Mammalian fertilization is still lacking a comprehensive understanding of gamete fusion and block to polyspermy mechanisms. One reason is that they are highly dynamic processes involving transient events that can only be revealed and characterized by direct observation. To extend while challenging existing knowledge, this study applies real time brightfield and confocal imaging to inseminated ZP-intact mouse oocytes and statistical analyses to establish an accurate dynamic picture of the cascade of events leading to fusion and prevention of polyspermy in conditions as close to physiology as possible. These observations allow to characterize the roles of the different components of the oocyte (i.e. zona pellucida (ZP), perivitelline space (PVS), oocyte plasma membrane (OPM)) and the spermatozoon (i.e. head, flagellum) in promoting fertilization and preventing polyspermy. The kinetics we have determined challenge dogmas by showing that: (i) the first sperm is not necessarily the one that fertilizes in mice pointing to the existence of other post-penetration fertilization factors, (ii) the ZP block resulting from the cortical reaction is too slow to contribute to the prevention of polyspermy in mice. On the other hand, it evidences that the ZP directly contributes to polyspermy block in two other ways: (a) as a naturally effective entry barrier for the spermatozoon (independent of any block caused by fertilization), (b) as an effective exit barrier for components released by the OPM, which may contribute to a fast PVS block to polyspermy through the neutralizing of unwanted spermatozoa in the PVS. Moreover, our observations reveal that the ZP plays a key role in fertilization itself by channeling the flagellar oscillations of spermatozoa in the PVS to make them conducive to fusion. ### Competing Interest Statement The authors have declared no competing interest.

Liquid Crystals (LC) are anisotropic liquids characterized by long-range molecular correlation. The LC anisotropy allows the detection of structural effects induced by a surface. The lipid order parameter increases in the neighborhood of the protein molecules from the third or fourth neighbor of the protein. The profile of lipid order parameter is symmetric between the two proteins and generates an attractive contribution to the protein-protein interaction. Liquid crystals can be modelled by a lattice and mean-field approximations. Stratification can also occur in thin free films of concentrated dodecylsulfate solutions containing a small percentage of dodecanol, as pointed out by Keuskamp and Lyklema and Manev et al.

Synaptotagmin interaction with anionic lipid (phosphatidylserine/phosphatidylinositol) containing membranes, both in the absence and presence of calcium ions (Ca2+), is critical to its central role in orchestrating neurotransmitter release. The molecular surfaces involved, namely the conserved polylysine motif in the C2B domain and Ca2+-binding aliphatic loops on both C2A and C2B domains, are known. Here we use surface force apparatus combined with systematic mutational analysis of the functional surfaces to directly measure Syt1-membrane interaction and fully map the site-binding energetics of Syt1 both in the absence and presence of Ca2+. By correlating energetics data with the molecular rearrangements measured during confinement, we find that both C2 domains cooperate in membrane binding, with the C2B domain functioning as the main energetic driver, and the C2A domain acting as a facilitator.

The protective function of biological surfaces that are exposed to the exterior of living organisms is the result of a complex arrangement and interaction of cellular components. This is the case for the most external cornified layer of skin, the stratum corneum (SC). This layer is made of corneocytes, the elementary 'flat bricks' that are held together through adhesive junctions. Despite the well-known protective role of the SC under high mechanical stresses and rapid cell turnover, the subtleties regarding the adhesion and mechanical interaction among the individual corneocytes are still poorly known. Here, we explore the adhesion of single corneocytes at different depths of the SC, by pulling them using glass microcantilevers, and measuring their detachment forces. We measured their interplanar adhesion between SC layers, and their peripheral adhesion among cells within a SC layer. Both adhesions increased considerably with depth. At the SC surface, with respect to adhesion, the corneocyte population exhibited a strong heterogeneity, where detachment forces differed by more than one order of magnitude for corneocytes located side by side. The measured detachment forces indicated that in the upper-middle layers of SC, the peripheral adhesion was stronger than the interplanar one. We conclude that the stronger peripheral adhesion of corneocytes in the SC favors an efficient barrier which would be able to resist strong stresses.

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.

Synaptotagmin-1 (Syt1) is the primary calcium sensor (Ca2+ ) that mediates neurotransmitter release at the synapse. The tandem C2 domains (C2A and C2B) of Syt1 exhibit functionally critical, Ca2+ -dependent interactions with the plasma membrane. With the surface forces apparatus, we directly measure the binding energy of membrane-anchored Syt1 to an anionic membrane and find that Syt1 binds with ~6 kB T in EGTA, ~10 kB T in Mg2+ and ~18 kB T in Ca2+ . Molecular rearrangements measured during confinement are more prevalent in Ca2+ and Mg2+ and suggest that Syt1 initially binds through C2B, then reorients the C2 domains into the preferred binding configuration. These results provide energetic and mechanistic details of the Syt1 Ca2+ -activation process in synaptic transmission.

Reflets de la Physique n° 51 2 La Société Française de Physique (SFP) souhaite créer un pont entre la physique académique et celle du monde de l'entreprise.Dans cet objectif, elle organise un nouvel événement destiné aux doctorants, postdoctorants et aux services de recherche et développement (R&D) des grands groupes, PME et start-up.Ce sera la rencontre de deux univers qui doivent apprendre à mieux se connaître et s'apprécier.La grande majorité des doctorants n'ont qu'une idée assez vague du monde de l'entreprise, même si de nombreux efforts ont été consacrés au rapprochement entre recherches publique et privée.C'est pourquoi la SFP cherche à renforcer les liens entre ces deux mondes, en organisant ces rencontres physique-entreprise-recherche.Celles-ci s'adresseront directement aux jeunes physiciens qui n'ont pas eu l'opportunité de franchir la barrière « virtuelle » entre leur laboratoire et ceux des entreprises.Elles contribueront à montrer que la physique dans le monde industriel est beaucoup plus connectée qu'on le croit à celle que l'on développe dans les universités et centres de recherche.Les jeunes chercheurs verront le caractère varié et vivant de la physique pratiquée dans les entreprises, source inépuisable de sujets de recherche originaux.Ces rencontres se tiendront dans le cadre prestigieux des salons de l'Hôtel de ville de Paris, le 10 mars 2017, avec la participation d'Albert Fert, prix Nobel de physique 2007 et de Mathias Fink, lauréat de la médaille de l'innovation du CNRS.Les jeunes chercheurs contribueront aux échanges sous des formes variées : quinze tables rondes, deux conférences plénières et huit conférences thématiques, ainsi que des ateliers au cours desquels ils se pencheront sur des problèmes proposés par les services R&D des entreprises participantes.Des itinéraires de physiciens ayant abouti à la création de start-up novatrices seront présentés, et montreront le côté « visionnaire » que donne la formation par la recherche.Un événement de ce type, portant sur autant de domaines de la physique (matériaux, optique, acoustique, énergie, mécanique...), n'a jamais été organisé à l'échelle nationale.Une soixantaine d'entreprises, des instituts de recherche publique, des universités et des écoles y prendront part.De plus, la Caisse des dépôts, la Mairie de Paris et bien d'autres organismes nous ont rejoints pour participer à cette journée et la financer.Nous espérons créer ainsi des échanges riches et spontanés entre les intervenants de la R&D des entreprises présentes et les doctorants et postdoctorants.Les étudiants sont tous invités moyennant une participation minime de 16 euros ; l'hébergement est offert aux étudiants de province.Les inscriptions sont ouvertes, et nous comptons sur toutes les sections locales de la SFP et sur nos adhérents pour diffuser l'information aux jeunes physiciens, le nombre de participants étant limité.

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.

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.

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.

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.

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.

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.

The Surface Force Apparatus (SFA) measures directly, and with nanoscale resolution, the interaction energy vs. distance profile 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.

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.

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 kBT. 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.

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 (∼2–4 nm), the SNAREpins remain partly unstructured in their membrane-proximal region. The energy stabilizing a single SNAREpin in this configuration (35 kBT) corresponds closely with the energy needed to fuse outer but not inner leaflets (hemifusion) of pure lipid bilayers (40–50 kBT).