Our understanding of synaptic structure and function has increased drastically through complementary approaches in the last decades, but some fundamental questions remain unanswered. Specifically, determining the exact location of key molecules and the composition of macromolecular complexes may allow a much more detailed and comprehensive view of the neuron. We use cryo-electron tomography (cryo-ET) to reveal the molecular architecture of neurons in native conditions, in order to clarify some of these issues. The use of cryo-preparation techniques allows investigations of fully-hydrated, vitrified synaptic complexes at the molecular resolution in their native cellular environment free of aggregation, chemical fixation and staining artifacts.
Sample preparation is arguably the critical step in cryo-ET experiments. Synaptosomal cellular fraction is enriched in isolated synapses, and allows functional studies on neurotransmitter release and pharmacological treatments. Dissociated neuronal cultures offer the possibility of studying the architecture of neuronal complexes in situ and correlating it with functional information obtained by light microscopy. Hippocampal slice cultures offer a direct view into nervous tissue, but the difficulties in their cryo-preparation limits their usability. In our opinion, the combination of findings obtained by these complementary preparations holds promise for future research.
Presynaptic terminals enclose neurotransmitter-filled synaptic vesicles embedded in a complex network of filaments. Many physiological roles have been attributed to this presynaptic cytomatrix, but its organization and precise functions in the synaptic vesicle cycle are not well understood.
We showed that the majority of synaptic vesicles within 45 nm of the active zone are linked to it via tethers of different length and that, the precise configuration of the tethering assembly indicates vesicle availability for release. Specifically, our results suggest that the readily releasable pool is formed by synaptic vesicles having multiple short tethers, whose formation is SNARE complex dependent. Furthermore, our results indicate that connectors that link the majority of synaptic vesicles play a major structural role in vesicle organization. These filaments are dynamic and can bind or unbind vesicles in a stimulationdependent manner, thus modulating vesicle recruitment for release. We speculate that the non-uniform vesicle distribution that we observe within the presynaptic terminal is generated by the opposing dynamics of tethers attracting vesicles to the active zone, and connectors pulling vesicles towards the large clusters of interconnected vesicles dominating the distal region of the presynaptic terminal.
Synaptic adhesion complexes
Synaptic adhesion complexes present in the synaptic cleft not only allow the maintenance of the structural integrity of a synapse, but are also involved in the synaptic signaling and synaptogenesis.
The cryo-ET reconstruction of synaptic adhesion complexes of the mammalian central nervous system in their native state showed that a layer of increased density is present in the central region of the cleft. Furthermore, our morphological characterization of these complexes benefited from a novel image segmentation method, and showed that synaptic adhesion complexes are extensively laterally connected, possessing non-trivial topology.
Multiscale imaging of neurons in culture
Cryo-ET of neuronal cultures offers the possibility of studying the architecture of neuronal complexes in situ. While intact neuronal cells are for the most part too thick to be imaged by transmission electron microscopy, the recent introduction of focused ion beam for thining of vitrified cellular samples allows us to alleviate this diffculty. Our current efforts are directed towards stremlining and this procedure.
Correlative microscopy is a hybrid method that allows imaging of the same feature over multiple length scales, combining light microscopy with the ability to obtain high resolution information in cryo-ET. In this way the structural electron microscopy picture can be correlated with functional information obtained by light microscopy. Our approach is entirely software-based, not requiring the use of visual cues, and is therefore applicable to complex cellular landscapes such as mature neuronal cultures. By using a suitable fluorescent marker, this technique allowed us to locate functional presynaptic terminals and visualize them at the molecular level, thus assigning functional information to the structures visualized in cryo-tomograms. Recently, we extended our correlative procedure to allow targeting of focused ion milling to regions identified by fluorescent labeling.
Systematic visual detection and identification of features from cellular cryo-tomograms is a daunting, if not impossible task. Clearly, advanced image processing methods are needed for the objective characterization of these features. To this end, we have developed automated image segmentation and analysis methods that allow comprehensive detection and analysis of complexes present at neuronal synapses.
In addition, we are involved in the improvement of the current denoising algorithms.
Fukuda Y., Laugks U., Lucic V., Baumeister W., Danev R.: Electron cryotomography of vitrified cells with a Volta phase plate. J Struct Biol 190(2):143-54 (2015)
Lucic V., Neuroscience: Towards unified endocytosis. Nature, 515(7526):207-8 (2014)
Martinez-Sanchez A., Garcia I., Asano S., Lucic V., Fernandez J.-J.: Robust membrane detection based on tensor voting for electron tomography, J Struct Biol., 186(1):49-61 (2014)
Fukuda Y., Schrod N., Schaffer M., Feng L.R., Baumeister W., Lucic V.: Coordinate transformation based cryo-correlative methods for electron tomography and focused ion beam milling, Ultramicroscopy 143:15-23 (2014)
Lucic V., Rigort A., Baumeister W.: Cryo-electron tomography: the challenge of doing structural biology in situ, J Cell Biol 202(3):407-19 (2013)
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Lucic V., Baumeister W.: 3D electron microscopy based on cryo-electron tomography, in: G. C. K. Roberts (Ed.), Encyclopedia of Biophysics, Springer-Verlag Berlin Heidelberg, pp. 7-10 (2013)
Fernandez-Busnadiego R., Lucic V.: The cell at molecular resolution: Principles and applications af cryoelectron tomography, in: F. G. Wouterlood (Ed.), Cellular Imaging Techniques for Neuroscience and Beyond, Elsevier, pp. 141-183 (2012)
Lucic V., Förster F., Baumeister W.: Studying the macromolecular machinery of cells in situ by cryoelectron tomography, in: S. P. Egelman, E.H. (Ed.), Comprehensive Biophysics 2, Biophysical Techniques for Characterization of Cells, Elsevier B. V. Academic Press, Oxford, pp. 59-89 (2012)
Fernandez-Busnadiego R., Schrod N., Kochovski Z., Asano S., Vanhecke D., Baumeister W., Lucic V.: Insights into the molecular organization of the neuron by cryo-electron tomography, J Electron Microsc (Tokyo) 60 Suppl 1 (2011)
Vanhecke D., Asano S., Kochovski Z., Fernandez-Busnadiego R., Schrod N., Baumeister W., Lucic V.: Cryo-electron tomography: methodology, developments and biological applications, J Microsc 242(3):221-7 (2011)
Fernandez-Busnadiego R., Zuber B., Maurer U. E., Cyrklaff M., Baumeister W., Lucic V.: Quantitative analysis of the native presynaptic cytomatrix by cryoelectron tomography., J Cell Biol 188(1):145-56 (2010)
Lucic V., Leis A., Baumeister W.: Cryo-electron tomography of cells: connecting structure and function, Histochem Cell Biol 130(2):185-96 (2008)
Beck M., Lucic V., Forster F., Baumeister W., Medalia O.: Snapshots of nuclear pore complexes in action captured by cryo-electron tomography, Nature 449(7162):611-5 (2007)
Fernandez J.-J., Li S., Lucic V.: Three-dimensional anisotropic noise reduction with automated parameter tuning: Application to electron cryotomography, in: Borrajo D.; Castillo L.; Corchado J.M. (Ed.), Current topics in artificial inteligence, Vol. 4788 of Lecture notes in computer science series. Lecture notes in artificial intelligence subseries., Springer Verlag, pp. 60-69 (2007)
Fernandez J.-J., Li S., Lucic V.: Structure-preserving noise reduction in biological imaging, in: Corchado E.; Corchado J.M.; Abraham A (Ed.), Innovation in hybrid intelligent systems, Vol. 44 of Advances in soft computing, Springer Verlag, pp. 385-392 (2007)
Lucic V., Kossel A. H., Yang T., Bonhoeffer T., Baumeister W., Sartori A.: Multiscale imaging of neurons grown in culture: from light microscopy to cryo-electron tomography, J Struct Biol 160(2):146-56 (2007)
Korkin D., Davis F. P., Alber F., Luong T., Shen M. Y., Lucic V., Kennedy M. B., Sali A., Structural modeling of protein interactions by analogy: application to PSD-95., PLoS Comput Biol 2 (11) (2006) e153.
Lucic V., Forster F., Baumeister W., Structural studies by electron tomography: from cells to molecules., Annu Rev Biochem 74 (2005) 833-65.
Lucic V., Yang T., Schweikert G., Forster F., Baumeister W., Morphological characterization of molecular
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