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

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

[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.

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.

Henss, Thilo et al. (2021) International Worm Meeting "Optogenetic tools for manipulation of cyclic nucleotides, functionally coupled to CNG-channels"

Nagel, Georg, Henss, Thilo, Hirschhauser, Alexander, Scheib, Ulrike, Schneider-Warme, Franziska, Hegemann, Peter, Gao, Shiqiang, Gottschalk, Alexander, Pieragnolo, Alessia, Nagpal, Jatin

[International Worm Meeting, 2021]

The cyclic nucleotides cAMP and cGMP are ubiquitous second messengers that regulate numerous biological processes by activating e.g. protein kinases (PKA and PKG) or cyclic nucleotide gated channels (CNGCs). In eukaryotic GPCR signalling, cAMP is generated predominantly by membrane-bound adenylyl cyclases (mbACs), which are located in microdomains together with GPCRs, PK(A) and their targets. The existing optogenetic toolbox in C. elegans is restricted to soluble adenylyl cyclases (i.e. microbial photoactivatable adenylyl cyclases (PACs) from Euglena (euPAC) and Beggiatoa (bPAC), and the synthetic phytochrome-linked cyclases IlaC22 k27 and PaaC), the membrane-bound Blastocladiella emersonii guanylyl cyclase opsin (BeCyclOp) and hyperpolarising rhodopsins (e.g. Natronomonas pharaonis halorhodopsin - NpHR). Yet missing are membrane-bound photoactivatable adenylyl cyclases (mbPACs) and hyperpolarizers based on K+-currents. To obtain mbPACs, we mutated 2-3 key amino acids in the active site of Blastocladiella and Catenaria CyclOps, which are particular in combining a rhodopsin and a guanylyl cyclase domain. For characterization of photoactivatable nucleotidyl cyclases, we expressed the proteins alone or in combination with CNGCs ("two-component optogenetics") in muscle cells and cholinergic motor neurons. To investigate the extent of optogenetic cNMP production and the ability of the systems to de- or hyperpolarise cells, we performed behavioural analyses (locomotion, muscle contraction), measured cNMP content in vitro, and compared in vivo expression levels. We implemented Catenaria CyclOp as a new tool for cGMP production, allowing fine-control of cGMP levels. We established the mbPACs YFP-BeCyclOp(A-2x) and YFP-CaCyclOp(A-2x), based on mutated versions ("A-2x") of Be and Ca CyclOp, enabling more efficient and specific cAMP signalling compared to soluble bPAC, despite lower overall cAMP production. For hyperpolarization of excitable cells by two-component optogenetics, we introduced the cAMP-gated K+-channel SthK from Spirochaeta thermophila and combined it with bPAC, BeCyclOp(A-2x), or YFP-BeCyclOp(A-2x). As an alternative, we implemented the Blastocladiella emersonii cGMP-gated K+-channel BeCNG1 together with BeCyclOp. In summary, we established a comprehensive suite of optogenetic tools for cNMP manipulation, useful for applications in many cell types, including sensory neurons, and for potent hyperpolarization.

Woldemariam, Sarah et al. (2017) International Worm Meeting "Robust and sensitive cGMP sensor for real time imaging of intact C. elegans."

Li, Joy, Shankar, Raakhee, VanHoven, Miri, Gottschalk, Alexander, L'Etoile, Noelle, Nagpal, Jatin, Krzyzanowski, Michelle, Barsi-Rhyne, Benjamin, Futey, Mary, Tran, Alan, Yu, Yanxun, Ferkey, Denise, Sengupta, Piali, Woldemariam, Sarah

[International Worm Meeting, 2017]

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 order to understand how cGMP dynamics in neurons are modulated, we need a tool to visualize cGMP. To this end, we are characterizing a EGFP based cGMP sensor codon optimized for use in C. elegans that will allow us to image cGMP dynamics in neurons in the living behaving animal. We measured its kinetics by coexpressing the sensor with the optogenetic guanylyl cyclase BeCyclOp in body wall muscle and measured its dynamics in response to numerous stimuli, including changing ion concentrations, noxious stimuli and temperature.