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MPI für Biochemie  

Molecular Structural Biology
Wolfgang Baumeister

Molecular Architecture of Synaptic Complexes

 

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Dr. Vladan Lučić

Phone: +49 - 89 - 8578 2647

Molecular Architecture of Synaptic Complexes

cleft

A large body of knowledge regarding the identity as well as the interaction patterns of the mainmolecules involved in synaptic transmission and signaling is available. However, the precise organization of these molecules is expected to play an important role in their function. We use cryo-electron tomography (cryo-ET) (rev. in 1) to obtain three dimensional images of macromolecular complexes from central nervous system synapses and to characterize their architecture. 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. Dissociated neuronal cultures are amenable to cryo-ET and have the advantage that complexes are investigated in intact cells. On the other hand, synaptosomal cellular fraction contain synapses that retain essential functional properties and allow reaching higher resolution in cryo-tomograms. In our opinion, the combination of findings obtained by these two complementary preparations holds promise for future research.

 

Correlative light microscopy and cryo-ET is a conceptually straightforward but challenging technique that allows detection of the same features in light micrographs and cryo-tomograms (rev. in 2). 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 (3), thus assigning functional information to the structures visualized in cryo-tomograms.

 

Synaptic adhesion complexes present in the synaptic cleft not only allow the maintenance of thestructural integrity of a synapse, but are also involved in the synaptic signaling and synaptogenesis. Our morphological characterization of complexes visualized in cryo-tomograms obtained from synaptosomes benefited from a novel image segmentation method, and showed that synaptic adhesion complexes are extensively laterally connected, possessing non-trivial topology (4). 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 (5).

Publications

  1. V. Lucic, F. Forster, and W. Baumeister. Structural studies by electron tomography: from cellsto molecules. Annu Rev Biochem, 74:833-65, 2005.
  2. V. Lucic, A. Leis, and W. Baumeister. Cryo-electron tomography of cells: connecting structureand function. Histochem Cell Biol, 130(2):185-96, 2008.
  3. V. Lucic, A. H. Kossel, T. Yang, T. Bonhoeffer, W. Baumeister, and A. Sartori. Multiscale imagingof neurons grown in culture: from light microscopy to cryo-electron tomography. J Struct Biol, 160(2):146-56, 2007.
  4. V. Lucic, T. Yang, G. Schweikert, F. Forster, and W. Baumeister. Morphological characterization of molecular complexes present in the synaptic cleft. Structure, 13(3):423-34, 2005.
  5. J.-J. Fernandez, S. Li, and V. Lucic. Three-dimensional anisotropic noise reduction with automatedparameter tuning: Application to electron cryotomography. In Borrajo D.; Castillo L.; Corchado J.M., editor, Current topics in artificial inteligence, volume 4788 of Lecture notes in computerscience series. Lecture notes in artificial intelligence subseries., pages 60-69. Springer Verlag, 2007.