Studies of exocytotic membrane fusion by single molecule FRET and single molecule fluorescence images

PHYS 503

Marcelle Koenig, mkoenig1@stanford.edu1, Suren Felekyan2, Ralf Kuehnemuth2, Christina Schuette3, Jerker Widengren4, Reinhard Jahn5, and Claus A. M. Seidel, cseidel@gwdg.de2. (1) Department of Chemistry, Stanford University, Stanford, CA 94305-5080, (2) Heinrich-Heine-University Duesseldorf, 40225 Duesseldorf, Germany, (3) ProSciencia GmbH & Co. KG, 23562 Luebeck, Germany, (4) Royal Institute of Technology, 106 91 Stockholm, Sweden, (5) MPI for Biophysical Chemistry, 37077 Goettingen, Germany
Exocytosis of synaptic vesicles is accomplished by specific SNARE proteins. The assembly of stable complexes from SNARE partners residing in different membranes leads to a tight connection between the membranes and initiates membrane fusion. Details of the precise mechanism of action are still unclear. We developed an assay for exocytotic membrane fusion in which corresponding sets of fluorescently labeled SNARE proteins are reconstituted into liposomes. Results obtained by the simultaneous measurement of independent fluorescence parameters of single liposomes in solution (Multiparameter Fluorescence Detection, MFD) will be presented. Furthermore we extended our assay by confocal scanning of immobilized liposomes. This method allows to simultaneously obtain all fluorescence parameters of single liposomes per pixel, including e.g. Fluorescence Lifetime Imaging (FLIM). The analysis of the obtained fluorescence images from single liposomes provides new insights into the biomechanical understanding of exocytotic membrane fusion.
 

Poster Session
7:30 PM-10:00 PM, Wednesday, 13 September 2006 Moscone Center -- Hall D, Poster

Sci-Mix
8:00 PM-10:00 PM, Monday, 11 September 2006 Moscone Center -- Hall D, Sci-Mix

Division of Physical Chemistry

The 232nd ACS National Meeting, San Francisco, CA, September 10-14, 2006