title: Snapshot of sequential SNARE assembling states between membranes shows that N-terminal transient assembly initializes fusion.
abstract: Many prominent biological processes are driven by protein assembling between membranes. Understanding the mechanisms then entails determining the assembling pathway of the involved proteins. Because the intermediates are by nature transient and located in the intermembrane space, this determination is generally a very difficult, not to say intractable, problem. Here, by designing a setup with sphere/plane geometry, we have been able to freeze one transient state in which the N-terminal domains of SNARE proteins are assembled. A single camera frame is sufficient to obtain the complete probability of this state with the transmembrane distance. We show that it forms when membranes are 20 nm apart and stabilizes by further assembling of the SNAREs at 8 nm. This setup that fixes the intermembrane distance, and thereby the transient states, while optically probing the level of molecular assembly by Förster resonance energy transfer (FRET) can be used to characterize any other transient transmembrane complexes.
authors.affiliation: Laboratoire de Physique Statistique, École Normale Superieure, L'université de recherche Paris Sciences et Lettres, CNRS UMR 8550, Sorbonne Universités, Université Pierre-et-Marie-Curie (UPMC) University of Paris 06, Université Paris Diderot, 75005 Paris, France;
authors.forename: Yong Jian
authors.initials: YJ
authors.lastname: Wang
authors.affiliation: Department of Cell Biology, School of Medicine, Nanobiology Institute, Yale University, West Haven, CT 06516;
authors.forename: Feng
authors.initials: F
authors.lastname: Li
authors.affiliation: Laboratoire des Biomolécules, Sorbonne Universités, UPMC University of Paris 06, École Normale Supérieure, Département de Chimie, L'université de recherche Paris Sciences et Lettres, CNRS UMR 7203, 75005 Paris, France;
authors.forename: Nicolas
authors.initials: N
authors.lastname: Rodriguez
authors.affiliation: Laboratoire de Photonique et de Nanostructures, Unité Propre de Recherche 20, CNRS, 91460 Marcoussis, France.
authors.forename: Xavier
authors.initials: X
authors.lastname: Lafosse
authors.affiliation: Laboratoire de Physique Statistique, École Normale Superieure, L'université de recherche Paris Sciences et Lettres, CNRS UMR 8550, Sorbonne Universités, Université Pierre-et-Marie-Curie (UPMC) University of Paris 06, Université Paris Diderot, 75005 Paris, France;
authors.forename: Christine
authors.initials: C
authors.lastname: Gourier
authors.affiliation: Laboratoire de Physique Statistique, École Normale Superieure, L'université de recherche Paris Sciences et Lettres, CNRS UMR 8550, Sorbonne Universités, Université Pierre-et-Marie-Curie (UPMC) University of Paris 06, Université Paris Diderot, 75005 Paris, France;
authors.forename: Eric
authors.initials: E
authors.lastname: Perez
authors.affiliation: Laboratoire de Physique Statistique, École Normale Superieure, L'université de recherche Paris Sciences et Lettres, CNRS UMR 8550, Sorbonne Universités, Université Pierre-et-Marie-Curie (UPMC) University of Paris 06, Université Paris Diderot, 75005 Paris, France; pincet@lps.ens.fr.
authors.forename: Frederic
authors.initials: F
authors.lastname: Pincet
mesh_terms: D000818:Animals; D004867:Equipment Design; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation; D008561:Membrane Fusion / Q000502:physiology; D051379:Mice; D008958:Models, Molecular; D052898:Optical Tweezers; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins
keywords: FRET; N-terminal assembly; SFA; SNARE; transient state