Investigating the regulation of SNARE mediated membrane fusion by complexin and synaptotagmin on a single vesicle level using TIRF microscopy

• Applicant: Dr. Andrea Gohlke
• Subject Area: Biochemistry
• Term: from 2011 to 2012
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 217141997

Calcium dependent synaptic vesicle fusion in neurons is mediated by the formation of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes. During the docking and subsequent fusion process, vesicle and target membrane are brought into close proximity by pairing of vesicle-associated v-SNAREs with cognate t-SNAREs on target membranes. In this tightly regulated process, complexin has been reported to act as both a promotor and inhibitor of SNAREpin assembly by facilitating the assembly process, but then clamping fusion halfway. Upon stimulation, the complexin clamp is believed to be released through the calcium sensor synaptotagmin. However, the exact function and interaction of complexin and synaptotagmin with the SNARE complex remain controversial. In this proposal, I aim to investigate the role of complexin and synaptotagmin during SNARE-mediated fusion using a high temporal/spatial resolution assay that combines TIRF (total internal reflection fluorescence) microscopy with a microfluidic flow system. In this assay docking and fusion of single vesicles with a planar supported bilayer will be monitored to provide information about the docking rate, fusion rate, and delay between docking and fusion. To study their structure-function relationships, I will determine the effect of different naturally occurring isoforms and mutations of complexin and synaptotagmin. In addition, I will analyze the influence of the membrane lipid composition towards fusion capability. These results will be supported by dynamic light scattering and surface plasmon resonance. Elucidating the detailed mechanism of synaptic vesicle fusion may ultimately contribute to improved treatment and therapy options for neurodegenerative diseases and other neuronal disorders.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor Dr. James Rothman