Schneider, Martin W. et al. (2019) International Worm Meeting "A Photoactivatable Botulinum Neurotoxin for Inducible Control of Neurotransmission."

Sinnen, Brooke L., Schneider, Martin W., Liu, Qi, Tucker, Chandra L., Kennedy, Matthew J., Gottschalk, Alexander

[International Worm Meeting, 2019]

Various biological processes from immune and endocrine signaling to synaptic transmission require regulated secretion. The most powerful and widely used tools to block those processes are clostridial neurotoxins, which specifically proteolyze conserved soluble N?ethylmaleimide?sensitive factor attachment protein receptor (SNARE) family proteins that are critical for vesicle docking and fusion. However, their applicability in the brain and elsewhere is restricted by controlling their activity with a high spatial and temporal resolution. Here we engineered botulinum neurotoxin serotype B (BoNT/B) light chain protease, which cleaves vesicle-associated membrane proteins (VAMPs) including the synaptic vesicle specific C. elegans homolog SNB-1, so that it can be activated with blue light. Therefore, a split protein complementation approach was used wherein the protein was cleaved into two fragments that could functionally reconstitute when fused to the improved light-induced dimerizer (iLID)-SspB photodimerizer system. We demonstrated that the photoactivatable BoNT/B (PA-BoNT) can persistently disrupt excitatory neurotransmission in a light-dependent manner. Thus we expressed different versions of the tool pan?neuronally in C. elegans to assess its effect on locomotion behavior. A partially automated video acquisition and analysis system was established for optogenetic behavioral assays of dozens of animals in parallel. We could show that one hour of pulsed blue light?activation suppressed coordinated swimming behavior by about 50%. This was reversible after 24h in the dark. Interestingly, fusing one part of the optogenetic tool to a synaptic vesicle localized membrane protein (SNG-1) was sufficient to increase its efficacy. Thus PA-BoNT fills a gap for localized and sustained silencing of neurons when compared to spatially diffusive chemogenetic (e.g. designer receptors exclusively activated by designer drugs - DREADDs) or temporally fast-acting optogenetic approaches (e.g. halorhodopsin - NpHR). This makes it particularly useful to study neuronal circuits in a behavioral or learning context. Comparable tools for inhibition of synapses use the photosensitizer miniature singlet oxygen generator (miniSOG) to generate damaging radicals that can have off-target effects. The defined molecular mechanism of action of PA-BoNT not only overcomes this limitation but allows shifting substrate specificity by applying the engineering strategy to other structurally conserved botulinum serotypes (and thus to different SNAREs like SNAP-25). In addition to blocking neurotransmitter release, this optogenetic tool will also have broad applicability to conditionally disrupting secretion of other bioactive molecules, e.g. neuromodulators or neuropeptides.

Bowerman BA et al. (1994) Worm Breeder's Gazette "The Maternal Gene skn-4 and the Specification of Ventral Blastomere Fates in the Early C. elegans Embryo"

Bowerman BA, Shelton CA, Thorpe CJ, Martin PR

[Worm Breeder's Gazette, 1994]

THE MATERNAL GENE SKN-4 AND THE SPECIFICATION OF VENTRAL BLASTOMERE FATES IN THE EARLY C. ELEGANS EMBRYO Bruce Bowerman, Paula R. Martin, Christopher J. Thorpe, and Christopher A. Shelton. The Institute of Molecular Biology, University of Oregon, Eugene, OR 97403.

Zimmer M (2009) Chem Soc Rev "GFP: from jellyfish to the Nobel prize and beyond."

Zimmer M

[Chem Soc Rev, 2009]

On December 10, 2008 Osamu Shimomura, Martin Chalfie and Roger Tsien were awarded the Nobel Prize in Chemistry for "the discovery and development of the green fluorescent protein, GFP". The path taken by this jellyfish protein to become one of the most useful tools in modern science and medicine is described. Osamu Shimomura painstakingly isolated GFP from hundreds of thousands of jellyfish, characterized the chromophore and elucidated the mechanism of Aequorean bioluminescence. Martin Chalfie expressed the protein in E. coli and C. elegans, and Roger Tsien developed a palette of fluorescent proteins that could be used in a myriad of applications.

Hall DH et al. (1994) Worm Breeder's Gazette "Cytology of Degenerin-Induced Cell Death in the PVM Neuron"

Driscoll MA, Chalfie M, Gu GQ, Hall DH, Gong L

[Worm Breeder's Gazette, 1994]

Cytology of degenerin-induced cell death in the PVM neuron David H. Hall, Guoqiang Gu+, Lei Gong#, Monica Driscoll#, and Martin Chalfie+, * Dept. Neuroscience, Albert Einstein College of Medicine, Bronx, N.Y. 10461 + Dept. Biological Sciences, Columbia University, New York, N.Y. 10027 # Dept. Molecular Biology and Biochemistry, Rutgers University, Piscataway, N.J. 08855

Ray C et al. (1990) Worm Breeder's Gazette "CORRECTION Isolation of a C. elegans Cysteine Protease Gene"

McKerrow JH, Ray C

[Worm Breeder's Gazette, 1990]

In the previous issue of the WBG (Vol. 11, #3, page 20) we reported the isolation of a cysteine protease clone from a mixed-stage C. elegans cDNA library. This library was originally obtained from Chris Martin and not from Cynthia Kenyon as was reported in the article. Our apologies for any misunderstanding caused by this oversight.

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.

Goto S (1999) Journal of Gerontology "Commentary on "Protein carbonyl accumulation in aging Dauer formation-defective (daf) mutants of Caenorhabditis elegans"."

Goto S

[Journal of Gerontology, 1999]

In recent years, oxidative damage to macromolecules has gained popularity as the basis of the molecular mechanism of aging. Martin proposes oxidative damage to macromolecules as one of the major public mechanisms of aging. Interest in modifications of protein by reactive oxygen species in aging was apparently introduced by Stadtman. Although various types of oxidative modifications can occur in proteins, carbonyl residues believed to be generated by metal catalyzed reaction or otherwise introduced by lysine, arginine and/or proline residues in vivo are often used as a marker of direct or

Fields CA (1990) Nucleic Acids Res "Information content of Caenorhabditis elegans splice site sequences varies with intron length."

Fields CA

[Nucleic Acids Res, 1990]

A database of sequences of 139 introns from the nematode Caenorhabditis elegans was analyzed using the information measure of Schneider et al. (1986) J. Mol. Biol. 128: 415-431. Statistically significant information is encoded by at least the first 30 nt and last 20 nt of C. elegans introns. Both the quantity and the distribution of information in the 5' splice site sequences differs between the typical short (length less than 75 nt) and rarer long (length greater than 75 nt) introns, with the 5 sites of long introns containing approximately one bit more information. 3' splice site sequences of long and short C. elegans introns differ significantly in the region between -20 and -10 nt.

Hampoelz B et al. (2004) Cell "Heterotrimeric G proteins: new tricks for an old dog."

Knoblich JA, Hampoelz B

[Cell, 2004]

Heterotrimeric G proteins are well known for their function in signal transduction downstream of seven transmembrane receptors. More recently, however, genetic analysis in C. elegans and in Drosophila has revealed a second, essential function of these molecules in positioning the mitotic spindle and attaching microtubules to the cell cortex. Five new publications in Cell (Afshar et al., 2004; Du and Macara, 2004 [this issue of Cell]; Hess et al., 2004), Developmental Cell (Martin-McCaffrey et al., 2004), and Current Biology (Couwenbergs et al., 2004) show that this function is conserved in vertebrates and-like the classical pathway- involves cycling of G proteins between GDP and GTP bound conformations.

Woldemariam, Sarah et al. (2015) International Worm Meeting "Elucidating the role of cGMP dynamics in behavioral plasticity."

Schneider, Martin, Krzyzanowski, Michelle, Bethke, Mary, Nagpal, Jatin, Gottschalk, Alexander, Woldemariam, Sarah, Ferkey, Denise, L'Etoile, Noelle

[International Worm Meeting, 2015]

cGMP is a ubiquitous second messenger implicated in many important biological processes. In neurons, cGMP dynamics can regulate the function of ion channels and kinases, resulting in physiological changes. In the context of learning and memory, these changes result in short-term and long-term behavioral changes based on the organism's experience. We attempt to understand the molecular basis for long-term plasticity by studying the behavioral responses of the nematode C. elegans. Along with our collaborator Michelle Krzyzanowski from the Denise Ferkey lab in SUNY Buffalo, we are interested in how food might modulate behaviors. One food-modulated behavior is repulsion from quinine. This repulsion is mediated by the ASH neuron and it is down regulated by food withdrawal and the cGMP-dependent protein kinase EGL-4. This poses a conundrum since no guanylyl cyclases, which produce cGMP, are expressed in ASH. Genetic evidence suggests that guanylyl cyclases in other neurons are required for the food-modulated repulsion from quinine in ASH and that gap junctions are required for the transmission of cGMP from these neurons to ASH. In order to understand how cGMP dynamics in these neurons are modulated, we need a tool to visualize cGMP. To this end, we are using a cGMP sensor that will allow us to image cGMP dynamics in ASH and other neurons in the living behaving animal in the presence and absence of food.