Reconstitution of calcium-triggered rapid neurotransmitter release

• Antragstellerin: Dr. Wensi Vennekate
• Fachliche Zuordnung: Molekulare Biologie und Physiologie von Nerven- und Gliazellen ; Biochemie
• Förderung: Förderung von 2013 bis 2015
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 234221592

Fusion of neurotransmitter-filled synaptic vesicles with the plasma membrane is a key step in the communication between neurons. Vesicles are docked at the plasma membrane but do not undergo fusion until an action potential causes a transient increase in the intracellular calcium concentration which triggers fusion. Upon fusion, neurotransmitters are released from the vesicular lumen to the synaptic cleft. Fusion occurs with sub-millisecond kinetics after the arrival of an action potential. Several key players of this fundamental process have been identified and broad functions to individual components have been assigned: a protein complex composed of vesicle (v-) and target membrane (t-) SNAREs (soluble N-ethylmaleimide-sensitive-factor attachment receptor) constitutes the core of the fusion machinery; complexins (CPX) belong to the late regulatory proteins: they bind the SNARE complex and could affect fusion; synaptotagmin (Syt) is the fast calcium sensor protein and is capable of accelerating SNARE-mediated membrane fusion by several orders of magnitude: it binds to anionic membranes containing phosphatidylserine (PS) or phospatidylinoditol-4,5-bisphosphate (PIP2) in both calcium-dependent and -independent ways. But how all these components work together in a synergistic and synchronized manner is still a mystery.Small liposomes reconstituted with v-SNAREs fuse with t-SNARE reconstituted ones in the absence of any other protein, suggesting SNAREs constitute the core of the fusion machinery. However, in contrast to the situation in vivo, this SNARE only reaction occurs constitutively and is calcium-independent. One of the grand challenges in cell biology and neurobiology today is to reconstitute the calcium sensitive fast membrane fusion so that its underlying molecular mechanisms can be dissected.I propose to reconstitute the process of vesicle docking and calcium-triggered fusion with single-molecule sensitivity and ~10 msec resolution. Using microfluidic channels we will measure single fusion events between v-SNARE reconstituted small unilamellar vesicles (SUVs) and supported bilayers (SBLs) containing cognate t-SNAREs. The SUV-SBL geometry is closer to the physiological vesicle-plasma membrane fusion. The proposed research will allow us to understand how the actions of the various molecular players are coordinated and synchronized. The project will combine my expertise of SNAREs and Synaptotagmin with that of the Physiology department of Yale University in the most synergistic manner.

• DFG-Verfahren: Forschungsstipendien
• Internationaler Bezug: USA
• Gastgeber: Erdem Karatekin, Ph.D.