laboratoire de physique statistique
laboratoire de physique statistique


Snapshot of sequential SNARE assembling states between membranes shows that N-terminal transient assembly initializes fusion - Wang, Yong Jian and Li, Feng and Rodriguez, Nicolas and Lafosse, Xavier and Gourier, Christine and Perez, Eric and Pincet, Frederic

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 Forster resonance energy transfer (FRET) can be used to characterize any other transient transmembrane complexes.
A specific flagellum beating mode for inducing fusion in mammalian fertilization and kinetics of sperm internalization - Ravaux, Benjamin and Garroum, Nabil and Perez, Eric and Willaime, Herve and Gourier, Christine

Abstract : The salient phases of fertilization are gamete adhesion, membrane fusion, and internalization of the spermatozoon into the oocyte but the precise timeline and the molecular, membrane and cell mechanisms underlying these highly dynamical events are far from being established. The high motility of the spermatozoa and the unpredictable location of sperm/egg fusion dramatically hinder the use of real time imaging optical techniques that should directly provide the dynamics of cell events. Using an approach based on microfluidics technology, the sperm/egg interaction zone was imaged with the best front view, and the timeline of the fertilization events was established with an unparalleled temporal accuracy from the onset of gamete contact to full sperm DNA decondensation. It reveals that a key element of the adhesion phase to initiate fusion is the oscillatory motion of the sperm head on the oocyte plasma membrane generated by a specific flagellum-beating mode. It also shows that the incorporation of the spermatozoon head is a two steps process that includes simultaneous diving, tilt, and plasma membrane degradation of the sperm head into the oocyte and subsequent DNA decondensation.
Binding of sperm protein Izumo1 and its egg receptor Juno drives Cd9 accumulation in the intercellular contact area prior to fusion during mammalian fertilization - Chalbi, Myriam and Barraud-Lange, Virginie and Ravaux, Benjamin and Howan, Kevin and Rodriguez, Nicolas and Soule, Pierre and Ndzoudi, Arnaud and Boucheix, Claude and Rubinstein, Eric and Wolf, Jean Philippe and Ziyyat, Ahmed and Perez, Eric and Pincet, Frederic and Gourier, Christine
DEVELOPMENT 1413732-3739 (2014) 

Abstract : Little is known about the molecular mechanisms that induce gamete fusion during mammalian fertilization. After initial contact, adhesion between gametes only leads to fusion in the presence of three membrane proteins that are necessary, but insufficient, for fusion: Izumo1 on sperm, its receptor Juno on egg and Cd9 on egg. What happens during this adhesion phase is a crucial issue. Here, we demonstrate that the intercellular adhesion that Izumo1 creates with Juno is conserved in mouse and human eggs. We show that, along with Izumo1, egg Cd9 concomitantly accumulates in the adhesion area. Without egg Cd9, the recruitment kinetics of Izumo1 are accelerated. Our results suggest that this process is conserved across species, as the adhesion partners, Izumo1 and its receptor, are interchangeable between mouse and human. Our findings suggest that Cd9 is a partner of Juno, and these discoveries allow us to propose a new model of the molecular mechanisms leading to gamete fusion, in which the adhesion-induced membrane organization assembles all key players of the fusion machinery.
CD9 tetraspanin generates fusion competent sites on the egg membrane for mammalian fertilization - Jegou, Antoine and Ziyyat, Ahmed and Barraud-Lange, Virginie and Perez, Eric and Wolf, Jean Philippe and Pincet, Frederic and Gourier, Christine

Abstract : CD9 tetraspanin is the only egg membrane protein known to be essential for fertilization. To investigate its role, we have measured, on a unique acrosome reacted sperm brought in contact with an egg, the adhesion probability and strength with a sensitivity of a single molecule attachment. Probing the binding events at different locations of wild-type egg we described different modes of interaction. Here, we show that more gamete adhesion events occur on Cd9 null eggs but that the strongest interaction mode disappears. We propose that sperm-egg fusion is a direct consequence of CD9 controlled sperm-egg adhesion properties. CD9 generates adhesion sites responsible for the strongest of the observed gamete interaction. These strong adhesion sites impose, during the whole interaction lifetime, a tight proximity of the gamete membranes, which is a requirement for fusion to take place. The CD9-induced adhesion sites would be the actual location where fusion occurs.
Complexin activates and clamps SNAREpins by a common mechanism involving an intermediate energetic state - Li, Feng and Pincet, Frederic and Perez, Eric and Giraudo, Claudio G. and Tareste, David and Rothman, James E.

Abstract : The core mechanism of intracellular vesicle fusion consists of SNAREpin zippering between vesicular and target membranes. Recent studies indicate that the same SNARE-binding protein, complexin (CPX), can act either as a facilitator or as an inhibitor of membrane fusion, constituting a controversial dilemma. Here we take energetic measurements with the surface force apparatus that reveal that CPX acts sequentially on assembling SNAREpins, first facilitating zippering by nearly doubling the distance at which v-and t-SNAREs can engage and then clamping them into a half-zippered fusion-incompetent state. Specifically, we find that the central helix of CPX allows SNAREs to form this intermediate energetic state at 9-15 nm but not when the bilayers are closer than 9 nm. Stabilizing the activated-clamped state at separations of less than 9 nm requires the accessory helix of CPX, which prevents membrane-proximal assembly of SNAREpins.
Two-dimensional crystallization of hard sphere particles at a liquid-liquid interface - Marnette, Olivier and Perez, Eric and Pincet, Frederic and Bryant, Gary

Abstract : A method for studying crystallization of hard sphere like particles in two dimensions is presented. The method involves trapping the particles at the interface between two immiscible liquids. Particles at the interface undergo 2D Brownian motion, and at sufficiently high densities crystallization is observed. The pseudo hard sphere nature of the particle interactions under these conditions is maintained, as demonstrated by the area density at which crystallization occurs. In contrast to established techniques for studying crystallization in pseudo 2D hard spheres, the particles trapped at the interface undergo no vertical motion, so the system is in principle closer to a true 2D system. The method is therefore amenable to the study of the effects of polydispersity on crystallization behaviour. The advantages and disadvantages of the method are discussed. (C) 2009 Elsevier B.V. All rights reserved.
Mapping mouse gamete interaction forces reveal several oocyte membrane regions with different mechanical and adhesive properties - Jegou, Antoine and Pincet, Frederic and Perez, Eric and Wolf, Jean Philippe and Ziyyat, Ahmed and Gourier, Christine
LANGMUIR 241451-1458 (2008) 

Abstract : This study focuses on the interaction involved in the adhesion of mouse gametes and on the mechanical properties of the oocyte membrane. The oocyte has an asymmetrical shape, and its membrane is composed of two distinct areas. One is rich in microvilli, and the other is smoother and without microvilli. With a biomembrane force probe (BFP) adapted to cell-cell measurements, we have quantified the separation forces between a spermatozoon and an oocyte. Microvillar and amicrovillar areas of the oocyte surface have been systematically probed and compared. In addition to a substantial difference in the elastic stiffness of these two regions, the experiments have revealed the presence of two types of membrane domains with different mechanical and adhesive properties, both distributed over the entire oocyte surface (i.e., in both microvillar and amicrovillar regions). If gamete contact occurs in the first type of domain, then the oocyte membrane deforms only elastically under traction. The pull-off forces in these domains are higher in the amicrovillar region. For a spermatozoon contact with the other type of domain, there can be a transition from the elastic to viscoelastic regime, and then tethers are extruded from the oocyte membrane.
The Surface Force Apparatus to Reveal the Energetics of Biomolecules Assembly. Application to DNA Bases Pairing and SNARE Fusion Proteins Folding - Perez, Eric and Li, Feng and Tareste, David and Pincet, Frederic

Abstract : The Surface Force Apparatus (SFA) measures directly, and with nanoscale resolution, the interaction energy vs. distance pro. le of planar arrays of biological molecules (e.g., lipids, polymers, or proteins). Through recent advances in the reconstitution and deposition of lipid bilayers, it is now possible to use SFA to study the interactions between membrane-incorporated biomolecules and to reveal any conformational changes and intermediate assembly states. Therein we describe two example systems. First, we show that using bilayers functionalized to carry DNA bases on their lipid headgroups, we can measure a macroscopic nucleoside nucleoside adhesion force, from which one can obtain a molecular binding energy. Second, we describe the use of the SFA to study the interaction between SNARE proteins, which are involved in most of intracellular fusion events. Membrane fusion occurs when SNARE proteins assemble between lipid bilayers in the form of SNAREpins. SFA measurements between SNAREs embedded in lipid bilayers allowed us to elucidate the energetics and dynamics of SNAREpin folding, and to capture an intermediate binding state in SNAREpin assembly.
Hydrophobic forces and hydrogen bonds in the adhesion between retinoid-coated surfaces - Tareste, David and Pincet, Frederic and Lebeau, Luc and Perez, Eric
LANGMUIR 233225-3229 (2007) 

Abstract : Interactions between hydrophobic chains of lipid monolayers and interactions between hydrophilic headgroups of lipid bilayers (with or without a molecular recognition step) are now well documented, especially for commonly used lipids. Here, we report force measurements between a new class of fluorinated lipid layers whose headgroups (synthetic ligands of retinoid receptors) display a very unusual polar/apolar character and can interact via a combination of hydrophobic forces and hydrogen bonds. Although these two interactions produce adhesion and are therefore not easily distinguishable, we show that it is possible to extract both contributions unambiguously. Experiments are performed both in pure water, where the adhesion is a combination of hydrophobic forces and hydrogen bonds, and in Tris buffer, where the hydrophobic effect is the dominant short-range attractive force. The contribution of hydrophobic forces scaled down to molecular interactions is deduced from force versus distance profiles, and the same value is found independently in pure water and Tris buffer, about 1 k(B)T. We also show that retinoid lipid layers attract each other through a very long-range (100 nm) exponential force, which is insensitive to the pH and the salinity. The origin of this long-range attraction is discussed on the basis of previously proposed mechanisms.
Energetics and dynamics of SNAREpin folding across lipid bilayers - Feng Li and Pincet, Frederic and Perez, Eric and Eng, William S. and Melia, Thomas J. and Rothman, James E. and Tareste, David

Abstract : Membrane fusion occurs when SNAREpins fold up between lipid bilayers. How much energy is generated during SNAREpin folding and how this energy is coupled to the fusion of apposing membranes is unknown. We have used a surface forces apparatus to determine the energetics and dynamics of SNAREpin formation and characterize the different intermediate structures sampled by cognate SNAREs in the course of their assembly. The interaction energy-versus-distance profiles of assembling SNAREpins reveal that SNARE motifs begin to interact when the membranes are 8 nm apart. Even after very close approach of the bilayers (similar to 2-4 nm), the SNAREpins remain partly unstructured in their membrane-proximal region. The energy stabilizing a single SNAREpin in this configuration (35 k(B)T) corresponds closely with the energy needed to fuse outer but not inner leaflets (hemifusion) of pure lipid bilayers (40-50 k(B)T).