Gottschalk, Alexander et al. (2019) International Worm Meeting "Synapsin is required for cAMP-dependent neuropeptide release."

Shao, Jiajie, Yu, Szi-Chieh, Liewald, Jana, Gottschalk, Alexander, Steuer Costa, Wagner

[International Worm Meeting, 2019]

Release of neuropeptide-containing dense core vesicles (DCVs) is an important process in neuromodulation and in the switching of brain states. By optogenetics, behavioral studies, electrophysiology, electron microscopy, and imaging, we previously showed (Steuer Costa et al., 2017) that cAMP increase in cholinergic motor neurons leads to mobilization and increased fusion of synaptic vesicles (SVs), as well as DCVs. The neuropeptides released caused an auto-regulatory increase in SV filling with acetylcholine, observable by visibly increased SV diameter and increased miniature post-synaptic current (mPSC) amplitudes. Here, we show that synapsin is required for the cAMP-dependent stimulation of neuropeptide release. Synapsin is a phosphoprotein that tethers SVs to the actin cytoskeleton, and forms liquid-liquid phase-separated SV clusters via its C-terminal intrinsically disordered region. In synapsin mutants, DCVs are largely absent from synaptic terminals, and are not released following light-induced cAMP increase. While SV release still occurs, mPSC amplitudes are not increased, and no increase in SV diameter is observed in synapsin mutants. Synapsin mutant terminals are largely devoid of DCVs, which instead accumulate in the neuronal processes and cell bodies. We also analyzed CRISPR-engineered mutants of the major PKA phosphorylation site in synapsin SNN-1B, S9. The non-phosphorylatable S9A mutant was essentially normal, while the phosphomimetic S9E was affected for DCV release, and accumulated DCVs in synapse-distal regions of the neurons. Thus, synapsin captures DCVs at terminals, making them available for release. We are currently analyzing DCV trafficking in the mutant neurons. Steuer Costa, Yu, Liewald, Gottschalk, 2017, Curr Biol 27: 495-507

Gottschalk, A. et al. (2019) International Worm Meeting "Optogenetic interrogation of a neuronal circuit orchestrating locomotion 'stop' using fast volumetric scanning and dual Ca2+ imaging."

Gottschalk, A., Bach, Maximilian, Van der Auwera, Petrus, Steuer Costa, Wagner, Aghigh, Arash

[International Worm Meeting, 2019]

Identifying specific neurons within a neuronal circuit and linking their activity to a certain behavior remains one of the most challenging tasks in the field of neurobiology. Using an optimized system for fast automated x-,y-tracking of a fluorescent marker in freely moving C. elegans, we found that the GABAergic interneuron RIS, which is known to promote sleep during larval development, is reutilized in adulthood for orchestrating locomotion 'stop'. We could show that RIS exhibits compartmentalized calcium dynamics by performing axonal calcium imaging. By further expanding our setup using a Piezo scanner for simultaneous volumetric scanning we are able to analyze the neuronal network of RIS by recording calcium activity from two distinct neurons within the network at the same time. This is achieved by expressing the genetically encoded calcium indicators (GECIs) GCaMP6 and jRCaMP1b cell specifically in RIS and the neurons upstream (i.e. CEPs) and downstream (i.e. RIB & RIM) of RIS according to its connectome. The observed, possibly linked activities are verified by optogenetic stimulation or inhibition of RIS or the CEPs using microbial rhodopsins, and assessing the correlation of calcium activity in the downstream neuron. The scanner in our system requires less than 200 ms per volume, moving in a triangular scanning pattern, and enabling to account for fast calcium dynamics. The data analysis is based on open source and easy-to-use image analysis software. Image registration is done with ImageJ software using simple stack-slice manipulations and the Scale Invariant Feature Transform (SIFT). Subsequently all the data from the calcium and behavioral imaging is evaluated using customized MATLAB and Mathematica scripts. Measuring spontaneous or evoked calcium activity in one or two distinct neurons at high spatiotemporal resolution in soma, ventral nerve cord and nerve ring processes and extracting behavioral parameters like bending angles, body length and speed will enable deeper insights into the architecture and functional interplay of cells of the described neuronal network.

Nagpal, Jatin et al. (2015) International Worm Meeting "Optogenetic control of cGMP mediated signal transduction ."

Steuer Costa, Wagner, Gao, Shiqiang, Bethke, Mary, Brueggemann, Chantal, L'Etoile, Noelle, Schneider, Martin, Woldemariam, Sarah, Nagpal, Jatin, Nagel, Georg, Gottschalk, Alexander

[International Worm Meeting, 2015]

Cyclic guanosine monophosphate (cGMP) is a widely used 2nd messenger in cellular signaling, acting via protein kinase G (PKG), or cyclic nucleotide gated (CNG) channels. In sensory neurons, cGMP allows for signal modulation and amplification, before depolarization. Manipulating cGMP levels is required to access this signalling and provide insights into signal encoding. We achieve this by implementing two photo-activatable guanylyl cyclases - 1) guanylyl cyclase rhodopsin from Blastocladiella emersonii (BeCyclOp) and 2) a mutated version of Beggiatoa sp. bacterial light-activated adenylyl cyclase (BlaC), with specificity for GTP, termed BlgC or bPGC (Beggiatoa photoactivated guanylyl cyclase). BeCyclOp enabled rapid and precise light-triggered cGMP increases in heterologous cells (Xenopus oocytes) and in C. elegans. BeCyclOp exhibits an unusual 8 transmembrane topology and cytosolic N-terminus. Oocyte experiments revealed light/dark activity ratio of ~5,000 and no cAMP production. Via co-expression of the TAX-2/-4 CNG channel in C. elegans body wall muscle, BeCyclOp photoactivation induced rapid light-driven depolarization and contraction of muscle cells. In C. elegans O2/CO2 sensory BAG neurons, BeCyclOp activation rapidly triggered slowing of locomotion, consistent with the normal sensory function of BAG, and in agreement with previous BAG activation by channelrhodopsin (ChR2). Interestingly, a quick 'recovery' of the slowing response was observed both in ChR2 and BeCyclOp stimulation, despite ongoing photostimulation, arguing that this apparent desensitization is neither mediated at the level of cGMP nor the CNG channel, but at the output synapses or in downstream networks.Light activation of bPGC expressed in muscle cells along with TAX-2 and TAX-4 caused a relatively slower and less pronounced contraction as compared to BeCyclOp. We could validate these differences in cGMP production from the two cyclases by directly imaging the cGMP rise, using a genetically encoded cGMP sensor, WincG2. WincG2 (or worm indicator of cGMP) is based on FlincG3, a circularly permutated EGFP fused to cGMP binding domain of PKG. Currently, we are expressing the cyclases in a variety of C. elegans sensory neurons that use cGMP as the 2nd messenger and are performing behavioural experiments that recapitulate cGMP mediated signal transduction in these sensory neurons using optogenetic activation of the cyclases.

Shao, Jiajie et al. (2021) International Worm Meeting "Synapsin is required for dense core vesicle capture and cAMP-dependent neuropeptide release"

Liewald, Jana, Costa, Wagner, Yu, Szi-Chieh, Gottschalk, Alexander, Shao, Jiajie

[International Worm Meeting, 2021]

Release of neuropeptides from dense core vesicles (DCVs) is essential for neuromodulation. Compared to the release of small neurotransmitters, much less is known about the mechanisms and proteins contributing to neuropeptide release. By optogenetics, behavioral analysis, electrophysiology, electron microscopy, and live imaging, we show that synapsin SNN-1 is required for cAMP-dependent neuropeptide release in Caenorhabditis elegans hermaphrodite cholinergic motor neurons. In synapsin mutants, behaviors induced by the photoactivated adenylyl cyclase bPAC, which we previously showed to depend on acetylcholine and neuropeptides (Steuer Costa et al., 2017), are altered like in animals with reduced cAMP. Synapsin mutants have slight alterations in synaptic vesicle (SV) distribution, however, a defect in SV mobilization was apparent after channelrhodopsin-based photostimulation. DCVs were largely affected in snn-1 mutants: DCVs were ~30% reduced in synaptic terminals, and not released following bPAC stimulation. Imaging axonal DCV trafficking, also in genome-engineered mutants in the serine-9 protein kinase A phosphorylation site, showed that synapsin captures DCVs at synapses, making them available for release. SNN-1 co-localized with immobile, captured DCVs. In synapsin deletion mutants, DCVs were more mobile and less likely to be caught at release sites, and in non-phosphorylatable SNN-1B(S9A) mutants, DCVs traffic less and accumulate, likely by enhanced SNN-1 dependent tethering. Our work establishes synapsin as a key mediator of neuropeptide release.

Husson, Steven J. et al. (2011) International Worm Meeting "Optogenetics dissection of the nociceptive PVD network: RNAi of PVD-specific genes reveals TRP channels as signal amplifiers."

Steuer Costa, Wagner, Husson, Steven J., Lu, Hang, Watson, Joseph D., Treinin, Millet, Miller III, David M., Stirman, Jeffrey N., Spencer, W. Clay, Gottschalk, Alexander

[International Worm Meeting, 2011]

Higher animals display myriads of neurons contributing to pain sensation, making it extremely difficult to study the neuronal circuits underlying nociception. In contrast, only a handful of neurons mediate nociception in C. elegans. Nociceptive neurons contain highly branched dendrites and trigger the sensation of pain in response to noxious insults. The FLP and PVD neurons adopt similar complex dendritic arbors and have been implicated in harsh mechanical touch responses, while other touch receptor neurons (TRNs) detect gentle mechanical stimuli. Studying the behavioral output of the mechanosensory modality of PVD or FLP cells has so far been complicated, because a harsh touch inevitably agitates the whole body of the animal, thus making it impossible to prevent contributions of other cells. In contrast, optogenetic tools like the blue light-activated depolarizing channelrhodopsin-2 (ChR2), allow non-invasive stimulation of the sensory neuron of interest. Expression of ChR2 in the PVD neurons, FLP neurons or downstream command interneurons and subsequent photoactivation, allows us to mimic the harsh touch response, without interfering signaling from the TRN neurons. We show that behavioral responses to harsh touch are shaped by both the FLP and PVD sensors to jointly determine the output response as forward or reverse escape. Molecular players required for PVD function were identified by microarray profiling of mRNAs specifically isolated from PVD. Candidate nociceptive genes were knocked down by RNAi and effects on photo-triggered, ChR2-mediated escape responses were quantified. We found the VGCC a- and b-subunits UNC-2 and CCB-1 to be required for chemical synaptic output from PVD. Targeting synaptobrevin by the Tetanus toxin in PVD to impair synaptic output abolished PVD-dependent behavior. Knockdown of regulators of PVD differentiation severely impaired PVD function, while a DEG/ENaC channel and a TRP channel act cell-autonomously in PVD to shape its dynamic range and amplify the output signal, uncovering a novel role of TRP channels downstream of primary sensor molecules.

Gattegno T et al. (1997) International C. elegans Meeting "A MUTATION AFFECTING CELL FUSIONS DURING ELONGATION OF THE EMBRYO"

Podbilewicz B, Gattegno T

[International C. elegans Meeting, 1997]

Cell fusion is a widespread process throughout development and evolution. Many organs in multicellular organisms contain multinucleate cells formed by cell fusion; e.g., in humans (placenta, bone and muscle) and in C. elegans (hypodermis, vulva, pharynx, uterus, and excretory gland). Enveloped viruses (e.g. HIV) fuse with cell membranes in order to invade target cells, and fertilization involves sperm-egg fusion. We have characterized the order of epithelial cell fusions in C. elegans (1) and have isolated mutations that disrupt cell fusions in the embryo. In our pilot screens we did not obtain any mutation with fewer fusions than in wild-type. The zygotic lethal mutation zu316 was isolated in a screen for mutations that arrest during embryonic elongation (2). In the strain JJ825 zu316/+; lin-2 (e1309) the dorsal hypodermal cells fail to fuse. This mutant was outcrossed and we are now characterizing zu316 by 4D microscopy and immunofluorescence using tissue-specific mAbs and confocal microscopy. The development of the mutant terminates between 1.5 to 2-fold stages of elongation but some embryos arrest earlier. The mutants appear normal up to the beginning of morphogenesis and then the dorsal hypodermal cells look round during elongation and do not get flattened as in N2. Using MH27 and anti-LIN26 antibodies (kindly provided by R. Waterston and M. Labouesse, respectively) we notice that in zu316 there are fewer cell fusions in the dorsal hyp6 and hyp7 than in wild-type embryos. 1. Podbilewicz B. and White J.G. (1994) Dev. Biol. 161, 408-424 2. Costa M. and Priess J. (1995) Worm Meeting Abstract, p. 165 We thank Mike Costa and Jim Priess for sending JJ825 to us.

Yu SC et al. (2021) J Neurosci "Synapsin is required for dense core vesicle capture and cAMP-dependent neuropeptide release."

Gottschalk A, Shao J, Liewald JF, Steuer Costa W, Yu SC

[J Neurosci, 2021]

Release of neuropeptides from dense core vesicles (DCVs) is essential for neuromodulation. Compared to the release of small neurotransmitters, much less is known about the mechanisms and proteins contributing to neuropeptide release. By optogenetics, behavioral analysis, electrophysiology, electron microscopy, and live imaging, we show that synapsin SNN-1 is required for cAMP-dependent neuropeptide release in <i>Caenorhabditis elegans</i> hermaphrodite cholinergic motor neurons. In synapsin mutants, behaviors induced by the photoactivated adenylyl cyclase bPAC, which we previously showed to depend on acetylcholine and neuropeptides (Steuer Costa et al., 2017), are altered like in animals with reduced cAMP. Synapsin mutants have slight alterations in synaptic vesicle (SV) distribution, however, a defect in SV mobilization was apparent after channelrhodopsin-based photostimulation. DCVs were largely affected in <i>snn-1</i> mutants: DCVs were 30% reduced in synaptic terminals, and not released following bPAC stimulation. Imaging axonal DCV trafficking, also in genome-engineered mutants in the serine-9 protein kinase A phosphorylation site, showed that synapsin captures DCVs at synapses, making them available for release. SNN-1 co-localized with immobile, captured DCVs. In synapsin deletion mutants, DCVs were more mobile and less likely to be caught at release sites, and in non-phosphorylatable SNN-1B(S9A) mutants, DCVs traffic less and accumulate, likely by enhanced SNN-1 dependent tethering. Our work establishes synapsin as a key mediator of neuropeptide release.<b>SIGNIFICANCE STATEMENT</b>Little is known about mechanisms that regulate how neuropeptide-containing dense core vesicles (DCVs) traffic along the axon, how neuropeptide release is orchestrated, and where. We found that one of the longest known synaptic proteins, required for the regulation of synaptic vesicles and their storage in nerve terminals, synapsin, is also essential for neuropeptide release. By electrophysiology, imaging and electron microscopy in <i>Caenorhabditis elegans</i>, we show that synapsin regulates this process by tethering the DCVs to the cytoskeleton in axonal regions where neuropeptides are to be released: Without synapsin, DCVs cannot be captured at the release sites and, consequently, cannot be fused with the membrane, and neuropeptides not released. We suggest that synapsin fulfils this role also in vertebrates, including humans.

Shane J Woods et al. (2005) International Worm Meeting "A genome-wide approach to examine genetic robustness through knock-down of duplicate genes using RNAi."

Anthony Rogers, Julie Ahringer, David M Rivers, Shane J Woods

[International Worm Meeting, 2005]

Redundant paralogs have been partially attributed to genetic robustness against null mutations (Gu et al., 2003). In an attempt to assess the genetic robustness of the C. elegans genome we have identified ~ 2000 putative duplicate gene pairs. We have optimized a double RNAi feeding method using our RNAi feeding library (Fraser et al., 2000; Kamath et al., 2003) and an RNAi-supersensitive worm strain to test candidate gene pairs for redundant functions. We will present the data from this screen, where we score embryonic lethality, sterility, and other directly observable phenotypes. This screen presents a resource for novel gene function complementing existing functional genomic screens. In addition, it will allow us to further understand how genomic redundancy is globally organised, as well as the process driving genetic backup. Previous studies of genome redundancy in C. elegans have relied on fitness data from single gene loss-of-function screens (Kamath et al., 2003; Conant & Wagner, 2004). This will be the first genome-wide screen in any higher order organism directly examining genetic robustness on a functional level.

Wieberg, Sydney et al. (2021) MicroPubl Biol

Euwer, Harper, Gerst, Anna, Wieberg, Sydney, Maiden, Stephanie L.

[MicroPubl Biol, 2021]

During embryonic development, changes to cell shape, structure, and location act together to construct an organism's overall form. Epidermal morphogenesis in Caenorhabditis elegans has been a well-studied model for these types of events as this epithelial tissue has substructures like cell-cell and cell-matrix adhesions similar to those in vertebrates (Hsiao and Chisholm 2012). Epidermal cells arise on the dorsal surface of the C. elegans embryo with different subsets of cells undergoing various morphogenetic changes to wrap the embryo in an epithelial sheet (Chisholm and Hardin 2005). During dorsal intercalation, two rows of epithelial cells interdigitate with one another to become a single row along the anterior-posterior axis (Priess and Hirsh 1986; Williams-Masson et al. 1998). Additional cells from either side of the embryo migrate towards the ventral midline, forming new cell-cell adhesions with their contralateral neighbors (Priess and Hirsh 1986; Williams-Masson et al. 1997; Costa et al. 1998). Contractions along circumferential F-actin bundles in the epidermis then change the shape of the cells to drive elongation of the animal along the anterior-posterior axis (Priess and Hirsh 1986). The spectrin cytoskeleton lies apically between the plasma membrane and circumferential F-actin bundles and is thought to be important in structurally supporting the F-actin network during these contractions (Praitis et al. 2005; Lardennois et al. 2019). Homozygous mutant animals of sma-1(ru18), a putative null allele of sma-1/beta(H)-spectrin, are shorter than wildtype with severe disruptions in F-actin bundles normally located at the apical surface (McKeown et al. 1998; Praitis et al. 2005). Since F-actin bundles are also anchored to cadherin-based adhesions at dorsal-ventral cell boundaries, disruption of HMR-1/cadherin, HMP-2/beta-catenin, or HMP-1/alpha-catenin also result in elongation defects and a loss of F-actin at adherens junctions (Costa et al. 1998).

Mondragon A et al. (1998) European Worm Meeting "Characterisation of the C. elegans cadherin superfamily"

Mondragon A, Pettitt J

[European Worm Meeting, 1998]

Cadherins are calcium dependent cell-surface molecules that have been implicated in cell recognition, adhesion, proliferation and morphogenesis both in vertebrates and the fruit fly Drosophila. The C. elegans Genome Sequencing Consortium has reported 10 genes encoding members of the cadherin superfamily, and it is likely that this represents the full complement of cadherins in C. elegans. Two C. elegans cadherins, HMR-1 and CDH-3, have been shown to be required for epithelial cell morphogenesis (1,2), and we would like to determine the roles played by the other cadherins. We are using both RNA interference assays and deletion screens to address this problem. We will present a progress report of this work. The long term aim is to obtain a comprehensive picture of how each cadherin contributes to C. elegans development. It is likely that this work will reveal fundamental aspects of cadherin function that will be applicable to other, more complex, metazoans. 1. Costa et al., (1998). J. Cell Biol. 141, 297-308 2. Pettitt et al., (1996). Development 122 4149-4157.