Repellent Molecules Research Articles

Behavioral and electrophysiological responses of Aedes albopictus to certain acids and alcohols present in human skin emanations

Human skin emanations attract hungry female mosquitoes toward their host for blood feeding. In this study, we report the flight orientation and electroantennogram response of Aedes albopictus females to certain unsaturated acids and alcohols found in human skin. In the Y-tube olfactometer, odors of lactic acid and 2-methyl-3-pentanol attracted 54-65% of Ae. albopictus females at all doses in a dose-dependent manner. However, at the highest dose (10(-2) g), the acids repelled 40-45% females. Attractancy (ca. 62-68%) at lower doses and repellency (ca. 30-45%) at higher doses were recorded for 3-methyl-3-pentanol and 1-octen-3-ol, while 5-hexen-1-ol, cis-2-hexen-1-ol, and trans 2-hexen-1-ol odor repelled ca. 55-65% of Ae. albopictus females at all doses. Antenna of female Ae. albopictus exhibited a dose-dependent EAG response up to 10(-3) g of L-lactic acid, trans-2-methyl-2-pentenoic acid, 2-octenoic acid, trans-2-hexen-1-ol and 1-octen-3-ol stimulations; however, the highest dose (10(-2) g) caused a little decline in the EAG response. EAG response of 9-10-fold was elicited by lactic acid, 2-octenoic acid, trans-2-hexenoic acid, and 3-methyl-3-pentanol, while cis-2-hexen-1-ol and trans-2-methyl pentenoic acid elicited 1-5-fold responses compared to solvent control. A blend of attractive compounds could be utilized in odor-baited trap for surveillance and repellent molecules with suitable formulation could be used to reduce the biting menace of mosquitoes.

Nano-droplet formation in water/ethanol or isopropanol/mosquito repellent formulations

It was recently demonstrated that nano-structures were present in water/ethanol/oil systems, where the oil was either octanol or fragrance molecules. The goal of the present work is to check if such structures exist also in other, related systems and if a general concept can be deduced from these observations. To this purpose, natural and synthetic mosquito repellent molecules were investigated, which represent nearly all repellents used on the market. For the ternary water/alcohol (ethanol or isopropanol)/repellent systems ternary phase diagrams were established. The presence and the ordering of the nano-droplets were checked and characterized with dynamic and static light scattering and conductivity measurements. Based on these results it can be concluded that a nano-ordering with generally an organic continuum exists in hydro-alcoholic commercial mosquito repellents, and thus that these systems are not simply molecular solutions. This might have a consequence for diffusion processes in the skin.

Multiple activities of insect repellents on odorant receptors in mosquitoes

Several lines of evidence suggest that insect repellent molecules reduce mosquito-host contacts by interacting with odorants and odorant receptors (ORs), thereby ultimately affecting olfactory-driven behaviours. We describe the molecular effects of 10 insect repellents and a pyrethroid insecticide with known repellent activity on two highly specific Aedes aegypti (Diptera: Culicidae) ORs, AaOR2 + AaOR7 and AaOR8 + AaOR7, exquisitely sensitive to key mosquito attractants indole and (R)-(-)-1-octen-3-ol, expressed in oocytes of Xenopus (Anura: Pipidae). Our study demonstrates that insect repellents can both inhibit odorant-evoked currents mediated by ORs and independently elicit currents in the absence of odorants. All of the repellents had effects on one or both ORs; most of these compounds were selective inhibitors and showed a high degree of specificity in their capacity to activate the two ORs. These results show that a range of insect repellents belonging to structurally diverse chemical classes modulate the function of mosquito ORs through multiple molecular mechanisms.

Molecular development of the extrinsic sensory innervation of the gastrointestinal tract

The extrinsic sensory innervation of the gastrointestinal tract is the conduit through which the gut and the central nervous system communicate. The hindbrain receives information directly from the bowel via the vagus nerve, while information from spinal afferents arrives in the central nervous system through the dorsal root ganglia. This review focuses on the molecular development of these vagal and spinal innervations, with an emphasis on mechanisms that involve axon guidance. During development, axons from both the nodose ganglia and dorsal root ganglia grow into the gut, innervate their appropriate enteric targets and avoid inappropriate cells in the gut wall. These developmental outcomes suggest that both attractive and repellent molecules are important in establishing the normal pattern of the extrinsic sensory innervation. In the fetal mouse gut, the guidance of vagal sensory axons is mediated by axon guidance molecules, such as netrin and the netrin receptor, deleted in colorectal cancer (DCC), as well as extracellular matrix molecules, such as laminin-111. Dorsal root ganglion neurons are known to express, and their axons to respond to, axon guidance molecules. The question of whether or not these molecules are involved in guiding spinal afferents to the bowel, however, has not yet been examined. It is anticipated that a better understanding of how vagal and spinal afferents innervate the fetal gut and reach specific enteric locations will provide deeper insights into the underlying mechanisms of normal and abnormal sensation from the gastrointestinal tract.

Electrically controlling cell adhesion, growth and migration

We have developed a neurochip to control the adhesion and outgrowth of individual neurons by electrochemical removal of protein repellent molecules from transparent electrodes. The neurochip architecture is based on three parallel indium-tin-oxide (ITO) electrodes on a SiO 2 substrate and a photoresist structure forming a landing spot for the neuron soma and two lateral outgrowth pathways for the neurites. The whole surface was turned protein and cell repellent with poly(ethylene glycol) grafted-poly( l-lysine) (PLL- g-PEG) before enabling neuron soma adhesion by selective PLL- g-PEG removal. After the neuron has settled down a potential was applied to the pathway electrodes to permit the neurite outgrowth along pathways formed by the SU8 structure. We also show the possibility to control cell migration by small pulsed currents. Myoblasts were therefore seeded on a chemical pattern of cell adhesive PLL and cell resistant PLL- g-PEG. The PLL- g-PEG was then removed electrochemically from the electrodes to permit migration onto the cell free electrodes. Electrodes without applied current were confluently overgrown within 24 h but a small pulsed current was able to inhibit cell growth on the bare ITO electrode for more than 72 h. With both techniques, cell adhesion, growth and migration can be controlled dynamically after the cells started to grow on the substrate. This opens new possibilities: we believe the key to control the development of topologically controlled neuron networks or more complex co-cultures is the combination of passive surface modifications and active control over the surface properties at any time of the experiment.

Efficacy of the botanical repellents geraniol, linalool, and citronella against mosquitoes

We determined the degree of personal protection provided by citronella, linalool, and geraniol in the form of commercially available candles or diffusers, both indoors and outdoors. Under the uniform conditions of the experiments, all substances repelled significantly more mosquitoes than the unprotected control. Furthermore, the repellents tested were more active when in the form of a continuous release diffuser than in candle form. All candles were 88 g containing 5% of the active ingredient and all diffusers contained 20 g of 100% active ingredient. Indoors, the repellency rate of citronella candles was only 14% while the repellency rate of citronella diffusers was 68%. The repellency of geraniol candles was 50% while the diffusers provided a repellency rate of 97%. No linalool candles were available for study but linalool diffusers repelled mosquitoes by 93%. Outdoors, citronella diffusers placed 6 m from mosquito traps repelled female mosquitoes by 22%, linalool repelled females by 58%, and geraniol repelled females by 75%. Trap catches were significantly reduced again when diffusers were placed 3 m from the traps. We concluded that geraniol had significantly more repellent activity than citronella or linalool in both indoor and outdoor settings.

Nucleation and Growth of Integrin Adhesions

We present a model that provides a mechanistic understanding of the processes that govern the formation of the earliest integrin adhesions ex novo from an approximately planar plasma membrane. Using an analytic analysis of the free energy of a dynamically deformable membrane containing freely diffusing receptors molecules and long repeller molecules that inhibit integrins from binding with ligands on the extracellular matrix, we predict that a coalescence of polymerizing actin filaments can deform the membrane toward the extracellular matrix and facilitate integrin binding. Monte Carlo simulations of this system show that thermally induced membrane fluctuations can either zip-up and increase the radius of a nucleated adhesion or unzip and shrink an adhesion, but the fluctuations cannot bend the ventral membrane to nucleate an adhesion. To distinguish this integrin adhesion from more mature adhesions, we refer to this early adhesion as a nouveau adhesion.

Modulation of synaptic transmission and plasticity by cell adhesion and repulsion molecules

Adhesive and repellent molecular cues guide migrating cells and growing neurites during development. They also contribute to synaptic function, learning and memory in adulthood. Here, we review the roles of cell adhesion molecules of the immunoglobulin superfamily (Ig-CAMs) and semaphorins (some of which also contain Ig-like domains) in regulation of synaptic transmission and plasticity. Interestingly, among the seven studied Ig-CAMs, the neuronal cell adhesion molecule proved to be important for all tested forms of hippocampal plasticity, while its associated unusual glycan polysialic acid is necessary and sufficient part for synaptic plasticity only at CA3-CA1 synapses. In contrast, Thy-1 and L1 specifically regulate long-term potentiation (LTP) at synapses formed by entorhinal axons in the dentate gyrus and cornu ammonis, respectively. Contactin-1 is important for long-term depression but not for LTP at CA3-CA1 synapses. Analysis of CHL1-deficient mice illustrates that at intermediate stages of development a deficit in a cell adhesion molecule is compensated but appears as impaired LTP during early and late postnatal development. The emerging mechanisms by which adhesive Ig-CAMs contribute to synaptic plasticity involve regulation of activities of NMDA receptors and L-type Ca2+ channels, signaling via mitogen-activated protein kinase p38, changes in GABAergic inhibition and motility of synaptic elements. Regarding repellent molecules, available data for semaphorins demonstrate their activity-dependent regulation in normal and pathological conditions, synaptic localization of their receptors and their potential to elevate or inhibit synaptic transmission either directly or indirectly.

Modulation of Semaphorin3A Activity by p75 Neurotrophin Receptor Influences Peripheral Axon Patterning

The p75 neurotrophin receptor (p75(NTR)) interacts with multiple ligands and coreceptors. It is thought to mediate myelin growth inhibition as part of the Nogo receptor complex, in addition to its other roles. Paradoxically, however, peripheral axons of p75(ExonIII-/-) mutant embryos are severely stunted. This inhibition of axon growth may be a result of neurite elongation defects in p75(NTR) mutant neurons. Here, we show that p75(ExonIII-/-) DRG neurons are hypersensitive to the repellent molecule Semaphorin3A (Sema3A). NGF modulates Sema3A activity equally well in both the p75(NTR) mutant and wild-type neurons, indicating that the hypersensitivity of p75(NTR) mutant neurons is probably not related to their NGF receptor activity. Neuropilin1 and p75(NTR) partially colocalize in DRG growth cones. After Sema3A stimulation, the degree of colocalization is dramatically increased, particularly in clusters associated with Sema3A receptor complex activation. Coimmunoprecipitation studies show that p75(NTR) interacts directly with the Sema3A receptors Neuropilin1 and PlexinA4. When coexpressed with both Neuropilin1 and PlexinA4, p75(NTR) reduces the interaction between these two receptor components. Finally, p75(NTR)/Sema3A double-mutant embryos show growth similar to that observed in Sema3A-null mice. These data indicate that p75(NTR) is an important functional modulator of Sema3A activity and that, in the absence of p75(NTR), oversensitivity to Sema3A leads to severe reduction in sensory innervation. Our results also suggest that while inhibition of p75(NTR) in CNS injury may enhance nerve regeneration resulting from the inhibition of myelin-associated protein, it may also inhibit nerve regeneration through its modulation of Sema3A.

Semaphorin3A regulates axon growth independently of growth cone repulsion via modulation of TrkA signaling

Regulation of axon growth is a critical event in neuronal development. Nerve growth factor (NGF) is a strong inducer of axon growth and survival in the dorsal root ganglia (DRG). Paradoxically, high concentrations of NGF are present in the target region where axon growth must slow down for axons to accurately identify their correct targets. Semaphorin3A (Sema3A), a powerful axonal repellent molecule for DRG neurons, is also situated in their target regions. NGF is a modulator of Sema3A-induced repulsion and death. We show that Sema3A is a regulator of NGF-induced neurite outgrowth via the TrkA receptor, independent of its growth cone repulsion activity. First, neurite outgrowth of DRG neurons is more sensitive to Sema3A than repulsion. Second, at concentrations sufficient to significantly inhibit Sema3A-induced repulsion, NGF has no effect on Sema3A-induced axon growth inhibition. Third, Sema3A-induced outgrowth inhibition, but not repulsion activity, is dependent on NGF stimulation. Fourth, Sema3A attenuates TrkA-mediated growth signaling, but not survival signaling, and over-expression of constitutively active TrkA blocks Sema3A-induced axon growth inhibition, suggesting that Sema3A activity is mediated via regulation of NGF/TrkA-induced growth. Finally, quantitative analysis of axon growth in vivo supports the possibility that Sema3A affects axon growth, in addition to its well-documented role in axon guidance. We suggest a model whereby NGF at high concentrations in the target region is important for survival, attraction and inhibition of Sema3A-induced repulsion, while Sema3A inhibits its growth-promoting activity. The combined and cross-modulatory effects of these two signaling molecules ensure the accuracy of the final stages in axon targeting.

Cell Adhesion as Dynamic Interplay of Lock-and-Key, Generic and Elastic Forces

The selectivity of cell-cell and cell-tissue adhesion is determined by specific short range forces between cell surface proteins. Long range entropic interfacial forces (mediated by repeller molecules and membrane undulations) and adhesion-induced elastic stresses in the cell envelope serve the fine control of the strength and duration of adhesion. The initial step of cell adhesion exhibits typical features of a first order wetting transition resulting in the formation of tight adhesion domains by lateral phase separation of receptors. External lift forces can cause shrinking and unbinding of adhesion sites if the receptors are immobile but induce domain growth if they are mobile. Strong adhesion domains (resisting nano-Newton forces) can form by commitment of some 10,000 receptors enabling cells to control adhesion strength rapidly by varying the receptor and repeller densities on cell surfaces through endocytosis and exocytosis. The adhesion domains can function as constraint reaction spaces facilitating the local assembly of actin stress fibers and control cell signalling processes as shown for the activation of immunological responses by immunological synapses.

Hedgehog regulated Slit expression determines commissure and glial cell position in the zebrafish forebrain

Three major axon pathways cross the midline of the vertebrate forebrain early in embryonic development: the postoptic commissure (POC), the anterior commissure (AC) and the optic nerve. We show that a small population of Gfap+ astroglia spans the midline of the zebrafish forebrain in the position of, and prior to, commissural and retinal axon crossing. These glial ;bridges' form in regions devoid of the guidance molecules slit2 and slit3, although a subset of these glial cells express slit1a. We show that Hh signaling is required for commissure formation, glial bridge formation, and the restricted expression of the guidance molecules slit1a, slit2, slit3 and sema3d, but that Hh does not appear to play a direct role in commissural and retinal axon guidance. Reducing Slit2 and/or Slit3 function expanded the glial bridges and caused defasciculation of the POC, consistent with a ;channeling' role for these repellent molecules. By contrast, reducing Slit1a function led to reduced midline axon crossing, suggesting a distinct role for Slit1a in midline axon guidance. Blocking Slit2 and Slit3, but not Slit1a, function in the Hh pathway mutant yot (gli2DR) dramatically rescued POC axon crossing and glial bridge formation at the midline, indicating that expanded Slit2 and Slit3 repellent function is largely responsible for the lack of midline crossing in these mutants. This analysis shows that Hh signaling helps to pattern the expression of Slit guidance molecules that then help to regulate glial cell position and axon guidance across the midline of the forebrain.

Domains in membranes and vesicles

Biomimetic membranes such as lipid bilayers with several molecular componentsexhibit intramembrane domains. These domains are formed after the membranesare quenched into a two-phase coexistence region. Solid domains tend to formfacets or cylindrical segments whereas liquid domains tend to form spherical buds.In the latter case, one can distinguish three tension regimes; budding alwaysoccurs in the low tension regime for which we present preliminary experimentalresults for vesicle membranes composed of phospholipid, sphingomyelin andcholesterol. A multicomponent vesicle which adheres to a substrate surfaceconsists of a bound and an unbound membrane segment which differ in theircompositions. This shift in composition can induce domains within the boundsegment. Such domains are also formed when membranes adhere via stickermolecules and can be driven (i) by the interplay of sticker adhesion and shapefluctuations and (ii) by the competition between sticker and repeller molecules.

The jing gene is required for embryonic brain development in Drosophila [corrected

Loss- and gain-of-function studies have demonstrated a crucial role for the jing zinc finger transcription factor in neuronal and glial differentiation and survival in the embryonic central nervous system midline of Drosophila. Here, we have studied the role of jing during embryonic brain development. Proper jing function is required for the formation of the primary brain axon scaffold. In homozygous jing (3) mutant embryos the preoral commissure is not pioneered and never forms. Other axon pathways are pioneered but subsequently do not form properly, including the postoral tritocerebral commissure, the circumesophageal connectives and the pathways that connect the brain with the ventral nerve cord. To understand the cellular basis of the axon phenotype the jing expression pattern in the brain was characterized using a jing-lacZ enhancer trap. jing-lacZ enhancer trap expression occurs in glia and neurons in the brain midline and lateral clusters as determined by co-localization of the lacZ gene product with Repo and Castor, respectively. In addition, the jing-lacZ enhancer trap and the basic helix-loop-helix-PAS gene, single-minded ( sim), are expressed in the only glial midline cluster present in stage-14 wild-type embryos. jing function is required for the differentiation of Repo-, Castor- and Sim-positive cells in the embryonic brain as each of these populations contain a reduced number of cells in homozygous jing (3) mutant embryos. We further find that jing is required for neuronal and glial survival as repo- and castor-expressing cells undergo cell death in homozygous jing (3) mutant embryos, as revealed by double labeling with Tunel. Expression of jing in sim-expressing cells in the brain disrupts the entire axon scaffold but most significantly results in loss of the preoral and postoral tritocerebral commissures. In addition, circumesophageal connectives are repelled after expression of two copies of UAS- jing in sim-expressing cells, suggesting the activation of axon repellent molecules. Over-expression of sim in the brain is also associated with loss of preoral and postoral tritocerebral commissures. Therefore, the proper dosage of jing and sim in the brain is critical for the formation of the primary axon scaffold. These results show that an important role for jing in the developing brain is the regulation of neuronal and glial differentiation and survival.

Cell adhesion as wetting transition?

Cell adhesion is controlled by a complex interplay of short range (lock-and-key) forces mediated by cell surface receptors, a phalanx of (short and long range) nonspecific (generic) interactions, and last but not least membrane elasticity. The physical basis of cell adhesion is explored by the design of simplified model systems, mimicking cell and tissue surfaces, enabling local measurements of cellular shape changes and adhesion forces by microinterferometry. Cell adhesion can be understood as first-order dewetting transition that results in the formation of adhesion plaques, such as focal adhesion sites of cells, which allow cell adhesion at astonishingly low receptor densities. The repeller molecules of the glycocalix play a key role for the control of the adhesion transition and the mechanical stability of the adhering cells by relaxing the strength of the binding forces. Stress fibers are postulated to be essential for the stabilization of adhesion domains against leverage through bending moments enforced by hydrodynamic shear forces.

Decorated surfaces by biofunctionalized gold beads: application to cell adhesion studies.

We describe a simple but versatile method to decorate solid surfaces randomly with colloidal gold particles to which ligands of cell receptors can be coupled to generate local attraction sites for the control of cell adhesion. A self-assembled monolayer of (3-mercaptopropyl)trimethoxysilane was deposited on glass slides. Gold beads were anchored to the functionalized surface through the sulfur group. We characterized the gold bead distribution on the functionalized surface with reflection interference contrast microscopy. The gold beads were functionalized with a disulfide-coupled cyclic pentapeptide containing an arginine-glycine-aspartic acid (RGD) tripeptide sequence which is selectively recognized by integrin receptors alpha(V)beta(3) of endothelial cells. A blocking layer of bovine serum albumin was adsorbed onto the surface to prevent non-specific binding of the cells. We demonstrate that the RGD-functionalized colloidal gold beads act as local attraction centers, mediating rapid cell anchoring on a substrate impeding cell adhesion in the absence of attraction centers. Surprisingly, microinterferometry shows that after a time delay of about 1 h, the regions of the cell surface between the gold beads form close contacts with the substrate, which is attributed to strong van der Waals attraction after escape of repeller molecules from the contact surface.

Developmental aspect of the gonadotropin-releasing hormone system

Gonadotropin-releasing hormone (GnRH) regulates the hypothalamo-pituitary-gonadal (HPG) axis in all vertebrates studied. GnRH neurons that regulate the HPG axis are primarily derived from progenitor cells in the nasal compartment (NC) and migrate along olfactory system derived fibers across the cribriform plate to destinations in the forebrain. Across their long and uncommon migratory route many factors are likely important for their successful development. Several classes of molecules are being studied for their potential influences on migration, including those related to cell surface interactions (membrane receptors, adhesion molecules, extracellular matrix (ECM) molecules, etc.) and those related to communication across distances (neurotransmitters, peptides, chemoattractant or repellent molecules). Of the classes of molecules associated with cell surface interactions, glycoconjugates with terminal galactose, are temporally and spatially expressed on olfactory fibers that guide GnRH neurons and may play role(s) in migration. Of the molecules associated with communication across distances, the neurotransmitter gamma-aminobutyric acid (GABA) is associated with the GnRH migration pathway and influences the position and organization of GnRH neurons in vitro and in vivo. Furthermore, galactose-containing glycoconjugates and GABA are associated with GnRH neurons in species ranging from humans to lamprey. In mice and rats, GABA is found transiently within a subpopulation of GnRH neurons as they migrate through the NC. One of the key elements in considering regulators of GnRH neuron migration is the diversity of GnRH synthesizing cells. For example, only subpopulations of GnRH neurons also contain GABA, specific GABA receptors, or select glycoconjugates. Similarly, treatments that influence GnRH neuronal migration may only affect specific subsets and not the entire population. It is likely that we will not be able to characterize the migration of all GnRH neurons by a single factor. By combining molecular inquiries with genetic models, single cell analyses, and an in vitro migration model, we are beginning to decipher one of the most critical events in the establishment of the reproductive axis.

Spinal Nerve Segmentation in the Chick Embryo: Analysis of Distinct Axon-Repulsive Systems

In higher vertebrates, the segmental organization of peripheral spinal nerves is established by a repulsive mechanism whereby sensory and motor axons are excluded from the posterior half-somite. A number of candidate axon repellents have been suggested to mediate this barrier to axon growth, including Sema3A, Ephrin-B, and peanut agglutinin (PNA)-binding proteins. We have tested the candidacy of these factors in vitro by examining their contribution to the growth cone collapse-inducing activity of somite-derived protein extracts on sensory, motor, and retinal axons. We find that Sema3A is unlikely to play a role in the segmentation of sensory or motor axons and that Ephrin-B may contribute to motor but not sensory axon segmentation. We also provide evidence that the only candidate molecule(s) that induces the growth cone collapse of both sensory and motor axons binds to PNA and is not Sema3A or Ephrin-B. By grafting primary sensory, motor, and quail retinal neurons into the chick trunk in vivo, we provide further evidence that the posterior half-somite represents a universal barrier to growing axons. Taken together, these results suggest that the mechanisms of peripheral nerve segmentation should be considered in terms of repellent molecules in addition to the identified molecules.

Slit Inhibition of Retinal Axon Growth and Its Role in Retinal Axon Pathfinding and Innervation Patterns in the Diencephalon

We have analyzed the role of the Slit family of repellent axon guidance molecules in the patterning of the axonal projections of retinal ganglion cells (RGCs) within the embryonic rat diencephalon and whether the slits can account for a repellent activity for retinal axons released by hypothalamus and epithalamus. At the time RGC axons extend over the diencephalon, slit1 and slit2 are expressed in hypothalamus and epithalamus but not in the lateral part of dorsal thalamus, a retinal target. slit3 expression is low or undetectable. The Slit receptors robo2, and to a limited extent robo1, are expressed in the RGC layer, as are slit1 and slit2. In collagen gels, axon outgrowth from rat retinal explants is biased away from slit2-transfected 293T cells, and the number and length of axons are decreased on the explant side facing the cells. In addition, in the presence of Slit2, overall axon outgrowth is decreased, and bundles of retinal axons are more tightly fasciculated. This action of Slit2 as a growth inhibitor of retinal axons and the expression patterns of slit1 and slit2 correlate with the fasciculation and innervation patterns of RGC axons within the diencephalon and implicate the Slits as components of the axon repellent activity associated with the hypothalamus and epithalamus. Our findings suggest that in vivo the Slits control RGC axon pathfinding and targeting within the diencephalon by regulating their fasciculation, preventing them or their branches from invading nontarget tissues, and steering them toward their most distal target, the superior colliculus.

Adhesive switching of membranes: experiment and theory.

We report on a study of a model bioadhesion system: giant vesicles in contact with a supported lipid bilayer. Embedded in both membranes are very low concentrations of homophilic recognition molecules (contact site A receptors) competing with higher concentrations of repeller molecules: polyethylene glycol (PEG) lipids. These repellers mimic the inhibiting effect of the cell glycocalyx on adhesion. The effective adhesive interaction between the two membranes is probed by interferometric analysis of thermal fluctuations. We find two competing states of adhesion: initial weak adhesion is followed by slower aggregation of the adhesion molecules into small, tightly bound clusters that coexist with the regions of weak adhesion. We interpret our results in terms of a double-well intermembrane potential, and we present a theoretical analysis of the intermembrane interaction in the presence of mobile repeller molecules at a fixed chemical potential that shows that the interaction potential indeed should have just such a double-well shape. At a fixed repeller concentration we recover a conventional purely repulsive potential. We discuss the implications of our findings in terms of a general amplification mechanism of the action of sparse adhesion molecules by a nonspecific double-well potential. We also discuss the important role of the Helfrich undulation force for the proposed scenario.