proteins could bind to G proteins to uncouple them from receptors, to uncouple them from their downstream effectors, to affect interactions between the various G protein subunits, or to activate the GTPase activity of G protein alpha subunits. To distinguish among these models, we have begun to express and purify recombinant forms of EGL-10 and GOA-1 and are investigating the properties and interactions of these proteins in vitro. Both proteins have been expressed in the baculovirus system, and progress in analysis of these proteins will be presented. 1. Koelle, M.R., and Horvitz, H.R (1996). Cell 84, 115-125. 2. Segalat, L., Elkes, D.A., and Kaplan, J.M. (1995). Science 267, 1648-1651. 3. Mendel, J.E., Korswagen, H.C., Liu, K.S., Hajdu-Cronin, Y.M., Simon, M.I., Plasterk, R.H, and Sternberg, P.W. (1995). Science 267, 1652-1655. 4. De Vries, L., Mousli, M., Wurmser, A., and Farquhar, M.G. (1995). Proc. Natl. Acad. Sci. USA 92, 11916-11920.

Brundage LA et al. (1996) West Coast Worm Meeting "MUTATIONS IN A C. ELEGANS Gq ALPHA GENE DISRUPT MOVEMENT, EGG LAYING AND VIABILITY"

Kim U, Simon MI, Avery L, Sternberg PW, Brundage LA, Mendel JE, Katz A

[West Coast Worm Meeting, 1996]

In mammals, Heterotrimeric G proteins convert signals from hundreds of extracellular receptors to a very limited set of intracellular responses. The biochemical properties of individual G proteins have been studied in detail, but the larger sense of how multiple G protein pathways are integrated to process information and ultimately regulate behavior is not understood. We hope to visualize this larger picture by studying how behaviors are regulated by individual G proteins in C. elegans. G proteins are composed of three subunits: the alpha subunit, which activates effectors directly when liganded with GTP, and beta and gamma subunits, which are released as heterodimers when G proteins are activated. G alpha subunits are grouped into four families based on their similarities in sequence and function: Gs alpha, Gi/Go alpha, G12 alpha and Gq alpha. We now know that C. elegans expresses at least one member of each of these families. Here we have examined the function of Gq alpha in C. elegans. We have found that egl-30 encodes a C. elegans alpha subunit which is more than 80% identical to Gq alpha family proteins (1). Two candidate null alleles of egl-30 contain amber codons, and are essentially lethal. Animals homozygous for these alleles hatch as larvae with extremely feeble muscle contraction. They are virtually paralyzed and their pharynxes pump weakly and only sporadically, leading to a starved appearance, extremely slow growth, and arrest throughout larval development. A small number of these hermaphrodites, however, develop into semi-fertile adults which can produce offspring of grossly normal morphology. Animals homozygous for six other egl-30 alleles are viable and fertile, but they move slowly, leave flattened tracks, and become bloated with late stage eggs. Hermaphrodites overexpressing the wild type egl-30 gene exhibit the opposite behavior: they leave tracks with bends on average 60% deeper than wild type, and lay eggs prematurely. Analysis of egl-30 mutants suggests that their phenotypes reflect defects in the muscle or neuromuscular junction. It is intriguing that Go alpha and Gq alpha alleles produce essentially opposite effects in C. elegans. Go alpha deficient animals lay eggs prematurely and produce deeper than normal sinusoidal movement (2, 3), like strains over-expressing egl-30. Go alpha gain-of-function alleles induce movement and egg laying defective phenotypes similar to the egl-30 reduction-of-function strains (2, 3). Preliminary analysis of a strain containing mutations in both genes suggests that egl-30 is epistatic to goa-1. Theoretically, Gq and Go activation could produce opposite effects through regulation of intracellular Ca++. Since fluxes of intracellular Ca++ are directly involved in muscle contraction, and egl-30 mutants are defective in muscle function, such a mechanism could be at work here. (1). Brundage, L ., Avery, L., Katz, A. Kim, U-J., Mendel, J.E. Sternberg, P.W., and Simon, M.I. (1996). Neuron in press (2). Mendel, J.E., Korswagen, H.C., Liu, K.S., Hajdu-Cronin, Y.M., Simon, M.I., Plasterk, R.H., and Sternberg, P.W. (1995). Science 267, 1652-1655. (3). Segalat, L., Elkes, D.A., and Kaplan, J.M. (1995). Science 267, 1648-1651.