Functional analysis of novel proteins associated with nicotinic acetylcholine receptors and synaptic vesicles in Caenorhabditis elegans
Zusammenfassung der Projektergebnisse
In this project, we studied synaptic transmission in the locomotion nervous system, in most cases the neuromuscular junction, of the nematode Caenorhabditis elegans, on both post- and presynaptic sides. We characterized a complex of two ER proteins, likely with chaperone functions that influence how heteropentameric nAChRs are assembled from a variety of co-expressed subunits. These proteins may have similar roles in mammalian neuronal nAChR biogenesis, and a human orthologue could partially rescue the loss of one of the C. elegans factors. We went on to take the nAChR subunits we previously identified by biochemical methods and proteomics in C. elegans body wall muscle, along with ancillary proteins as well as NRA-2/-4, into a heterologous system, the Xenopus oocyte. Using 11 cRNAs that were co-expressed, we could essentially recapitulate the findings made in vivo, and also determine that one nAChR subunit can functionally substitute for another one, generating a receptor with different functional properties, that may be preferentially formed to evade excitotoxicity, as this channel has a reduced Ca2+ conductivity. We attempted capitalize on our biochemical expertise to purify synaptic vesicles, and to determine their proteome in C. elegans. This approach could have generated new insights into the SV release machinery and SV biogenesis, and provided a way to do so in a cell-type specific manner. However, our tandem-affinity purification (TAP) approach, which worked well on solubilized nAChR complexes (with one copy of the TAP tag per complex), failed to allow isolation of organelles (which contain multiple copies of the affinity tag) in sufficient purity to define a “clean” proteome of the C. elegans SV. We thus turned back to purifying complexes, and currently concentrate on the SNARE complex, with the intention to find novel regulators of formation of this complex or in its recycling. First results are promising, but the project could not yet be finalized within the running period of this proposal. Last, we established and used optogenetic methods for in vivo stimulation and analysis of neurotransmitter release, in a cell-type specific manner, based on the microbial rhodopsin channelrhodopsin-2, a light-gated cation channel, and on the photoactivated adenylyl cyclase PAC, a light-driven enzyme generating the 2nd messenger cAMP. We devised methodology to analyze pre-synaptic mutants using “Optogenetic Investigation of Neurotransmission – OptIoN”, both by behavior, and assisted by a microfluidic high-throughput system, as well as by electrophysiology. Furthermore, we analyzed a GABAB receptor, acting as a feedback system for cholinergic neurotransmission, in cholinergic motor neurons. The project was covered widely in lay-scientific media and even general newspapers, radio and TV programs (for a list of links, please see http://www.biochem.unifrankfurt.de/index.php?id=238).
Projektbezogene Publikationen (Auswahl)
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(2009) The Conserved RIC-3 Coiled-Coil Domain Mediates Receptor specific Interactions with Nicotinic Acetylcholine Receptors. Mol. Biol. Cell. 20: 1419-27
Biala Y, Liewald JF, Ben-Ami HC, Elishevitz E, Shteingauz A, Gottschalk A, Treinin M
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(2008) Optogenetic analysis of synaptic function. Nat. Meth. 5: 895-902
Liewald, J.F., Brauner, M., Stephens, G., Bouhours, M., Schultheis, C., Zhen, M. and Gottschalk, A.
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(2009) An ER-resident membrane protein complex of NRA-2 and NRA-4 regulates nicotinic acetylcholine receptor subunit composition in Caenorhabditis elegans. EMBO J. 28: 2636–2649
Almedom, R., Liewald, J.F., Hernando, G., Schultheis, C., Rayes, D., Pan, J., Schedletzky, T., Hutter, H., Bouzat, C., and Gottschalk, A.
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(2010) High-throughput study of synaptic transmission at the neuromuscular junction enabled by optogenetics and microfluidics. J. Neurosci. Meth. 191: 90-3
Stirman J, Brauner M, Gottschalk A, Lu H
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(2011) Optogenetic analysis of GABAB receptor signalling in Caenorhabditis elegans motor neurons. J. Neurophysiol. 106: 817-827
Schultheis, C., Brauner, M., Liewald, J.F., and Gottschalk A.
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(2011) PACα - an optogenetic tool for in vivo manipulation of cellular cAMP levels, neurotransmitter release, and behavior in Caenorhabditis elegans. J. Neurochem. 116: 616-625
Weissenberger, S., Schultheis, C., Liewald, J.F., Erbguth, K., Nagel, G., and Gottschalk, A.
