Laura C Manning et al. (2003) International Worm Meeting "Dissecting the role of alpha-catenin in C. elegans adherens junctions"

Laura C Manning, Jonathan Pettitt

[International Worm Meeting, 2003]

Cadherin based adherens junctions (AJ) are important in epithelial morphogenesis and maintenance of epithelial shape. The absence of cadherin junction components has been associated with many epithelial cancers. One component of the cadherin-catenin AJ is alpha-catenin. Alpha-catenin acts to anchor the complex to the actin cytoskeleton; in addition it is also able to bind other proteins that are known to interact with both the AJ and the actin cytoskeleton. As a result alpha-catenin most likely acts to recruit additional proteins necessary for adhesion to the AJ. Due to this central role within the cadherin-catenin complex alpha-catenin is a potential site of AJ regulation. A further understanding of the pathways regulating the stability of these junctions could provide further insight into the mechanisms by which cancer cells become invasive. The C. elegans alpha-catenin homologue, hmp-1, is essential for ventral epidermal enclosure and elongation of the embryo. Embryos lacking zygotic hmp-1 expression are unable to elongate, resulting in a humpback (Hmp) phenotype. We have previously isolated a mutation of hmp-1(fe4) which appears to perturb, but not completely abolish HMP-1 function in C. elegans.Some genes involved in cadherin junction formation and regulation have been previously identified in C. elegans and other model systems. For example, unc-34 is the only member of the Ena/VASP family in C. elegans. This family of proteins act to regulate actin remodeling and localise to newly formed cadherin junctions. Loss of unc-34 does not result in a significant epithelial phenotype in wild type worms, but in the presence of the fe4 mutation embryos either fail to ventrally enclose or arrest as Hmp embryos, thus indicating a role for unc-34 within cadherin-mediated adhesion. Evidence has also been presented that the small GTPases; Rho, Rac and Cdc42; along with their associated signaling molecules could also have a role in the regulation of cadherin-mediated adhesion. We are performing non-biased genetic screens to identify additional suppressors and enhancers of the fe4 mutant phenotype. Our screening strategy allows the identification of mutations that genetically interact with hmp-1, but do not affect epithelial development in a wild-type hmp-1 background. The results of these screens will be an increased understanding of the composition of the C. elegans AJ, as well as the regulatory role that alpha-catenin and its interacting partners play within cadherin-catenin adhesion complexes.

Sasidharan, S. et al. (2017) International Worm Meeting "Interdependent accumulation of Cadherin-Catenin complexes and WAVE/SCAR branched actin regulators at the apical junction promotes epithelial morphogenesis."

Sasidharan, S., Larsen, E, Soto, M, Borinskaya, S

[International Worm Meeting, 2017]

Our lab studies embryonic morphogenesis in C. elegans. Our earlier work showed that branched actin, powered by the WAVE/SCAR and Arp2/3 complexes, promotes polarized events during epithelial tissue development. These events include epidermal cell migration during ventral enclosure, and the polarized formation of the embryonic intestine. Actin is an integral component of the epithelial apical junction, yet the interactions of branched actin regulators with apical junction components are still not clear. We used live imaging of C. elegans embryos and newly available CRISPR-tagged strains (from our lab and the Goldstein lab) to define the developmental time course of accumulation of apical junction components relative to the branched actin regulators WVE-1 and ARP-2/ARX-2 as the embryonic intestinal epithelium becomes polarized. Live imaging of apical F-actin accumulation in the developing intestine showed that the two main apical junction complexes, namely Cadherin Catenin Complex (CCC) and DLG-1 AJM-1 complex (DAC), branched actin regulators, and non-muscle myosin were essential for apical actin enrichment. Biochemical data have shown that alpha catenin (a CCC component) inhibits Arp2/3-dependent branched actin, which suggests branched actin is only needed at the earliest stages of apical junction development. We found that loss of WVE-1 led to increased levels of CCC components after junction formation. The CCC components, in turn, regulated the levels of WAVE. Intriguingly, loss of two components of the CCC, E-Cdh/HMR-1 and beta -catenin/HMP-2, reduced levels of WVE-1 at apical junctions whereas loss of a-catenin/HMP-1 led to elevated apical WVE-1. Due to the interdependency in the localization of CCC and WVE-1 at the apical junction (AJ) and the temporal analysis of CCC and WAVE complex accumulation, we conclude that the CCC accumulates at AJ first and aids in recruitment of WAVE complex. Later CCC and DAC help in the maintenance of WAVE complex levels at AJ. WVE-1 in turn regulates HMR-1 turnover and shows inversely complemental levels with respect to a-catenin at the AJ. Therefore branched actin regulators are continually recruited to junctions to set up dynamic junction formation and maintenance of the AJ. Our on-going studies are investigating the mechanism for the interdependency of WAVE and CCC components at the apical junction.W

Borinskaya, S. et al. (2017) International Worm Meeting "Branched actin defines Cadherin receptor dynamics in C. elegans embryonic epithelia."

Sasidharan, S., Borinskaya, S., Larsen, E., Soto, M.

[International Worm Meeting, 2017]

During embryonic morphogenesis organs form and migrate to their final positions. This process requires continuous assembly and remodeling of adherens junctions (AJ). Our studies have identified Arp2/3 (branched actin nucleator) and its nucleation promoting factor, WAVE/Scar, as essential regulators of epithelial junctions. E-Cadherin/HMR-1 is a transmembrane receptor and an essential component of the AJ. Recent studies have shown that the turnover of Cadherin at the AJ is much more dynamic than previously thought. Adherens junctions rely heavily on the architecture and dynamics of the actin cytoskeleton. However, determining the function of the branched actin network at the junctions has proven elusive. Biochemical data have shown that AJ component a-catenin/HMP-1 inhibits Arp2/3-dependent branched actin, which suggested that branched actin is only needed at the earliest stages of apical junction development. We have addressed the role of branched actin at epithelial junctions using Transmission Electron Microscopy and live imaging. We found that branched actin is continuously recruited during both the dynamic process of junction formation and the later process of junction maintenance. Our results demonstrated an essential role for branched actin regulators in apical junction maintenance, and interdependence between the branched actin regulators and the Cadherin-Catenin complex (CCC). Therefore, we propose that there is a feedback mechanism present in which AJ-to-WAVE crosstalk dictates how the branched actin network is functioning. To our surprise, the three main components of the CCC complex, including Cadherin/HMR-1, accumulated to higher levels in embryos depleted of WAVE. We turned to FRAP imaging technique and biochemical approaches on C. elegans embryos to address this observation. We predicted that the increased levels of HMR-1::GFP in WAVE mutants resulted from slower turnover of HMR-1 at the membrane. Instead, we observed higher HMR-1::GFP mobility in the intestinal and epidermal junctions of WAVE-defective embryos. Several competitive models could be used to explain these results. Our goal is to determine which of the proposed mechanisms or Cadherin turnover, lateral-to-apical diffusion or vesicular trafficking, is significantly disrupted in WAVE mutants. We will interpret the fluorescence recovery traces by kinetic modeling. In addition we will quantify HMR-1 levels and mobility in embryos that are devoid of trafficking proteins that regulate distinct trafficking steps. These studies will advance understanding of the branched actin role at the forming and established adherens junctions. We will elucidate how branched actin affects Cadherin turnover at the apical cell-cell contacts in an in vivo system.

Van Buskirk, Cheryl (2017) International Worm Meeting "A forward genetic screen for mutants defective in recovery sleep."

Van Buskirk, Cheryl

[International Worm Meeting, 2017]

In response to damaging conditions such as noxious heat or UV exposure, C. elegans enters a period of behavioral quiescence known as stress-induced sleep or recovery sleep (RS), during which sensory responses are dampened and feeding and movement cease1. Recovery sleep is mediated by activation of EGF receptors on the peptidergic ALA interneuron and subsequent release of a collection of neuropeptides1-3. At this time it is not known how cellular damage leads to the initiation of EGF signaling, and gaps remain in our understanding of signal transduction events within ALA as well as in the target tissues affected by ALA peptides. In order to uncover genes required for recovery sleep we have initiated an EMS screen for sleepless F2 animals, using the pore-forming toxin Cry5B as our damage-inducing agent. Progeny of sleepless candidates are tested for responses to other known sleep-inducing stressors, such as heat and UV light. Mutants that are found to be generally defective in recovery sleep are kept for SNP mapping, complementation as needed, and eventual whole-genome sequencing. We will present our detailed methods, some obstacles that have been overcome in our mapping of sleep mutants, and information on candidate identity if available. We also describe how this project has been implemented within the context of an undergraduate laboratory course called BIOL447 FIRE: Full Immersion Research Experience. 1. Hill AJ, Mansfield R, Lopez JMNG, Raizen DM, Van Buskirk C. 2014. Cellular Stress Induces a Protective Sleep-like State in C. elegans. Curr. Biol. 24, 2399-2405. 2. Nelson MD, Lee KH, Churgin MA, Hill AJ, Van Buskirk C, Fang-Yen C, Raizen DM. 2014. FMRFamide-like FLP-13 neuropeptides promote quiescence following heat stress in Caenorhabditis elegans. Curr. Biol. 24:2406-2410. 3. Nath RD, Chow ES, Wang H, Schwarz EM, Sternberg PW. 2016. C. elegans stress- induced sleep emerges from the collective action of multiple neuropeptides. Curr. Bio.l 26:2446-2455.

Josefin Nystrom-Friberg et al. (2003) International Worm Meeting "Two new loci affecting body length in C. elegans."

Simon Tuck, Armand Leroi, Josefin Nystrom-Friberg, Richard W Padgett, Zai-Zhong Shen

[International Worm Meeting, 2003]

Mutations in genes encoding a signal transduction pathway activated by a ligand in the TGF- faimly, DBL-1 can profoundly affect body size in C. elegans. Loss of function mutations in genes in the pathway causes a Sma phenotype characterized by a marked reduction in body size whereas constitutive activation of the pathway increases body length (Lon). Worms displaying the Sma or Lon phenotypes appear to have the same numbers of cells as wild type suggesting that the pathway affects body size by affecting cell size rather than cell number. The pathway may excert its efffects in part at least by affecting endoreduplicaton of hypodermal nuclei, and in part by regulating expression of components of the cuticle. Two targets of the pathway have been identified, lon-1which encodes a PR-related protein and lon-3 which encodes a cuticle collagen1, 2, 3, 4. Mutations in lon-1 affect both endoreduplication and body length, whereas mutations in lon-3 affect length without affecting endoreduplication. In a screen for new mutations causing a Lon phenope we have isolated 2 alleles, sp10 and sp11, of a locus on X, lying between unc-9 and lin-15. The mutations are all recessive and 100% penetrant; homozygous mutants are approximately 23 % longer than wild type but display no other obvious defects. Morphometric analysis indicates that the phenotype caused by these mutations is very similar to that caused by overexpression of DBL-1. We have identified a cosmid that rescues the mutant phenotype. The screen also identified a single allele, sp9, of a locus between unc-3 and lin-15 on X. This mutation causes an incompletely penetrant Lon phenotype; homozygous mutants are also slightly Egl. 1. Morita K, Flemming AJ, Sugihara Y, Mochii M, Suzuki Y, Yoshida S, Wood WB, Kohara Y, Leroi AM, Ueno N, EMBO 21: 1063-1073 (2002); 2. Maduzia LL, Gumienny TL, Zimmerman CM, Wang H, Shetgiri P, Krishna S, Roberts AF, Padgett RW, Developmental Biology 246: 418-428 (2002); 3. Nystrom J, Shen ZZ, Aili M, Flemming AJ, Leroi A, Tuck S, Genetics 161: 83-97 (2002); 4. Suzuki Y, Morris GA, Han M, Wood WB, Genetics 162:1631-1639 (2002).

Andrew Bretscher et al. (2009) European Worm Neurobiology Meeting "Sensory neurons regulating C. elegans carbon dioxide avoidance behaviour"

Eiji Kodama, Andrew Bretscher, Mario De Bono

[European Worm Neurobiology Meeting, 2009]

CO2 comprises only ~0.038% of earth.s atmosphere but local CO2 concentrations can rise much higher in the presence of aerobically respiring organisms. Previous data have shown that well-fed C. elegans avoid CO2 levels greater than 0.5% 1,2. This worm can alter behaviour in response to both changes in CO2 levels, d[C]/dt, and to absolute CO2 concentrations [C]. These responses are highly regulated. For example starvation strongly suppresses CO2 avoidance as does exposure to hypoxia. We are investigating how the C. elegans nervous system coordinates responses to CO2. Recent laser ablation studies show that the anteriorly located BAG neurons are required for acute CO2 avoidance 1. BAG neurons also respond to changes in O2 3 and we have shown that O2 levels can modulate CO2 avoidance 2. These data raise two alternatives: 1) BAG responses to O2 modulate neural circuits mediating CO2 responses; 2) BAG neurons respond to both O2 and CO2. To test whether the BAG neurons are CO2 sensors, we applied CO2 to N2 animals expressing the high-affinity genetically encoded calcium sensor YC3.60 in this neuron. The neurons responded with a large change in YFP/CFP fluorescence, indicative of a rise in calcium. Calcium transients were observed as long as CO2 levels remained high and peak activity coincided with CO2 increases. Concentrations as low as 0.25% elicited robust CO2 responses. unc-13 and unc-31 mutants expressing pBAG::YC3.60 retained N2-like responses to CO2. This suggests that pre-synaptic input is not required for CO2 responses and that BAG neurons are primary CO2 sensors. CO2-induced activity in BAG requires the cGMP-gated ion channel subunits TAX-2 and TAX-4. At a behavioural level TAX-2 and TAX-4 promote CO2 avoidance, but other pathways are also important. Employing in vivo calcium imaging to test hypotheses based on earlier genetic work we are currently seeking other sensory neurons that display pronounced CO2 sensitivity. References 1. Hallem, E. A. & Sternberg, P. W. Acute carbon dioxide avoidance in Caenorhabditis elegans. Proc Natl Acad Sci U S A 105, 8038-8043 (2008). 2. Bretscher AJ, Busch KE, de Bono M. Carbon dioxide avoidance behavior is integrated with responses to ambient oxygen and food in Caenorhabditis elegans. Proc Natl Acad Sci U S A 105, 8044-9 (2008). 3. Zimmer M, Gray JM, Pokala N, Chang AJ, Karow DS, Marletta MA, Hudson ML, Morton DB, Chronis N, Bargmann CI. Neurons detect increases and decreases in oxygen levels using distinct guanylate cyclases. Neuron 61, 865-79 (2009).

Mansfield, R. et al. (2015) International Worm Meeting "Disruption of cellular proteostasis drives sleep behavior in C. elegans."

Mansfield, R., Van Buskirk, C.

[International Worm Meeting, 2015]

Sleep is widely regarded to be a restorative state, and the physiological perturbations associated with sleep deprivation are extensive. Despite intensive study, none of these perturbations has in turn been shown to drive sleep. Recently our lab has shown that in C. elegans, environmental stressors such as heat, cold and toxins induce a sleep-like state (Hill et al., 2014). As noxious environmental stressors are known to interrupt normal proteostasis, the existence of a subsequent sleep state suggests that sleep serves to assist in the restoration of normal proteostasis. This idea is supported by our observation that sleepless animals have impaired survival following severe stress (Hill et al., 2014). Further, we have found that chaperone response defective mutants display exaggerated sleep responses. These mutants include animals lacking the stress-induced transcription factors hsf-1/HSF-1, daf-16/FOXO and a component of the endoplasmic reticulum stress-response pathway hsp-4/BiP. We are testing site-of-action for this effect by examining strains with tissue-specific rescue of HSF-1. In addition, we are using pharmacological inhibitors of the proteasome to test whether direct inhibition of the proteostasis machinery can trigger sleep. Last, we are performing RNAi against both positive and negative regulators of the heat shock transcriptional response, and screening for sleep phenotypes. The results of these efforts will be presented.Hill AJ, Mansfield R, Lopez JMG, Raizen DM, Van Buskirk C (2014) Cellular stress induces a protective sleep-like state in C. elegans. Curr Bio 24:1-7.Zimmerman JE, Naidoo N, Raizen DM, Pack AI (2008) Conservation of sleep: insights from non-mammalian model systems. Trends Neurosci 31:371-6.

Scott Parker et al. (2004) European Worm Meeting "THE ROLE OF CAVEOLIN-1 AND -2 IN IP3 MEDIATED CALCIUM RELEASE IN CAENORHABDITIS ELEGANS."

Howard Baylis, Scott Parker

[European Worm Meeting, 2004]

The production of the second messenger inositol 1,4,5-trisphosphate (IP3) through the hydrolysis of PIP2 is a key mechanism by which cell surface stimuli can regulate intracellular calcium levels. It has been shown that, in the model organism Caenorhabditis elegans, the IP3 receptor (IP3R) is widely expressed and is involved in the regulation of a wide range of developmental and physiological processes (1). Previous work has shown that calcium signals are associated with caveolae (2) and that the components of IP3 signalling are also localised within caveolae (3). We therefore set out to establish whether caveolae or lipid rafts play a role in the modulation of IP3 and calcium-mediated signalling in C. elegans. There are two caveolin genes in C.elegans (4) of which cav-1 has been implicated in the regulation of meiotic cell progression (5). To establish the expression pattern of both caveolins we have made transgenic animals carrying the cav-1 and cav-2 genes fused to gfp. cav-1 is widely expressed in embryos and the nervous system, while cav-2 is only expressed in gut cells. Interestingly, no co-localisation occurs. To test the role of cav-1 and cav-2 in IP3-mediated processes we are examining the effects of knocking down caveolins using RNA-mediated interference (RNAi) and gene knock-outs. Furthermore, overexpression of caveolins have revealed interesting phenotypes. 1.Walker DS, Gower NJ, Ly S, Bradley GL, Baylis HA (2002) Mol Biol Cell 13, 1329-37. 2.Isshiki M, Ando J, Yamamoto K, Fujita T, Ying Y, Anderson RG (2002) J Cell Sci 115, 475-84. 3.Isshiki M, Anderson, RG (1999) Cell Calcium 26, 201-8. 4.Tang Z, Okamoto T, Boontrakulpoontawee P, Katada T, Otsuka AJ, Lisanti MP (1997) J Biol Chem 272, 2437-45. 5.Scheel J, Srinivasan J, Honnert U, Henske A, Kurzchalia TV (1999) Nat Cell Biol 1, 127-9.

Aamodt EJ et al. (1991) International C. elegans Meeting "GUT-SPECIFIC GENE EXPRESSION DURING C. elegans DEVELOPMENT."

Chung MA, Aamodt EJ, McGhee JD

[International C. elegans Meeting, 1991]

The ges-1 gene codes for a non-specific carboxylesterase that is expressed only in cells of the gut lineage. We are taking several approaches to investigate the control of ges-1 expression: deletion analysis, in VIVO competition, and cross-species transformation. Deletion analysis: Transformation of ges-1(0J mutants with a 5'deletion series of the ges-1 gene reveals two unexpected behaviours: (1) the pattern of ges-1 expression switches from the gut into muscle cells of the (sister) MS-lineage or into muscle and hypodermis cells of the (cousin) C-lineage, depending on the particular deletion. These switches in expression pattern indicate that gut-specific gene expression in C. elegans involves not only gut-specific activators but also multiple repressors that are present within particular non-gut lineages. (2) Most deletions are expressed in more than one pattern with either one pattern or the other expressed in individual embryos. This either/or choice suggests that the expression pattern is chosen before the different cell lineages diverge. In vivo competition: Injection of excess control region DNA into wild-type worms can lead to extinction of normal ges-1 expression in the gut and can also lead to expression of ges-1 outside the gut, consistent with the idea that ges-1 expression involves both activators and repressors. Crossspecies- analysis: We have compared the control of ges-1 in C. eleqans and C. briaqsae. The Mello et aJ. transformation procedure to make transformed lines of C. elegans also works to make transformed lines of C. brigasae, showing that the C. elegans rol-6 gene is expressed in the hypodermal cells of C. briqqsae. The C. elegans ges-1 gene was transformed into C. brigqsae and the C. brigqsae ges-1 gene was transformed into C. eleaans. The results show that the ges-1 control circuits have been accurately maintained between these two species, even though flanking sequences of the ges-1 genes have diverged considerably.

Xie, Y. et al. (2011) International Worm Meeting "The RFX Transcription Factor DAF-19 Couples Toll-interleukin 1 Receptor Domain Protein TIR-1 Signaling to Regulation of Neuronal Activities in C. elegans."

Moussaif, M., Xie, Y., Choi, S., Sze, J.

[International Worm Meeting, 2011]

Prior studies from our laboratory and others have demonstrated two layers of transcriptional regulation of the serotonin-biosynthesis enzyme tryptophan hydroxylase tph-1 in the ciliated chemosensory neurons ADF. The OCR-2/OSM-9 TRPV channel regulates the basal tph-1 expression under optimal growth conditions (Zhang et al., 2004). Stress conditions or mutations of the sensory cilia further upregulate tph-1 expression in ADFs (Zhang et al., 2005; Chang et al., 2006; Moussaif and Sze, 2009; Shivers et al., 2009). To identify new genes involved in the regulation of tph-1 expression in the ADF neurons, two genetic screens were carried out. We identified that yz58, a gain-of-function allele of the Toll-interleukin 1 receptor domain protein TIR-1, restored tph-1 expression in the ocr-2 mutant background. Characterization of mutations that suppress tph-1 upregulation by tir-1 (yz58) led to the identification of two daf-19 alleles. Interestingly, both mutations are located in the DAF-19 dimerization domain. Mutations in DAF-19 do not affect the expression of CAT-1/VMAT, indicating that the mutations do not transform the ADF serotonergic cell fate. Further analysis suggests that DAF-19 also interacts with TIR-1 signaling in other cellular and physiological functions. References Chang AJ, Chronis N, Karow DS, Marletta MA, Bargmann CI. 2006. A distributed chemosensory circuit for oxygen preference in C. elegans. PLoS Biol 4:e274. Moussaif M, Sze JY. 2009. Intraflagellar transport/Hedgehog-related signaling components couple sensory cilium morphology and serotonin biosynthesis in Caenorhabditis elegans. J Neurosci 29:4065-75. Shivers RP, Kooistra T, Chu SW, Pagano DJ, Kim DH. 2009. Tissue-specific activities of an immune signaling module regulate physiological responses to pathogenic and nutritional bacteria in C. elegans. Cell Host Microbe 6:321-30. Zhang S, Sokolchik I, Blanco G, Sze JY. 2004. Caenorhabditis elegans TRPV ion channel regulates 5HT biosynthesis in chemosensory neurons. Development 131:1629-38. Zhang Y, Lu H, Bargmann CI. 2005. Pathogenic bacteria induce aversive olfactory learning in Caenorhabditis elegans. Nature 438:179-84.