Presynaptic tethers: molecular composition and functional role
Final Report Abstract
Here we obtained comprehensive, in situ, single nanometer resolution images of complexes involved in SV priming and neurotransmitter release by cryo-ET of synaptosomes and neuronal cultures. We simultaneously imaged multiple complexes in their native composition, conformation and environment, and within entire presynaptic terminals, resulting in a precise localization of complexes and determination of their spatial organization. Furthermore, a concurrent detection of alterations caused by genetic and pharmacological perturbations allowed us to identify some of the presynaptic complexes and led us to propose a comprehensive structural model of priming and release. Our cryo-ET results showed that Munc13- and SNAP25-dependent tethers localize vesicles with a single nanometer precision, thus defining sequential vesicle states that immediately precede neurotransmitter release. The transition between the states involves two parallel biochemical pathways and is associated with a conversion to SNARE complex-dependent tethers and a loss of protein connectors that interlink vesicles. These results provide an example of a cellular process carried out by a concerted action of multiple spatially separated and molecularly diverse complexes comprising a large, non-periodic, molecular assembly and provides a framework for further exploration of such assemblies. Using transgenic mice expressing either a genetically encoded fluorescent marker or the biotin ligase BioID specifically either in the dentate gyrus or the CA3 region of the hippocampus we have obtained data about the abundance of the mRNAs and proteins as well as of alternatively spliced variants present in these two functionally distinct neuron types. We are currently finalizing the bioinformatic comparative analysis of these data sets. These data sets constitute a comprehensive resource that provides, to our knowledge for the first time, corresponding information about the abundance and the alternative splicing of mRNA and protein from these neuron types isolated from the same Cre-driver lines. Thereby, it will form the basis for further studies into the functional relevance of the distinct composition of the release machinery in different neuron types.
Publications
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(2021) Trans-synaptic assemblies link synaptic vesicles and neuroreceptors. Sci Adv 7(10):eabe6204
Martinez-Sanchez A, Laugks U, Kochovski Z, Papantoniou C, Zinzula L, Baumeister W, Lučić V
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(2022) A presynaptic phosphosignaling hub for lasting homeostatic plasticity. Cell Reports 39(3):110696
Müller JA, Betzin J, Santos-Tejedor J, Mayer A, Oprişoreanu A-M, Engholm-Keller K, Paulußen I, Gulakova P, McGovern TD, Gschossman LJ, Schönhense E, Wark JR, Lamprecht A, Becker AJ, Waardenberg AJ, Graham ME, Dietrich D, Schoch S
