Molecular Architecture of Synaptic Complexes
Phone: +49 - 89 - 8578 2647
Alumni:
Dr. Ruben Fernandez, Yale University Dr. Dimitri Vanhecke, University of Bern Goeffrey Kong, Ph.D., University of Melbourne Dr. Ting YangOur 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) [1, 2] 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 these 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 diffculties 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 [3]. 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 stimulation-dependent 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 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 [4]. 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.
Cryo-ET of cultured neurons offers the possibility of studying the architecture of neuronal complexes in situ and correlating it with functional information obtained by light microscopy. 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 [5]. The correlative light microscopy and cryo-ET approach allows the minimization of sample irradiation during the search for features of interest in vitrified neuronal cultures, a process usually hindered by the dense network of neuronal processes and their sensitivity to the electron irradiation. 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 [6], thus assigning functional information to the structures visualized in cryo-tomograms.
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 [4, 3]. In addition, we are involved in the improvement of the current denoising algorithms [7].
rss
Dr. Vladan Lucic
Top
Print