Pause, Arnim et al. (2015) International Worm Meeting "Loss of Folliculin confers osmotic stress resistance via AMPK-dependent remodeling of carbohydrate stores in C. elegans."

Coull, Barry, Flamand, Mathieu, Possik, Elite, Vijayaraghavan, Tarika, Ajisebutu, Andrew, Manteghi, Sanaz, Hall, David, Pause, Arnim, van Stensel, Maurice

[International Worm Meeting, 2015]

Mechanisms of adaptation to environmental changes in osmolarity are fundamental for cellular life. When exposed to hyperosmotic stress, cells and organisms utilize conserved strategies to prevent water loss and maintain cellular integrity and viability. Here we identify a novel AMPK-dependent pathway of resistance to hypertonic stress mediated by the AMPK regulator flcn-1 in C. elegans. FLCN-1 is the worm homologue of the tumor suppressor Folliculin (FLCN), responsible for the Birt-Hogg-Dube hereditary cancer disorder. We show that loss of flcn-1 increases glycogen stores in an AMPK dependent manner and that the glycogen reserves are rapidly degraded upon hypertonic stress, leading to a remarkable accumulation of the organic osmolyte glycerol, promoting resistance to hyperosmotic stress. Importantly, loss of AMPK, glycogen synthase or glycogen phospharylase, which are critical enzymes in glycogen metabolism, strongly suppressed the increased osmotic stress resistance in flcn mutant animals. We further demonstrate that glycerol 3-phosphate dehydrogenase-1 is strongly induced in flcn-1 animals upon hyperosmotic stress and that simultaneous loss of gpdh-1 and gpdh-2 abolished the flcn-1/AMPK-mediated osmotic stress phenotype. Importantly, we show that glycogen accumulates in kidneys from mice lacking FLCN and in kidneys and renal tumors from a BHD patient. Since BHD is a renal hyperproliferation disorder, a mechanism of osmotic stress resistance in kidney hyperosmotic environments might explain tumorigenesis in BHD patients. Overall, our data indicate that FLCN is an evolutionary conserved regulator of glycogen metabolism, that might be acting as a tumor suppressor via AMPK-dependent accumulation of glycogen and organic osmolytes, resulting in an advantageous increase in proliferation and survival in hyperosmotic environments. .

Maxwell, Colin S. et al. (2013) International Worm Meeting "Poising and pausing of Pol II during starvation."

Lewarch, Caitlin L., Core, Leighton J., Waters, Colin T., Kruesi, William S., Antoshechkin, Igor, Kurhanewicz, Nicole, Maxwell, Colin S., Baugh, L. Ryan, Meyer, Barbara J., Lis, John T.

[International Worm Meeting, 2013]

In order to respond appropriately to changing environments, organisms must quickly alter the genes they express. Arrested L1 larvae rapidly alter gene expression in response to feeding, providing an attractive model to study the mechanisms of rapid gene induction. We previously showed that RNA Polymerase II (Pol II) is regulated at a post-recruitment step during L1 arrest. This regulation correlates with genes up-regulated by feeding, suggesting that it promotes rapid gene induction. We hypothesized that this regulation was mechanistically related to Pol II pausing, which has been proposed to allow the rapid induction of genes. To address this, we located elongation complexes genome-wide during starvation by sequencing short nascent RNAs as well as by using global nuclear run-on sequencing. We show here that Pol II is regulated during early elongation (pausing). Analysis of a TFIIS mutant reveals mechanistic similarities to pausing in other systems. However, Pol II pausing is associated with active stress-response genes that are actually down-regulated upon feeding. In addition to pausing, we show that 'poised' Pol II accumulates without initiating upstream of repressed growth genes that are up-regulated upon feeding. Poised Pol II and paused Pol II are associated with distinct core promoter architectures, suggesting alternative pathways for pre-initiation complex formation. Both growth and stress genes are regulated post-recruitment during starvation, but during initiation and elongation, respectively. Our work sheds light on the mechanisms organisms use to cope with changing environments.

Katz, Menachem et al. (2011) International Worm Meeting "C. elegans locomotory pattern, pausing frequency and speed is regulated by the CEP sheath glia."

Corson, Francis, Shaham, Shai, Katz, Menachem

[International Worm Meeting, 2011]

Neuronal circuits are tightly regulated to achieve proper animal behavior. Recent studies suggest that glial cells can influence neuronal functions and synaptic activities, and may, thus, contribute to behavior. However, the precise contributions of synaptic glia to animal behavior are poorly understood. C. elegans glia share morphological, functional, and genetic features with their vertebrate counterparts. However, in contrast to vertebrate glia, C. elegans glia are not essential for neuronal survival, offering a unique arena for exploring the involvement of glia in neuronal functions in a live animal. The CEP sheath glia (CEPsh) are bipolar cells that ensheath the dendrites of CEP neurons, and envelope the nerve ring. Reminiscent of mammalian astrocytes, these cells extend processes that abut specific synapses within the nerve ring. We showed that ablation of the CEPsh glia during the first larval stage results in abnormal locomotory behavior. CEPsh-ablated animals display reduced locomotion speed and extended locomotory pausing, as well as exaggerated small-angle turns and frequent reversals that limit their dispersal. To understand how these behaviors are regulated by CEPsh glia, we focused on the ALA-AVE synapse ensheathed by these glia (White et al., 1986). In line with the previously described role of ALA in behavioral quiescence and locomotory pausing (Van Buskirk and Sternberg, 2008), we found that inactivation of ALA reduces the pausing frequencies of glia-ablated animals. In addition, the dispersal of these animals and their speed of locomotion are improved. Inactivation of AVE in wild-type animals, induces extended pausing periods comparable to those seen in glia ablated animals, and animal speed is reduced. Our results suggest that AVE is a key regulator of speed and pausing frequency in the C. elegans nervous system, and that ALA functions to inhibit the activity of AVE. Moreover, our results indicate that the CEPsh glia provide important negative regulation on the activity of this tripartite synapse. To understand the molecular basis of CEPsh glia function we have begun to identify genes expressed in these cells using an RNA-tagging method, with the aim of examining the roles of enriched genes in C. elegans locomotory behavior.

Katz M et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Synaptic Activity Controlling Locomotory Pattern, Pausing Frequency and Speed is Regulated by C. elegans CEPsh Glia"

Katz M, Shaham S

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Information processing by the nervous system, leading to precise animal behavior, is complex. Recent studies suggest the possibility that glial cells participate in controlling synaptic activities, and may thus contribute to behavior. However, the precise role of synaptic glia in control of animal behavior is poorly understood. C. elegans glia share morphological, functional and genetic features with their vertebrate counterparts. However, in contrast to vertebrate glia, C. elegans glia are not essential for neuronal survival, offering a unique arena for exploring the involvement of glia in neuronal functions in a live animal. C. elegans CEP sheath glia (CEPsh) are bipolar cells that ensheath the dendrites of CEP neurons and envelope the nerve ring. Reminiscent of mammalian astrocytes, CEPsh extend processes that abut synapses within the nerve ring. Here we demonstrate that animals in which the CEPsh glia are genetically ablated during the first larva stage display behavioral defects including reduced locomotory speed and extended pausing. Ablated animals also display exaggerated small-angle turns and frequent reversals that limit their dispersal. To understand how these locomotory behaviors are regulated by CEPsh glia, we focused on a synapse between the ALA and AVE neurons, which is ensheathed by these glia (White et al., 1986). In line with the previously described role of ALA in behavioral quiescence and locomotory pausing (Van Buskirk and Sternberg, 2008), we found that inactivation of ALA reduces the pausing frequencies of glia-ablated animals. Inactivation of AVE in wild type animals, induced extended pausing periods similar to those seen in glia ablated animals. Our results suggest that AVE is a key regulator of speed and pausing frequency in the C. elegans nervous system, and that ALA functions to inhibit the activity of AVE. Moreover, our results indicate that the CEPsh glia provide important negative regulation on the activity of this tripartite synapse. To understand the molecular basis of CEPsh glia function we have begun to identify genes expressed in these cells using an mRNA-tagging method, with the aim of examining the roles of enriched genes in C. elegans locomotory behavior.

Shawn Lockery et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "A Predictive Model of Locomotory State Transitions in C. elegans"

Steven Augustine, Rebecca Anderson, Shawn Lockery, Kristy Lawton

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Locomotory state in C. elegans is regulated by a network of command neurons in two reciprocally connected pools-the forward neurons AVB and PVC and the reverse neurons AVA and AVD-but how this network functions is poorly understood. We propose a model in which the network functions as a stochastic, bi-stable switch. The model makes three simple assumptions: (A) forward command neurons act as a single unit, as do reverse command neurons; (B) unit activation switches stochastically between two states: 0 (off) and 1 (on); (C) the stochastic processes underlying the state changes of the forward and reverse units are uncorrelated. Accordingly, the network can exist in four states: both units off (00); forward on (10), reverse on (01), and both on (11). Previous neuronal ablations suggest that states 10, 01, and 00 correspond, respectively, to forward locomotion, reverse locomotion, and a pause state; we propose that state 11 is also a pause state because co-activation of forward and reverse motor systems could cause the body musculature to lock up. A key consequence of (C) is that only one unit changes state at a time. This means that transitions between the forward and reverse states (01 to 10, or 10 to 01) must pass through the intermediate state 00 or 11. Thus the model predicts pauses during transitions between forward and reverse locomotion. The model also predicts pauses during apparently continuous bouts of forward or reverse locomotion. To test these predictions, we constructed a novel tracking system capable of recording the worm's speed with a precision of 19 um/sec at a rate of 30 samples/sec. We found clear evidence for pauses, which were typified by a precipitous drop in speed and, after a variable delay, a precipitous rise in speed. Mean dwell time in the pause state was 0.15 sec (SEM = 0.01). Pauses were observed during all transitions between forward and reverse, and also during bouts of forward and reverse locomotion that appeared continuous to the naked eye. These results support the stochastic switch model and suggest revised definitions of fundamental locomotory states in C. elegans.

Shawn R Lockery et al. (2005) International Worm Meeting "Kinetic analysis of locomotory switching in C. elegans"

Shawn R Lockery, Steven B Augustine, Kristy J Lawton

[International Worm Meeting, 2005]

Locomotory state in C. elegans is regulated by a network of command neurons in two functional pools: the forward neurons AVB and PVC, and the reverse neurons AVA and AVD. These two pools are linked by reciprocal synaptic connections, but how this network functions to regulate locomotion is not well understood.In one model, the network functions as a stochastic, bi-stable switch. This model embodies two main assumptions: (1) Forward command neurons act as a single unit, as do reverse command neurons, (2) Units switch stochastically between the on (1) and off (0) states. According to these assumptions, the network can exist in four different states: both units off (00); forward unit on, reverse unit off (10); forward unit off, reverse unit on (01); both units on (11). Previous neuronal ablation studies suggest that state 10 is forward locomotion, state 01 is reverse locomotion, and state 00 is a pause state. Here it is further assumed that state 11 is also a pause state, because co-activation of forward and reverse motor systems might cause the body musculature to lock-up.A key consequence of assumption (2) is that only one unit changes state at a time, because the probability of simultaneous random events is zero. This means that transitions in either direction between the forward and reverse state (10<sym17>01 and 01<sym17>10, respectively) must involve the intermediate states 00 or 11. Thus the model predicts that a worm pauses briefly during transitions between forward and reverse. The model also predicts that brief pauses occur during apparently continuous bouts of forward or reverse locomotion.To test these predictions we videotaped wild type worms moving on foodless agar plates. Frame-by-frame analysis (30 frames/sec) revealed the presence of brief but measurable pauses (mean SD

Huang MM et al. (1998) Midwest Worm Meeting "Analysis of a C. elegans homologue of the Von Hippel Lindau Tumor Suppressor Gene"

Huang MM, Barstead RJ, Pause A, Takagi Y, Conaway RC, Conaway JW, Moulder GL

[Midwest Worm Meeting, 1998]

von Hippel-Lindau (VHL) disease is a dominantly inherited complex cancer syndrome in which patients develop a number of tumors in specific target organs (1). Moreover, mutations at the VHL locus may account for up to 90% of sporadic cases of clear cell renal carcinoma. In mammalian cells the protein product of the VHL gene is found in a complex with Elongins B and C, positive regulatory subunits of the heterotrimeric RNA polymerase II elongation factor Elongin (2). A C. elegans gene with significant sequence homology to the human VHL gene is found on cosmid F08G12. The protein encoded by this C. elegans homologue interacts in vitro with the mammalian Elongin BC heterodimer. The C. elegans gene is expressed in nearly all somatic cells based on experiments with a GFP reporter construct containing about 2 KBp of upstream DNA sequence. From a PCR screen of a large bank of mutagenized C. elegans strains we identified and recovered animals carrying a deletion within the C. elegans VHL homologue. Animals homozygous for the deletion were viable but exhibited defects in several tissue types. We will present a detailed phenotypic analysis of the mutant. 1. Gnarra, J. R. et al. Biochim Biophys Acta 1242, 201-10 (1996). 2. Takagi, Y., Pause, A., Conaway, R. C. & Conaway, J. W. J Biol Chem 272, 27444-9 (1997).

Anne-Francoise RUAUD et al. (2005) International Worm Meeting "A nicotinic agonist unmasks a neuronal control of developmental timing before the dauer decision."

Anne-Francoise RUAUD, Jean-Louis BESSEREAU

[International Worm Meeting, 2005]

In C. elegans, the nervous system plays a critical role in the binary decision to proceed either to reproductive development or to dauer larva in response to environmental conditions. Using the nicotinic agonist DMPP (DiMethyl Phenyl Piperazinium), we demonstrated that the nervous system can also control the timing of developmental events. Specifically, we observed that C. elegans development can be paused at the early L2 larval stage before the dauer decision. DMPP partially mimics this previously undescribed L2 developmental pause. At the beginning of the L2 stage, the hypodermal seam cells undergo an equational division rapidly followed by a stem cell division. On DMPP, the equational division is delayed and the second division never occurs. However, the timing of the following L2/L3 molt is not affected. Soon after leaving their L2 cuticle animals get vacuolated and die rapidly. Electron microscopy shows structural defects of the L3 cuticle, most probably explaining DMPP-induced lethality by the inability of this defective L3 cuticle to insulate the worm from the outside environment. Thus DMPP is able to specifically uncouple hypodermis morphogenesis from the molting cycle at the L2 stage. We demonstrated that genetic ablation of 8 sensory neurons is sufficient to confer resistance to DMPP. Moreover, mutants of unc-63, a gene encoding an AChR subunit, are partially resistant to DMPP. Therefore, DMPP-induced lethality may be due to overstimulation of a neuronal AChR containing the UNC-63 subunit. In order to identify the genes that are required for DMPP-induced lethality, we performed a Mos1-mediated screen for mutants that can grow on DMPP. Six genes were identified including daf-12. We demonstrated that daf-12 null alleles are resistant to DMPP. daf-12 is a key regulator of the dauer versus reproductive L3 decision. Interestingly, the DMPP toxicity period spans a short period ranging from mid-L1 to the L2/L3 molt, which also corresponds to the time of the dauer versus reproductive decision. We recently observed that worms grown on restricted amount of food in a non-crowded environment pause development at early L2 stage: most seam cells arrest before asymmetric division similar to what is observed with DMPP, molt does not proceed and animals can survive for up to 8 days. When food is provided, reproductive development resumes. This previously undescribed pausing might represent a 'wait-and-see' stage, when environmental cues are in-between reproductive and dauer formation, before taking an irrevocable decision to proceed to further development.

Banse, S.A. et al. (2017) International Worm Meeting "Fine scale electrophysiological analysis of pharyngeal aging and a transition-state model of activity."

Lockery, S.R., Banse, S.A., Weeks, J.C., Willis, J.H., Phillips, P.C., Robinson, K.J., Blue, B.W.

[International Worm Meeting, 2017]

Because age-associated loss of neuromuscular function severely impacts quality of life, research models capable of identifying the etiology of lost function are particularly valuable. The C. elegans pharynx is a neuromuscular model whose visually observed age-dependent decline in contraction frequency is used as an experimental health marker. Although informative about overall health state, contraction frequency alone provides little information about the underlying cause for lost function. Fine scale analysis of electropharyngeograms recorded from aging animals provides an alternative assay more amenable to mapping age-related changes to the underlying neuromuscular circuit. We find that decreased pump frequency is associated with bursts of activity punctuated by increased frequency of pauses. Interestingly, although inter-pump interval during an activity bout changes with age, pause duration remains relatively unchanged. This suggests a decreased frequency or efficacy of cholinergic signaling during an activity bout. This reduced efficacy in neuronal modulation of pumping contrasts with M3 related glutamatergic signaling efficacy which holds longer into adulthood. This difference is visible both in the duration of pumps, as well as in average waveform shapes associated with the beginning and end of a pump. We present an activity-state based model to explain the observed decreased pump frequency. We argue that this new approach is an information dense measure that holds promise in mapping age-dependent changes to the functional subunits of pharyngeal activity.

Adi Albeg et al. (2008) European Worm Meeting "Multi-dendritic nociceptors suppress dwelling"

Millet Treinin, Adi Albeg, Dror Feitelson

[European Worm Meeting, 2008]

PVDs and FLPs are both multi-dendritic neurons that together cover the. adults with a complex network of branches, with PVD branches covering the. body region and FLPs the head region. Both PVDs and FLPs were shown to. function as mechanoreceptors. In particular, PVDs together with the gentle. touch receptors mediate the response to high threshold (noxious) mechanical. stimuli to the body. To better understand the function of these two pairs. of neurons we generated animals in which different combinations of PVDs,. FLPs, and touch receptor neurons degenerate; the resulting strains were. analyzed for movement on food. This analysis shows that animals lacking. PVDs and FLPs are markedly slower, make more pauses, and spend more time in. backward movement (longer reversals). Ablation of PVDs is sufficient for. the effects on speed and the number of pauses. However, the increased. duration of backward movement requires ablation of both PVDs and FLPs.. Overall, the effects of ablating PVDs and FLPs is to increase dwelling. within a restricted area. These results suggest that basal activity of PVDs. and FLPs suppresses dwelling leading to increased roaming. Our results when. combined with previous research from other labs suggests that behavior of. C. elegans is a delicate balance between activity of sensory neurons. sensitive to positive (food) signals that promote dwelling and sensory. neurons sensitive to noxious signals that suppress dwelling.