laboratoire de physique statistique
laboratoire de physique statistique


Remodeling of Gamete Membrane during Mammalian Fertilization - Ravaux, Benjamin and Gourier, Christine
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.
Interfacial pressure and phospholipid density at emulsion droplet interface using fluorescence microscopy - Delacotte, Jerome and Gourier, Christine and Pincet, Frederic

Abstract : Phospholipids are widely used to stabilize oil in water micron size emulsion droplets; the interfacial phospholipid density and tension of such droplets are difficult to estimate. In the present paper, we describe a simple approach by which the measurement of a micron size oil droplet interface fluorescence intensity provides directly both the interfacial phospholipid density and the interfacial tension. This method relies on two prior calibration steps: (i) the quantitative variation of the interfacial tension with fluorescence intensity at droplets interface through micro-manipulation techniques; (ii) the variation of interfacial tension with phospholipid density through monolayer isotherm. Here, we show the validity of this approach with the example of micron size oil droplets stabilized with a phosphatidylcholine phospholipid, in aqueous buffer. (C) 2013 Elsevier B.V. All rights reserved.
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.
Homotypic and Heterotypic Adhesion Induced by Odorant Receptors and the beta 2-Adrenergic Receptor - Richard, Marion and Jamet, Sophie and Fouquet, Coralie and Dubacq, Caroline and Boggetto, Nicole and Pincet, Frederic and Gourier, Christine and Trembleau, Alain
PLOS ONE 8 (2013) 

Abstract : In the mouse olfactory system regulated expression of a large family of G Protein-Coupled Receptors (GPCRs), the Odorant Receptors (ORs), provides each sensory neuron with a single OR identity. In the wiring of the olfactory sensory neuron projections, a complex axon sorting process ensures the segregation of >1,000 subpopulations of axons of the same OR identity into homogeneously innervated glomeruli. ORs are critical determinants in axon sorting, and their presence on olfactory axons raises the intriguing possibility that they may participate in axonal wiring through direct or indirect trans-interactions mediating adhesion or repulsion between axons. In the present work, we used a biophysical assay to test the capacity of ORs to induce adhesion of cell doublets overexpressing these receptors. We also tested the beta 2 Adrenergic Receptor, a non-OR GPCR known to recapitulate the functions of ORs in olfactory axon sorting. We report here the first evidence for homo-and heterotypic adhesion between cells overexpressing the ORs MOR256-17 or M71, supporting the hypothesis that ORs may contribute to olfactory axon sorting by mediating differential adhesion between axons.
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.
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.
A Nanospring Named Erythrocyte. The Biomembrane Force Probe - Gourier, Christine and Jegou, Antoine and Husson, Julien and Pincet, Frederic

Abstract : The Biomembrane Force Probe, BFP, is a sensitive technique that allows the quanti. cation of single molecular bonds. It is a versatile tool that can be used in a wide range of forces (0.1 pN to 1 nN) and loading rates (1-10(6) pN/s). This article describes the principle of the BFP technique, how to set it up and its various advantages. In order to show that this technique is a powerful tool that can be used on a wide range of systems, two different types of applications are presented. The. first example shows how the energy landscape of a single bond can be deduced from the measurements on a well defined pair: the streptavidin-biotin couple. The second example presents a case where cell-cell interactions can be probed at the molecular level: mammalian gametes interactions.
Creation of intercellular bonds by anchoring protein ligands to membranes using the diphtheria toxin T domain - Perier, Aurelie and Gourier, Christine and Pichard, Sylvain and Husson, Julien and Lajeunesse, Evelyne and Babon, Aurelie and Menez, Andre and Gillet, Daniel
FEBS LETTERS 5815480-5484 (2007) 

Abstract : We describe the creation of cell adhesion mediated by cell surface engineering. The Flt3-ligand was fused to a membrane anchor made of the diphtheria toxin translocation domain. The fusion protein was attached to the surface of a cell by an acid pulse. Contact with another cell expressing the receptor Flt3 lead to its activation. This activity involved direct cell-cell contact. A mean force of 20 nN was needed to separate functionalized cells after 5 min of contact. Overall, we showed that it is possible to promote specific cell-cell adhesion by attaching protein ligands at the surface of cells. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.