RoR2 functions as a noncanonical Wnt receptor that regulates NMDAR-mediated synaptic transmission

Wnt signaling has a well-established role as a regulator of nervous system development, but its role in the maintenance and regulation of established synapses in the mature brain remains poorly understood. At excitatory glutamatergic synapses, NMDA receptors (NMDARs) have a fundamental role in synaptogenesis, synaptic plasticity, and learning and memory; however, it is not known what controls their number and subunit composition. Here we show that the receptor tyrosine kinase-like orphan receptor 2 (RoR2) functions as a Wnt receptor required to maintain basal NMDAR-mediated synaptic transmission. In addition, RoR2 activation by a noncanonical Wnt ligand activates PKC and JNK and acutely enhances NMDAR synaptic responses. Regulation of a key component of glutamatergic synapses through RoR2 provides a mechanism for Wnt signaling to modulate synaptic transmission, synaptic plasticity, and brain function acutely beyond embryonic development.Wnt ligands are highly conserved secreted glycoproteins responsible for important developmental and homeostatic processes throughout the animal kingdom (1, 2). They play a key role in morphogenesis, patterning, and lineage decision during central and peripheral nervous system development (3). Wnt ligands control gene expression (4), and their dysregulation has been implicated in cancer and major neuropathologies (5–9).The sustained expression of Wnt ligands and Wnt signaling components in the mature mammalian CNS and their involvement in neuropathologies suggest that these signaling cascades might also play a part in synaptic maintenance and function beyond embryonic development (10, 11). However, because of the pleiotropy and complexity of Wnt signaling, it has been difficult to dissect the components of Wnt signaling present in mature neurons and their role, if any, in the regulation of established synaptic connections and synaptic transmission.Although most excitatory glutamatergic neurotransmission in the brain is mediated by AMPA-type glutamate receptors [i.e., AMPA receptors (AMPARs)], unique properties allow the NMDA-type glutamate receptors [i.e., NMDA receptors (NMDARs)] to play a critical role in synaptic plasticity, learning and memory, and the establishment and maturation of functional neural circuits (12–14). Despite their importance, it is not known what controls the number and subunit composition of synaptic NMDARs. Dysfunction of NMDARs has been implicated in numerous diseases, including Huntington disease, Parkinson disease, depression, bipolar disorder, and schizophrenia (14, 15), in which a deficit in NMDAR mediated neurotransmission may be central (16). Interestingly, NMDAR-mediated currents can be acutely and specifically up-regulated by Wnt5a, a noncanonical Wnt ligand (17), but little is known regarding the signaling pathway and mechanisms involved in such regulation.The receptor tyrosine kinase-like orphan receptor 2 (RoR2) is part of a conserved family of tyrosine kinase-like receptors that have been proposed to serve as a receptor for noncanonical Wnt ligands, participating in developmental processes like cell movement and cell polarity (18, 19). Although RoR2 protein has been detected in mammalian neurons (20), its function and signaling pathways are not known. Here we show that RoR2 acts as a receptor for noncanonical Wnt ligands capable of regulating synaptic NMDARs. In hippocampal neurons, activation of RoR2 by noncanonical Wnt ligand Wnt5a activates PKC and JNK, two kinases involved in the regulation of NMDAR currents. In addition, we show that signaling through RoR2 is necessary for the maintenance of basal NMDAR-mediated synaptic transmission and the acute regulation of NMDAR synaptic responses by Wnt5a.Identification of RoR2 as a Wnt receptor that regulates synaptic NMDARs provides a mechanism for Wnt signaling to control synaptic transmission and synaptic plasticity acutely, and is a critical first step toward understanding the role played by Wnt signaling in the regulation of glutamatergic synaptic function under normal or pathological conditions.     

Notch inhibition induces mitotically generated hair cells in mammalian cochleae via activating the Wnt pathway

The activation of cochlear progenitor cells is a promising approach for hair cell (HC) regeneration and hearing recovery. The mechanisms underlying the initiation of proliferation of postnatal cochlear progenitor cells and their transdifferentiation to HCs remain to be determined. We show that Notch inhibition initiates proliferation of supporting cells (SCs) and mitotic regeneration of HCs in neonatal mouse cochlea in vivo and in vitro. Through lineage tracing, we identify that a majority of the proliferating SCs and mitotic-generated HCs induced by Notch inhibition are derived from the Wnt-responsive leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5⁺) progenitor cells. We demonstrate that Notch inhibition removes the brakes on the canonical Wnt signaling and promotes Lgr5⁺ progenitor cells to mitotically generate new HCs. Our study reveals a new function of Notch signaling in limiting proliferation and regeneration potential of postnatal cochlear progenitor cells, and provides a new route to regenerate HCs from progenitor cells by interrupting the interaction between the Notch and Wnt pathways.Sensory hair cell (HC) loss is the major cause of hearing loss and balance disorder. In nonmammalian vertebrates, HCs are regenerated in both auditory and vestibular systems after HC loss, leading to functional recovery of hearing and balance function (1–3). In mammals, limited spontaneous HC regeneration occurs in the vestibular system (4–8). In the adult mammalian vestibular sensory epithelium, inner ear stem cells were isolated with the capacity to differentiate into HCs and other inner ear cell types (9). In contrast, only neonatal mammalian cochleae have limited HC regeneration capacity in vivo, and harbor stem cells or progenitor cells that could proliferate and regenerate new HCs (10–12); however, no spontaneous HC regeneration has been observed in the mature cochlea (13).Recent studies reported that in neonatal mouse cochlea, Wnt-responsive leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5⁺) cells are the progenitors with the capacity to regenerate HCs under certain condition (11, 12, 14–17). However, endogenous Lgr5⁺ progenitors maintain mitotic quiescence in neonatal mouse cochlea, suggesting the existence of negative regulators that inhibit the proliferation of those progenitors. Overexpressing β-catenin in the Lgr5⁺ or Sox2⁺ [SRY (sex determining region Y)-box 2] cells initiates proliferation by forming BrdU⁺ foci adjacent to HCs (14, 16), serving as a potential approach to overcome the nonproliferative barrier of progenitors in the cochlear sensory epithelium. Alternatively, the identification and removal of the negative regulators could provide a new route to activate cochlear progenitor proliferation to achieve HC generation.Inner ear sensory epithelium consists of a mosaic of HCs and supporting cells (SCs), generated from the same precursor pool in the prosensory domain during development (18, 19). The formation of the mosaic HC and SC pattern is mediated by lateral inhibition through the Notch signaling pathway (20, 21). Evidence from birds and mice suggests that Notch signaling negatively regulates the formation of HCs and the loss of Notch signaling generates supernumerary ectopic HCs at the expense of SCs (22–26). During early embryonic development, it has been shown that inhibition of Notch/JAG2 and DLL1 may prolong the proliferation process of the prosensory cells in the inner ear (27, 28). Notch signaling may play an important role maintaining the homeostasis of cochlear sensory epithelium on cell number and structures.Although Wnt and Notch signaling are two fundamental pathways that regulate progenitor cell proliferation and determine the cell fate in the inner ear, their relationship remains largely unclear in the postnatal mouse cochlea. Here, by inhibiting the Notch signaling using Notch1 conditional knockout (KO) mice in vivo and by γ-secretase inhibitor IX (DAPT) treatment in vitro, we found that both inhibitions led to the proliferation of SCs and mitotic generation of HCs in the postnatal cochlear sensory epithelium. Lineage tracing demonstrated that a majority of the proliferating SCs and mitotically generated HCs were of Lgr5⁺ lineage. In addition, we showed that Notch inhibition resulted in β-catenin up-regulation in the Sox2⁺ SCs, whereas inhibition of Wnt signaling significantly decreased SCs proliferation and mitotic generation of HCs induced by Notch inhibition.     

Axin2 marks quiescent hair follicle bulge stem cells that are maintained by autocrine Wnt/β-catenin signaling

How stem cells maintain their identity and potency as tissues change during growth is not well understood. In mammalian hair, it is unclear how hair follicle stem cells can enter an extended period of quiescence during the resting phase but retain stem cell potential and be subsequently activated for growth. Here, we use lineage tracing and gene expression mapping to show that the Wnt target gene Axin2 is constantly expressed throughout the hair cycle quiescent phase in outer bulge stem cells that produce their own Wnt signals. Ablating Wnt signaling in the bulge cells causes them to lose their stem cell potency to contribute to hair growth and undergo premature differentiation instead. Bulge cells express secreted Wnt inhibitors, including Dickkopf (Dkk) and secreted frizzled-related protein 1 (Sfrp1). However, the Dickkopf 3 (Dkk3) protein becomes localized to the Wnt-inactive inner bulge that contains differentiated cells. We find that Axin2 expression remains confined to the outer bulge, whereas Dkk3 continues to be localized to the inner bulge during the hair cycle growth phase. Our data suggest that autocrine Wnt signaling in the outer bulge maintains stem cell potency throughout hair cycle quiescence and growth, whereas paracrine Wnt inhibition of inner bulge cells reinforces differentiation.The hair follicle is a complex miniorgan that repeatedly cycles through stages of rest (telogen), growth (anagen), and destruction (catagen) throughout life (1). During anagen, growing hair follicles emerge adjacent to the old telogen hair follicles that remain there throughout the cycle and create an epithelial protrusion known as the “bulge.” At the end of the hair cycle, in catagen, cells from the follicle migrate along the retracting epithelial strand and join the two epithelial layers of the telogen bulge—the inner and outer bulge layers—surrounding the club hair shaft (2).Several studies have established that stem cells residing in the outer bulge are the source of the regenerative capacity of the cycling hair follicle (3–5). During telogen, these stem cells are thought to be generally quiescent (6). In response to signals from their microenvironment during anagen, the stem cells divide and produce proliferative progeny that participate in the growth of the new follicle (7). Some of these activated stem cells and their progeny are believed to migrate away from the bulge, but are subsequently able to rejoin it after anagen is complete (2, 5). Cells that return to the outer bulge take on a follicular stem cell identity, ready to divide and participate in the next hair cycle (2, 8). Conversely, cells returning to the inner bulge do not divide and, instead, form an inner bulge niche of differentiated cells for the outer bulge cells (2). Stem cells remain quiescent during telogen for an extended period, and the identity of signals that maintain stem cell identity during this time are poorly understood.In the hair, Wnt/β-catenin signaling is required right from the earliest stages of development, for the initiation of hair placode formation (9). Wnt signals are needed later during postnatal homeostasis as well, for the initiation of anagen in postnatal hair (10). Therefore, in view of their well-established importance for stem cell maintenance in multiple adult tissues, including the skin (11), Wnts are candidate hair follicle stem cell (HFSC)-maintaining signals. However, Wnt signaling is generally believed to be inactive in the telogen bulge (8, 10, 12), which is thought to be quiescent. Wnt signaling becomes strongly elevated when bulge cells are “activated” to undergo the transition from telogen to anagen (13, 14). During anagen, Wnt signaling has been described to primarily specify differentiated cell fates in the anagen follicle (12, 15). As anagen proceeds and the follicle enters catagen and telogen again, the bulge is thought to revert to a Wnt-inhibited state (12, 13, 16, 17).Conversely, there is evidence for a functional requirement of Wnt/β-catenin signaling in the bulge other than initiating anagen and specifying differentiation during anagen. For instance, postnatal deletion of β-catenin in outer bulge cells results in the loss of label-retention and HFSC markers, suggesting that β-catenin is required for maintenance of HFSC identity (10).Here, beyond its role in hair differentiation and anagen initiation, we sought to determine whether Wnt/β-catenin signaling is also involved in HFSC maintenance during telogen. We found that Axin2 expression persists in HFSCs in the outer bulge throughout telogen and anagen, suggesting that active Wnt signaling is a consistent feature of bulge stem cells. Furthermore, these hair outer bulge stem cells produce autocrine Wnts and paracrine-acting Wnt inhibitors that may specify the positional identity of cells residing within the bulge niche.     

Parameter-free methods distinguish Wnt pathway models and guide design of experiments

The canonical Wnt signaling pathway, mediated by β-catenin, is crucially involved in development, adult stem cell tissue maintenance, and a host of diseases including cancer. We analyze existing mathematical models of Wnt and compare them to a new Wnt signaling model that targets spatial localization; our aim is to distinguish between the models and distill biological insight from them. Using Bayesian methods we infer parameters for each model from mammalian Wnt signaling data and find that all models can fit this time course. We appeal to algebraic methods (concepts from chemical reaction network theory and matroid theory) to analyze the models without recourse to specific parameter values. These approaches provide insight into aspects of Wnt regulation: the new model, via control of shuttling and degradation parameters, permits multiple stable steady states corresponding to stem-like vs. committed cell states in the differentiation hierarchy. Our analysis also identifies groups of variables that should be measured to fully characterize and discriminate between competing models, and thus serves as a guide for performing minimal experiments for model comparison.The Wnt signaling pathway plays a key role in essential cellular processes ranging from proliferation and cell specification during development to adult stem cell maintenance and wound repair (1). Dysfunction of Wnt signaling is implicated in many pathological conditions, including degenerative diseases and cancer (2–4). Despite many molecular advances, the pathway dynamics are still not well understood. Theoretical investigations of the Wnt/β-catenin pathway serve as testbeds for working hypotheses (5–12).We focus on models of canonical Wnt pathway processes with the aim of elucidating mechanisms, predicting function, and identifying key pathway components in adult tissues, such as colonic crypts. We compare four preexisting ordinary differential equation models (5–8) and find, using injectivity theory, that for any given conditions and parameter values, none of the models is capable of multiple cellular responses.In many tissues Wnt plays a crucial role in cell fate specification (3). At the base of colonic crypts, cells exist in a stem-like, proliferative phenotype in the presence of Wnt. As these cells’ progeny move up the crypt axis they enter a Wnt-low environment and change fate (perhaps reversibly), becoming differentiated, specialized gut cells (13). In neuronal and endocrinal tissues, Wnt/β-catenin data suggest cell fate plasticity under different environmental conditions (14, 15). Here, we introduce a new model motivated by experimental findings not described in previous models (16–18) to investigate bistable switching in the Wnt pathway. We find the new model to be capable of multiple cellular responses; furthermore, our parameter-free techniques identify that molecular shuttling (between cytoplasm and nucleus) and degradation together may serve as a possible mechanism for governing bistability in the pathway, corresponding to, for example, a committed cell state and a stem-like cell state.Comparison of models (and mechanisms) requires data; the type of comparison performed depends on the data at hand. If data show bistability (two distinct response states), then we could rule out all models that preclude bistability; however the converse is not true (a graded response may be compatible with all models). Experimental studies in Xenopus extracts have been performed to validate a model of Wnt signaling (5), with further pathway elucidation in refs. 19, 20; however, the parameters identified in these studies may differ markedly from those involved in mammalian Wnt signaling (21, 22). With the aim of discriminating between models, we present the five Wnt models under a unifying framework, with standardized notation to facilitate comparison. We fit parameters to recently published mammalian β-catenin signaling time-course data using Bayesian inference (22) and find that all of the studied models can describe the data well, demonstrating that additional data are required to compare models.To determine which sets of protein species should be measured for carrying out a comparison between models and data, we introduce matroid theory to systems biology. A matroid is a combinatorial structure from mathematics, and in our case, it provides all of the steady-state invariants (23, 24) that have minimal sets of variables. The algebraic matroid associated with the steady-state ideal determines specific sets of species that should be measured to perform model discrimination without knowledge of parameter values. We demonstrate this parameter-free analysis with experimental data for two Wnt models.In the next section, we introduce the previous models and new shuttle model. We perform injectivity–multistability analysis and classify the shuttle model as the only one capable of multistability. Next we infer the parameters of five competing models for time-course β-catenin data, revealing that all of the models fit the data. Finally we introduce algebraic matroids to inform experimental design for discriminating between models and data.     

Dissecting neural pathways for forgetting in Drosophila olfactory aversive memory

Recent studies have identified molecular pathways driving forgetting and supported the notion that forgetting is a biologically active process. The circuit mechanisms of forgetting, however, remain largely unknown. Here we report two sets of Drosophila neurons that account for the rapid forgetting of early olfactory aversive memory. We show that inactivating these neurons inhibits memory decay without altering learning, whereas activating them promotes forgetting. These neurons, including a cluster of dopaminergic neurons (PAM-β′1) and a pair of glutamatergic neurons (MBON-γ4>γ1γ2), terminate in distinct subdomains in the mushroom body and represent parallel neural pathways for regulating forgetting. Interestingly, although activity of these neurons is required for memory decay over time, they are not required for acute forgetting during reversal learning. Our results thus not only establish the presence of multiple neural pathways for forgetting in Drosophila but also suggest the existence of diverse circuit mechanisms of forgetting in different contexts.Although forgetting commonly has a negative connotation, it is a functional process that shapes memory and cognition (1–4). Recent studies, including work in relatively simple invertebrate models, have started to reveal basic biological mechanisms underlying forgetting (5–15). In Drosophila, single-session Pavlovian conditioning by pairing an odor (conditioned stimulus, CS) with electric shock (unconditioned stimulus, US) induces aversive memories that are short-lasting (16). The memory performance of fruit flies is observed to drop to a negligible level within 24 h, decaying rapidly early after training and slowing down thereafter (17). Memory decay or forgetting requires the activation of the small G protein Rac, a signaling protein involved in actin remodeling, in the mushroom body (MB) intrinsic neurons (6). These so-called Kenyon cells (KCs) are the neurons that integrate CS–US information (18, 19) and support aversive memory formation and retrieval (20–22). In addition to Rac, forgetting also requires the DAMB dopamine receptor (7), which has highly enriched expression in the MB (23). Evidence suggests that the dopamine-mediated forgetting signal is conveyed to the MB by dopamine neurons (DANs) in the protocerebral posterior lateral 1 (PPL1) cluster (7, 24). Therefore, forgetting of olfactory aversive memory in Drosophila depends on a particular set of intracellular molecular pathways within KCs, involving Rac, DAMB, and possibly others (25), and also receives modulation from extrinsic neurons. Although important cellular evidence supporting the hypothesis that memory traces are erased under these circumstances is still lacking, these findings lend support to the notion that forgetting is an active, biologically regulated process (17, 26).Although existing studies point to the MB circuit as essential for forgetting, several questions remain to be answered. First, whereas the molecular pathways for learning and forgetting of olfactory aversive memory are distinct and separable (6, 7), the neural circuits seem to overlap. Rac-mediated forgetting has been localized to a large population of KCs (6), including the γ-subset, which is also critical for initial memory formation (21, 27). The site of action of DAMB for forgetting has yet to be established; however, the subgroups of PPL1-DANs implicated in forgetting are the same as those that signal aversive reinforcement and are required for learning (28–30). It leaves open the question of whether the brain circuitry underlying forgetting and learning is dissociable, or whether forgetting and learning share the same circuit but are driven by distinct activity patterns and molecular machinery (26). Second, shock reinforcement elicits multiple memory traces through at least three dopamine pathways to different subdomains in the MB lobes (28, 29). Functional imaging studies have also revealed Ca²⁺-based memory traces in different KC populations (31). It is poorly understood how forgetting of these memory traces differs, and it remains unknown whether there are multiple regulatory neural pathways. Notably, when PPL1-DANs are inactivated, forgetting still occurs, albeit at a lower rate (7). This incomplete block suggests the existence of an additional pathway(s) that conveys forgetting signals to the MB. Third, other than memory decay over time, forgetting is also observed through interference (32, 33), when new learning or reversal learning is introduced after training (6, 34, 35). Time-based and interference-based forgetting shares a similar dependence on Rac and DAMB (6, 7). However, it is not known whether distinct circuits underlie forgetting in these different contexts.In the current study, we focus on the diverse set of MB extrinsic neurons (MBENs) that interconnect the MB lobes with other brain regions, which include 34 MB output neurons (MBONs) of 21 types and ∼130 dopaminergic neurons of 20 types in the PPL1 and protocerebral anterior medial (PAM) clusters (36, 37). These neurons have been intensively studied in olfactory memory formation, consolidation, and retrieval in recent years (e.g., 24, 28–30, 38–48); however, their roles in forgetting have not been characterized except for the aforementioned PPL1-DANs. In a functional screen, we unexpectedly found that several Gal4 driver lines of MBENs showed significantly better 3-h memory retention when the Gal4-expressing cells were inactivated. The screen has thus led us to identify two types of MBENs that are not involved in initial learning but play important and additive roles in mediating memory decay. Furthermore, neither of these MBEN types is required for reversal learning, supporting the notion that there is a diversity of neural circuits that drive different forms of forgetting.     

Structural interactions of a voltage sensor toxin with lipid membranes

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.Protein toxins from venomous organisms have been invaluable tools for studying the ion channel proteins they target. For example, in the case of voltage-activated potassium (Kv) channels, pore-blocking scorpion toxins were used to identify the pore-forming region of the channel (1, 2), and gating modifier tarantula toxins that bind to S1–S4 voltage-sensing domains have helped to identify structural motifs that move at the protein–lipid interface (3–5). In many instances, these toxin–channel interactions are highly specific, allowing them to be used in target validation and drug development (6–8).Tarantula toxins are a particularly interesting class of protein toxins that have been found to target all three families of voltage-activated cation channels (3, 9–12), stretch-activated cation channels (13–15), as well as ligand-gated ion channels as diverse as acid-sensing ion channels (ASIC) (16–21) and transient receptor potential (TRP) channels (22, 23). The tarantula toxins targeting these ion channels belong to the inhibitor cystine knot (ICK) family of venom toxins that are stabilized by three disulfide bonds at the core of the molecule (16, 17, 24–31). Although conventional tarantula toxins vary in length from 30 to 40 aa and contain one ICK motif, the recently discovered double-knot toxin (DkTx) that specifically targets TRPV1 channels contains two separable lobes, each containing its own ICK motif (22, 23).One unifying feature of all tarantula toxins studied thus far is that they act on ion channels by modifying the gating properties of the channel. The best studied of these are the tarantula toxins targeting voltage-activated cation channels, where the toxins bind to the S3b–S4 voltage sensor paddle motif (5, 32–36), a helix-turn-helix motif within S1–S4 voltage-sensing domains that moves in response to changes in membrane voltage (37–41). Toxins binding to S3b–S4 motifs can influence voltage sensor activation, opening and closing of the pore, or the process of inactivation (4, 5, 36, 42–46). The tarantula toxin PcTx1 can promote opening of ASIC channels at neutral pH (16, 18), and DkTx opens TRPV1 in the absence of other stimuli (22, 23), suggesting that these toxin stabilize open states of their target channels.For many of these tarantula toxins, the lipid membrane plays a key role in the mechanism of inhibition. Strong membrane partitioning has been demonstrated for a range of toxins targeting S1–S4 domains in voltage-activated channels (27, 44, 47–50), and for GsMTx4 (14, 50), a tarantula toxin that inhibits opening of stretch-activated cation channels in astrocytes, as well as the cloned stretch-activated Piezo1 channel (13, 15). In experiments on stretch-activated channels, both the d- and l-enantiomers of GsMTx4 are active (14, 50), implying that the toxin may not bind directly to the channel. In addition, both forms of the toxin alter the conductance and lifetimes of gramicidin channels (14), suggesting that the toxin inhibits stretch-activated channels by perturbing the interface between the membrane and the channel. In the case of Kv channels, the S1–S4 domains are embedded in the lipid bilayer and interact intimately with lipids (48, 51, 52) and modification in the lipid composition can dramatically alter gating of the channel (48, 53–56). In one study on the gating of the Kv2.1/Kv1.2 paddle chimera (53), the tarantula toxin VSTx1 was proposed to inhibit Kv channels by modifying the forces acting between the channel and the membrane. Although these studies implicate a key role for the membrane in the activity of Kv and stretch-activated channels, and for the action of tarantula toxins, the influence of the toxin on membrane structure and dynamics have not been directly examined. The goal of the present study was to localize a tarantula toxin in membranes using structural approaches and to investigate the influence of the toxin on the structure of the lipid bilayer.