SNARE proteinsinduce the fusion of synaptic vesicles with the presynaptic membrane. We will try to understand how the SNAREs mechanically pull the membranes toward fusion.
Synaptic transmission occurs when synaptic vesicles fuse with the presynaptic membrane, inducing neurtransmitter release. This fusion is due to the SNARE proteins. v-SNAREs are present on the synaptic vesicle and t-SNAREs on the neuron membrane. v and t-SNAREs assemble to pull the membranes togethertoward fusion. The structure of the resulting SNARE complex is known but the forces applied to the membranes and the degree of assembly of the proteins to trigger fusion with the intermembrane distance remain unknown. During this project, we will use a new experimental setup (see below) to measure forces and degree of assembly. The result will be a better understanding of the mechanical action of the SNAREs and, eventually, to directly act on them to modify or contro neurotransmission. The influence of regulatory factors will also be probed.
For this project, we will use a novel technique developped in the laboratory, the surface force apparatus - Förster Resonnance Energy Transfer (SFA-FRET). This setup allows the measurement of forces between membranes and the degree of assembly of protein complexes while imposing the intermembrane distance.
This work will provide a nanometer scale view of the interactions of membranes decorated with SNAREs and the degree of assembly of the SNARE complex with the intermembrane distance. The exact role of each domain of the SNARE proteins will thereby be decyphered.
This project will enhance a international partnership that is already very protuctive between the cell biology department at Yale university and the Physics department of the Ecole Normale Superieure in Paris.
After the project, the role of several regulatory factors and thir action on neurotransmission will be studied.
No scientific production after 6 months.
This project is the continuation of the ANR PCV project entitled “IntermeSNARE” funded in 2008 and that will end in 2012. IntermeSNARE led to three articles in “Nature Structural and Molecular Biology” that were highlighted by a “News and Views” in the same journal. SNARE proteins are the engines that drive fusion of synaptic vesicles with pre-synaptic plasma membranes to release neurotransmitters for synaptic transmission. Upon calcium signal, this fusion must be extremely fast (< 100 µs). To prepare synaptic vesicles to fusion so that they can snap fuse the membranes, SNAREs must be preassembled and their assembly must be clamped. In these articles, we present the molecular, energetic and dynamic bases allowing this “preparation” of the synaptic vesicles. On the technical front, we introduced a technique simultaneously providing the measurement of intermembrane forces and the observation of molecular arrangements in situ between the membranes by FRET (“Förster Resonance Energy Transfer”) with a resolution of a few Angstroms. This setup is based on the Surface Force Apparatus (SFA). No other technique is able to freeze the intermembrane distance and allow the direct observation in the space between the membranes. Thus, we wish to pursue our fruitful collaboration by moving to a higher level and showing the potential of our SFA/FRET setup on the case of SNARE assembly by establishing their energy and molecular landscapes. Indeed, understanding the molecular mechanisms involved in membrane fusion, and how they are controlled by regulatory proteins such as complexin, entails understanding the pathway of folding of the SNARE proteins involved, i.e. at which intermembrane distance each part of the molecular complex binds (=molecular landscape) and what energy is released during this binding (=energy landscape). Because folding/assembly intermediates are by nature transient, this is generally a very difficult or even intractable problem. However, in the case of folding/assembly between surfaces, exemplified by SNARE proteins, fixing the separation will freeze intermediate structures and make them accessible to structural and thermodynamic investigation. The result will be better understanding of a key physiological process that is altered in many functional disorders of the nervous system. The proposed research also introduces a powerful new technology platform to the study of membrane protein folding generally.
Monsieur Frédéric Pincet (Laboratoire de Physique Statistique de l'Ecole Normale Supérieure) – firstname.lastname@example.org
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
CNRS-LPS-ENS Laboratoire de Physique Statistique de l'Ecole Normale Supérieure
Help of the ANR 350,000 euros
Beginning and duration of the scientific project: December 2012 - 48 Months