Christine Gourier

CD9 tetraspanin generates fusion competent sites on the egg membrane for mammalian fertilization

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.

Specific and non specific interactions involving Le X determinant quantified by lipid vesicle micromanipulation

Carbohydate-carbohydrate recognition is emerging today as an important type of interaction in cell adhesion. One Ca2+mediated homotypic interaction between two LewisX determinants (LeX) has been proposed to drive cell adhesion in murine embryogenesis. Here, the adhesion energies of lipid vesicles functionalised with glycolipids bearing monomeric or dimeric LeX determinants were measured in NaCl or CaCl2 media with the micropipette aspiration technique. These experiments on LeX with an environment akin to that provided by biological membrane confirmed the existence of this specific calcium dependant interaction of monomeric LeX. In contrast, dimeric LeX produced a repulsive contribution. By using a simple model involving the various contributions to the adhesion free energy, specific and non specific interactions could be separated and quantified. The involvement of calcium ions has been discussed in the monomeric and dimeric LeX lipids. Published in 2004.

Binding of sperm protein Izumo1 and its egg receptor Juno drives Cd9 accumulation in the intercellular contact area prior to fusion during mammalian fertilization

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.

Snapshot of sequential SNARE assembling states between membranes shows that N-terminal transient assembly initializes fusion.

Significance Membrane fusion is the key step in cellular traffic, which is induced by the assembly of membrane protein, namely SNARE. How the protein assembly induces membrane fusion remains unknown. Answering this question requires knowledge of the assembly intermediates, which cannot be accessed by conventional methods. We developed an instrument not only to freeze a continuous series of intermediates of SNARE assembly but also to monitor the formation of these domains. Here, we demonstrate that the N-terminal assembly is the initializing step prior to fusion. 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.

Homotypic and Heterotypic Adhesion Induced by Odorant Receptors and the β2-Adrenergic Receptor

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 β2 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.

A specific flagellum beating mode for inducing fusion in mammalian fertilization and kinetics of sperm internalization

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

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.

Creation of intercellular bonds by anchoring protein ligands to membranes using the diphtheria toxin T domain

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.

Egg CD9 protein tides correlated with sperm oscillations tune the gamete fusion ability in mammal

Mammalian fertilization involves membrane events-adhesion, fusion, sperm engulfment, membrane block to polyspermy-whose causes remain largely unknown. Recently, specific oscillations of the sperm in contact with the egg were shown to be necessary for fusion. Using a microfluidic chip to impose the venue for the encounter of two gametes allowed real-time observation of the membrane remodelling occurring at the sperm/egg interface. The spatiotemporal mapping of egg CD9 revealed that this protein concentrates at the egg/sperm interface as a result of sperm oscillations, until a CD9-rich platform is nucleated on which fusion immediately takes place. Within 2-5 min after fusion, most of the CD9 leaves the egg for the external aqueous medium. Then an egg membrane wave engulfs the sperm head in ~25 min. These results show that sperm oscillations initiate the CD9 recruitment that causes gamete fusion after which CD9 and associated proteins leave the membrane in a process likely to contribute to block polyspermy. They highlight that the gamete fusion story in mammals is an unexpected interplay between mechanical constraints and proteins.