Electron cryo-tomography of excitatory synapses and the structural characterisation of the postsynaptic molecular complexes
Zusammenfassung der Projektergebnisse
Synapses are functional connection points between neurons, transmitting a presynaptic electrical signal (action potential) across the synaptic cleft to the postsynaptic neuron. Many molecules have been shown to be necessary for different aspects of synaptic transmission. However, much less is known about the organization of these molecules within synapses which has already been shown to be crucial for some synaptic processes. Cryo-electron tomography (cryo-ET) is a method that allows three-dimensional (3D) imaging of vitrified (frozen hydrated) biological material, within cells and cellular compartments. The material remains in “close-to-physiological” state, that is without artefacts that are often induces by chemical fixation, dehydration and heavy metal staining. In this project, we employed cryo-ET to study the structure of synapses at the molecular level. Our cryo-electron tomography experiments of the presynaptic terminal showed that these terminals contain a complex network of filaments. Our data indicate that these structures play a prominent role in synaptic vesicle release. Docked synaptic vesicles did not make membrane to membrane contact with the active zone but were instead linked to it by tethers of different length. Our observations allowed us to propose a model of synaptic vesicle organization and release based on these filaments. On the technical side, this analysis included a rigorous statistical analysis of results from more than thirty cryo-tomograms, obtained under six different experimental conditions. Up to our knowledge this is the first study of this scale in cryo-ET. The combination of faithful preservation and imaging by cryo-ET, and automated segmentation and analysis procedures that we developed was indispensable here. In a related project, which was the basis for the above presynaptic work, we showed the existence of a net-like layer in the synaptic cleft from isolated synapses from synaptosomal cellular fraction. We also found out that synaptic adhesion complexes are extensively laterally connected and possess complex topology. Furthermore we obtained several cryo-tomograms from dissociated neuronal cultures, confirming many of the observations from synaptosomes. Several technical developments and feasibility studies were included in these projects. We showed the feasibility of cryo-ET for investigations on synaptosomes and neuronal cultures, and optimized some of the current procedures involved. Furthermore we developed an automated procedure for segmentation of membrane-bound molecular complexes and implemented procedures for various forms of analysis (grey-scale, morphological, localization) of segmented complexes. Finally, we developed a correlative light microscopy and cryo-ET approach that is suitable for application to dense cellular environments such as mature neuronal cultures. We would like to point out that we were focused on solving questions related to the synaptic function. However, we started with a novel application of a relatively new technique and consequently we had to adapt to the circumstances to some extent and pursue different directions. The technical developments mentioned were always directly related to biological problems, however they were often used and further developed for other projects. Even though the work we have done within DFG SPP1128 program yielded insights into structural 2organisation and function of synapses, we consider it to be a large feasibility study. The full potential of these developments is yet to be realized.
Projektbezogene Publikationen (Auswahl)
-
(2005). Morphological charac- terization of molecular complexes present in the synaptic cleft. Structure 13, 423–34
Lucic, V., Yang, T., Schweikert, G., Forster, F., and Baumeister, W.
-
(2005). Structural studies by electron tomography: from cells to molecules. Annu Rev Biochem 74, 833–65
Lucic, V., Forster, F., and Baumeister, W.
-
(2007). Multiscale imaging of neurons grown in culture: from light microscopy to cryoelectron tomography. J Struct Biol 160, 146–56
Lucic, V., Kossel, A. H., Yang, T., Bonhoeffer, T., Baumeister, W., and Sartori, A.
-
(2008). Cryo-electron tomography of cells: connecting structure and function. Histochem Cell Biol 130, 185–96
Lucic, V., Leis, A., and Baumeister, W.
-
Cyrklaff, M., Baumeister, W., and Lucic, V. (2010). Quantitative analysis of the native presynaptic cytomatrix by cryoelectron tomography. J Cell Biol 188, 145–56
Fernandez-Busnadiego, R., Zuber, B., Maurer, U. E.
