Vettkotter, Dennis et al. (2021) International Worm Meeting "Introducing optoSynC - a novel optogenetic tool for synaptic silencing"

Liewald, Jana F., Gottschalk, Alexander, Schneider, Martin W., Vettkotter, Dennis

[International Worm Meeting, 2021]

The investigation of neuronal circuits as well as molecular and cellular functions of neurons requires the control of activity in a spatio-temporal precise manner. The field of optogenetics opens the pathway to stimulate or inhibit specific types of neurons with light. In the last years, several tools have been developed that allow inhibition of specific neurons on time scales from milliseconds to minutes or long-term silencing. However, these optogenetic tools come at the cost of fast induction or reversibility of the altered neuronal function. Thus, a tool which allows for fast, long-term and spatially restricted neuronal silencing, while still allowing for fast reversibility, is of substantial need. Here, we developed an approach to achieve these goals. Using the ability of the Arabidopsis thaliana cryptochrome 2 (CRY2) to form homo-oligomers, we designed an optogenetic tool to cluster synaptic vesicles (SVs) and thus inhibit their function acutely. The tool, called OptoSynC (optogenetic synaptic vesicle clustering), comprises CRY2, fused to the synaptic vesicle intrinsic membrane protein synaptogyrin (SNG-1). The efficiency of OptoSynC was evaluated at the behavioral level. Blue light illumination of pan-neuronally expressed OptoSynC significantly reduced swimming cycles by 80% within seconds. Termination of blue light for more than 15 minutes allowed worms to recover their initial swimming behavior. In addition to behavioral assays, inhibition of synaptic transmission could be demonstrated by electrophysiology experiments. Using a combination of optogenetic activation of neurons with the red-light activated channel Chrimson and blue-light activated inhibition using OptoSynC, we could show the effect even in a single neuron, PVD, required for nociception. While behaviorally, OptoSynC evokes drastic effects, its mode of action has yet to be revealed, as it either inhibits synaptic transmission by SV clustering in the reserve pool, or it might clog-up release sites at the presynaptic terminal due to clustering of SNG-1 protein already present in the plasma membrane. Therefore, we currently employ electron microscopy to shed light on this mechanistic detail. With further optimization of this tool and the knowledge of its underlying mechanism OptoSynC can be a potent tool for synaptic silencing, that might also be used for the research of how SVs are guided to the plasma membrane or in which precise sequence of events SV recycling proceeds.

J Grabitzki et al. (2004) European Worm Meeting "THE PCOME OF CAENORHABDITIS ELEGANS IDENTIFICATION OF PHOSPHORYLCHOLINE-SUBSTITUTED PROTEINS AND COMPARISON OF DEVELOPMENTAL CHANGES IN THE EXPRESSION OF THIS EPITOPE"

R Geyer, G Lochnit, J Grabitzki

[European Worm Meeting, 2004]

Caenorhabditis elegans has been found to be good model system for parasitic nematodes, drug screening and developmental studies. Like the respective parasitic worms, C. elegans expresses glycosphingolipids and glycoproteins, carrying, in part, phosphorylcholine (PC) substitutents, which might play important roles in nematode development, fertility and, at least in the case of parasites, the survival within the host (1). With the exception of a major secretory/ excretory product from Achanthocheilonema viteae (ES-62) (2) and the aspartyl-protease ASP-6 (3), no other proteins carrying this epitope has been identified and studied in detail yet. For C. elegans two N-linked PC-epitopes have been reported so far: (I) a pentamannosyl-core structure carrying three PC-residues (4) and (II) a trimannosyl-core species elongated by a N-acetylglucosamine substituted at C-6 with PC (5). Furthermore, in Dauer larvae of C. elegans there was evidence for the presence of glycans with the composition PC1Hex3HexNAc3 to PC2dHex2Hex4HexNAc7 (6). Here we present the 2D-electrophoretic separation of C. elegans proteins, the comparison of the PC-substitution pattern in distinct developmental stages and the mass spectrometric identification of PC-modified proteins. References: 1.Lochnit, G., Dennis, R. D., and Geyer, R. (2000) Biol Chem 381, 839-847 2.Harnett, W., Harnett, M. M., and Byron, O. (2003) Curr Protein Pept Sci 4, 59-71 3.Lochnit, G., Grabitzki, J., Henkel, B., and Geyer, R. (2003) Biochemical Journal submitted 4.Cipollo, J. F., Costello, C. E., and Hirschberg, C. B. (2002) J Biol Chem 277, 49143-49157 5.Haslam, S. M., Gems, D., Morris, H. R., and Dell, A. (2002) Biochem. Soc. Symp. 69, 117-134 6.Cipollo, J. F., Awad, A., Costello, C. E., Robbins, P. W., and Hirschberg, C. B. (2004) Proc Natl Acad Sci U S A 101, 3404-3408

Jane SD et al. (1997) International C. elegans Meeting "OPTICAL IMAGING OF VIABLE, IDENTIFIABLE NEURONES IN THE ANTERIOR NERVE RING OF CAENORHABDITIS ELEGANS."

Franks CJ, Holden-Dye L, Jane SD, Chad JE, Walker RJ, Wallach LP

[International C. elegans Meeting, 1997]

Here we demonstrate the feasibility of using confocal calcium imaging in identifiable neurones as a refined phenotypic analysis for C.elegans. Experiments were performed on the anterior region of a transgenic strain of C.elegans expressing green fluorescent protein (GFP) in cholinergic neurones. Nematodes were transected at the level of the pharyngeal-intestinal valve. The semi-intact preparation was placed in a chamber on a confocal microscope stage containing modified Dent's saline. In the first series of experiments, dual confocal imaging of GFP (ex.488;em.>520>560) and propidium iodide (ex.568; em. >585) was used to assess the viability of the neurones. GFP fluorescence was observed in neurones within the nerve ring. There was no co-localisation of labelling following incubation with the membrane impermeant nucleic acid intercalalting dye, propidium iodide (15 !M). Subsequent addition of 1% TritonX-100 resulted in a loss of the GFP signal from neurones and a concomitant appearance of propidium iodide staining. Further confirmation of neuronal viability was obtained in a second series of experiments by loading with the acetoxy-methyl ester of calcium orange (10 !M). Differential fluorescence loading was observed in the nerve ring (ex.488;em>585) which was co-localised with GFP fluorescence, implying neuronal distribution. Following wash-off of the loading solution, the calcium orange fluorescence was stable for up to 30 mins, and addition of the calcium mobilising drug ryanodine (10 !M) produced an increased fluorescence within neurones (n=3 preparations; 60% increase in fluorescence). Addition of ionomycin (5 !M) elicited a further increase in pixel intensity indicating that the dye (Kd185nM) was not fully saturated even in the presence of ryanodine. From these data, we infer that in this semi-intact preparation, the neurones can be visualised, have intact cell membranes and maintain Ca++ homeostasis. We thank James Rand and Dennis Frisby for the gift of unc-17:GFP transgenic C.elegans

Julia Grabitzki et al. (2006) European Worm Meeting "Phosphorylcholine-Modified Proteins of Caenorhabditis elegans are Specific to Developmental Stages"

Michael Ahrend, Gunter Lochnit, Julia Grabitzki, Rudolf Geyer

[European Worm Meeting, 2006]

Julia Grabitzki, Michael Ahrend, Rudolf Geyer and Gunter Lochnit. The free-living nematode Caenorhabditis elegans has been found to be an excellent model system for developmental studies [1] investigating parasitic nematodes [2] and drug screening [3]. Structural analyses of glycoconjugates derived from this organism revealed the presence of nematode specific glycosphingolipids of the arthro-series, carrying, in part, phosphorylcholine (PC) substituents [2]. PC, a small haptenic molecule, is found in a wide variety of prokaryotic organisms, i. e. bacteria, and in eukaryotic parasites such as nematodes. There is evidence that PC-substituted proteins glycolipids are assumed to be responsible for a variety of immunological effects including invasion mechanisms and long-term persistence of parasites within the host [4]. In contrast to PC-modified glycosphingolipids [5], only a limited number of PC-carrying (glyco)proteins were identified so far [6-9]. We have analysed the expression of PC-modified proteins of C. elegans during developmental stages using two dimensional SDS-Page separation, 2D-Western-blot and MALDI-TOF mass spectrometry. The pattern of PC-modified proteins was found to be stage specific. The PC-modification on proteins was most abundant in the egg and dauer larvae-stages followed by the adult-stage and L4. Only small amounts of the PC-substitution were found in L3 and L2. In L1 we couldnt detect any PC-Modification. The prediction of the cellular localisation of the identified proteins revealed a predominant cytosolic and mitochondrial occurrence of the PC- modification. Most of the identified proteins are involved in metabolism or in protein synthesis.. 1.. Brenner, S., Genetics, 1974. 77(1): p. 71-94.. 2.. Lochnit, G., R.D. Dennis, and R. Geyer, Biol Chem, 2000. 381(9-10): p. 839-47.. 3.. Lochnit, G., R. Bongaarts, and R. Geyer, Int J Parasitol, 2005. 35(8): p. 911-23.. 4.. Harnett, W. and M.M. Harnett, Mod. Asp. Immunobiol., 2000. 1(2): p. 40-42.. 5.. Friedl, C.H., G. Lochnit, R. Geyer, M. Karas, and U. Bahr, Anal Biochem, 2000. 284(2): p. 279-87.. 6.. Haslam, S.M., H.R. Morris, and A. Dell, Trends Parasitol, 2001. 17(5): p. 231-5.. 7.. Cipollo, J.F., C.E. Costello, and C.B. Hirschberg, J Biol Chem, 2002. 277(51): p. 49143-57.. 8.. Cipollo, J.F., A.M. Awad, C.E. Costello, and C.B. Hirschberg, J Biol Chem, 2005. 280(28): p. 26063-72.

Pemberton DJ et al. (1998) European Worm Meeting "Calcium imaging of viable, isolated anterior region of C. elegans."

Bascal ZA, Walker RJ, Chad JE, Pemberton DJ, Holden-Dye L

[European Worm Meeting, 1998]

The full potential of C. elegans for molecular analysis of neuronal function has not been realised due to the absence, until recently of either electrophysiological or calcium imaging of identifiable neurones in this important model system. In this study, we first have assessed viability of C. elegans dissected preparations following exposure to modified Dent!s ringer solution with osmolarity from 330 to 450 mOsM. We then went on to determine the ability of the pharyngeal muscle and neurones to take up esters of the fluorescent calcium indicators fluo-3, calcium orange and calcium crimson and the ability to measure calcium-dependent changes in fluorescence was tested by treatment with ionomycin and ethylene glycol-bis (b-aminoethyl ether) N,N,N!,N!-tetraacetic acid, (EGTA). Isolated anterior regions of C. elegans expressing green fluorescent protein (GFP) in cholinergic neurones were incubated in modified Dent!s saline of a known osmolarity together with the nucleic acid stain, propidium iodide (15mM). The preparations were then intermittently viewed under a Leitz inverted microscope with epi-fluorescence capabilities to assess the extent of cellular damage. Compromised neurones were seen as those which had exchanged GFP for propidium iodide fluorescence. Increasing the osmolarity of Dent!s saline with glucosamine had no significant effect on the time course of neuronal damage and retained GFP could clearly be seen in approximately 70% of neurones following 24 hours incubation. Intense propidium iodide fluorescence was observed at the point of dissection and within a few unidentifiable cells within the nerve ring. Subsequent exposure of the preparations to distilled water resulted in propidium iodide fluorescence and a loss of GFP, thus indicating rapid membrane disruption. The pharyngeal muscle and neurones took up all the calcium indicators tested, though the different dyes showed evidence of different subcellular distribution. Fluorescence images were collected following treatment with the calcium ionophore, ionomycin (5mM) followed by the calcium chelator, EGTA (10mM). Ionomycin produced a significant rise in terminal bulb fluorescence thus indicating a rise in intracellular calcium levels. Subsequent treatment with EGTA caused a strong reduction in emitted fluorescence which is likely to be the result of the chelation of free intracellular calcium. We conclude that propidium iodide exclusion and GFP retention indicate intact cell membranes. Further, that the neuronal capability to de-esterify fluorescent calcium indicators shows neuronal viability and that the positive controls tested show that intracellular calcium in identifiable neurones can be measured. Supported by the BBSRC and the University of Southampton. Thanks to James Rand and Dennis Frisby for the use of the unc-17:GFP strain.