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Acta Physiologica 2013; Volume 207, Supplement 694
92nd Annual Meeting of the German Physiological Society
3/2/2013-3/5/2013
Heidelberg, Germany
EXTRACTING SINGLE MOLECULE INFORMATION FROM SUPER-RESOLVED NANOSTRUCTURES OF ACTIVE ZONES
Abstract number: P187
Ehmann
1
*N.
, van de Linde
2
S., Ljaschenko
1
D., Keung
1
X.Z., Holm
2
T., Weyhersmüller
3
A., DiAntonio
4
A., Hallermann
3
S., Heckmann
1
M., Sauer
2
M., Kittel
1
R.J.
1
University of Würzburg, Institute of Physiology, Department of Neurophysiology, Würzburg, Germany
2
University of Würzburg, Department of Biotechnology & Biophysics, Würzburg, Germany
3
University of Göttingen, European Neuroscience Institute, Göttingen, Germany
4
Washington University, Department of Developmental Biology, Saint Louis, United States
A fundamental question in biological sciences is how structure is connected to function (Crick and Watson, 1953).
In the present study, we set out to decode structure-function relationships by correlating super-resolution images with functional determinants of short-term synaptic plasticity. To achieve this, we combined the excellent spatial resolution of dSTORM (direct Stochastic Optical Reconstruction Microscopy; Heilemann et al., 2008; van de Linde et al., 2011) with electrophysiology in the genetically accessible organism Drosophila melanogaster. By focusing on a key player at active zones (AZs), Bruchpilot (BRP; Kittel et al., 2006; Wagh et al., 2006), we could decipher that transmitter release probability, the number of release sites and vesicle trafficking speed is encoded in the quantity as well as the nanoscopic organisation of BRP. Taking advantage of the single molecule sensitivity of dSTORM, we put forward an experimental procedure to calibrate super resolution images, resulting in the ability to count single molecules in their native environment. Thus, we observed that AZs are modular, with each module containing on average 128 BRP molecules.
This analytical approach and further progress, e.g. 2-colour dSTORM and application to other samples, will likely help to understand essential relationships that are indispensible for the proper function of neuronal communication.
Crick F, Watson J (1953) Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature 171:737-738
Heilemann M, van de Linde S, Schüttpelz M, Kasper R, Seefeldt B, Mukherjee A, Tinnefeld P, Sauer M (2008) Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angewandte Chemie 47:6172-6176
Kittel RJ, Wichmann C, Rasse TM, Fouquet W, Schmidt M, Schmid A, Wagh D a, Pawlu C, Kellner RR, Willig KI, Hell SW, Buchner E, Heckmann M, Sigrist SJ (2006) Bruchpilot promotes active zone assembly, Ca2+ channel clustering, and vesicle release. Science 312:1051-1054
van de Linde S, Löschberger A, Klein T, Heidbreder M, Wolter S, Heilemann M, Sauer M (2011) Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nature protocols 6:991-1009
Wagh DA, Rasse TM, Asan E, Hofbauer A, Schwenkert I, Dürrbeck H, Buchner S, Dabauvalle M-C, Schmidt M, Qin G, Wichmann C, Kittel R, Sigrist SJ, Buchner E (2006) Bruchpilot, a protein with homology to ELKS/CAST, is required for structural integrity and function of synaptic active zones in Drosophila. Neuron 49:833-844
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Acta Physiologica 2013; Volume 207, Supplement 694 :P187