Like most invasive species, cane toads have attracted less research in their native range than in invaded areas. We radio-tracked 34 free-ranging toads in French Guiana, a source region for most invasive populations, across two coastal and two rainforest sites. Coastal toads generally sheltered in pools of fresh or brackish water but nocturnally foraged on beaches, whereas rainforest toads sheltered in forested habitats, moving into open areas at night. Over five days of monitoring, native toads frequently re-used shelters and moved little between days (means = 10–63 m/site) compared to invasion-front toads from Australia (~ 250 m). Larger toads moved less between days, but displaced in more consistent directions. At night, foraging toads travelled up to 200 m before returning to shelters. Foraging distance was related to body condition at coastal sites, with toads in poorer body condition travelling farther. Rain increased the probability of coastal toads sheltering in the dry habitats where they foraged. Dispersal and rainfall were lower at coastal sites, and the strategies utilized by coastal toads to minimize water loss resembled those of invasive toads in semi-desert habitats. This global invader already exhibits a broad environmental niche and substantial behavioural flexibility within its native range. Unsurprisingly, most of that research has been conducted in places where the species involved causes problems—that is, in regions that have been invaded—rather than in the species’ native range5. The majority of research on the biology of “pest species” in their countries of origin has been to identify natural enemies  that might be used for biocontrol in the invaded regions6. As a result, most of what we know about the biological attributes of invasive species tends to be derived from the invaded rather than native range of those taxa.The ecological and economic impacts of invasive species can be massiveRhinella marina), a large bufonid anuran native to South America but translocated to many countries around the world in a futile attempt at biocontrol of insect pests in commercial plantations8. This toxic toad’s devastating impact on Australian predators has stimulated hundreds of studies, including several radiotelemetry-based projects on the movement patterns of free-ranging toads within Australia26 and one study on invasive populations in Hawai’i27. In combination, that work has documented strong geographic variation in spatial ecology within the toads’ extensive introduced range. In particular, the extremely high dispersal abilities of Australian cane toads from invasion-front populations relative to long-established populations have highlighted how the rapid evolution of dispersal abilities can accelerate the invasion process21. However, such a high level of variation among populations makes it difficult to clarify the ancestral condition of movement patterns for the species. Toads were serially translocated from their native range in the Guianas, to the Caribbean, then to Hawai’i (in 1932) before being brought to Australia in 19357. Thus, the attributes of modern-day Hawai’ian toads27 may reflect adaptations to island life rather than traits present in the original colonists from South America, much as dispersal rates in Australia have evolved during the invasion of the Australian continent. As a result, the best way to identify the likely ancestral condition for spatial ecology of invasive populations of cane toads is to study the species in its native range—preferably, the region from which toads were taken for those international translocations. We have conducted such a study.That geographic focus holds true for cane toads  is a large bufonid anuran species that has been introduced to > 100 countries and islands worldwide7. All of the introduced populations of this global invader across the Caribbean and Pacific were originally sourced from the Guianas 7, and mitochondrial studies identify French Guiana as the source of the toads that were brought to Australia via Hawai’i and Puerto Rico28. The native range of this species is in areas east of the Andes in South America; west-of-Andes populations and Central American populations previously referred to this species have now been allocated to a separate taxon, R. horribilis29. Cane toads are generally diurnal during postmetamorphic life, but shift to primarily nocturnal activity as they grow larger30. They typically spend the day inside damp terrestrial retreat-sites, the use of which reduces water loss32, but emerge at night to forage for prey . Toads lack specialized physiological adaptations for reducing water loss in arid environments, but use behavioural mechanisms to maintain moisture34; in dry weather cane toads need to rehydrate frequently and usually do so by lying in pools or on damp ground35. Although the permeable skin of amphibians also renders them sensitive to osmotically stressful environments, cane toads have physiological strategies for regulating salts and are relatively tolerant of saltwater36. In seasonally arid regions cane toads are active primarily during wetter times of year, but movement and breeding occurs year-round in suitably warm damp habitats38. Toads generally avoid breeding in waterbodies surrounded by dense vegetation, instead preferentially breeding in temporary pools in open habitats39.40. To include that variation, we tracked toads at two sites in coastal beach habitats . Our telemetry was conducted in August  and September (Regina Wash) 2017. These sites are described briefly below, with additional details available in the Supplementary Methods.In French Guiana > 95% of the land area is forested, but cane toads occupy a range of habitat types from dense rainforest to coastal beachesats Fig.  and two ats Fig. . The cli2 pool surrounded by sand and, along one edge, tall, dense grass. A second pool ~ 250 m to the east was also used by toads, as were other, smaller sources of fresh or brackish water . Six male toads and four female toads were radio-tracked at this site.Gosselin is a sandy beach in Rémire-Montjoly backed by a narrow band of forest. This site contains a 1500 m2 rock pool 50 m from the beach. Many toads  were found gathered around this pool . Four male toads and six female toads were radio-tracked at this site.The eastern edge of this beach (~ 3 km N of Gosselin) abuts a rock outcrop with ephemeral pools, the largest of which is a 17 mThis tropical rainforest site is ~ 28 km S of Gosselin and surrounds a permanent pond in the Kaw forest. Three male toads and one female were radio-tracked at this site.2) within the tropical rainforest is located ~ 30 km S of the Kaw site and surrounds a slowly flowing stream. Ten male toads were radio-tracked at this site. Only one female was observed at this site, and this occurred after the tracking period.This open wash  to cotton twine waist-belts, and re-released each animal at its point of capture within 10 min of capture. The majority of toads (26) were initially captured at night. However, we also sought to determine whether nocturnal collection biased our movement estimates towards active individuals. We therefore also located eight toads within their diurnal shelters and attached their radio-transmitters during the day. Locating new toads during the day was not possible at the rainforest sites, where toads were dispersed throughout the forest, so seven of the eight “diurnal start” toads were from coastal habitats. The only “diurnal start” toad from the rainforest was the only rainforest female ever observed during the tracking period; a large 690 g (210 mm SVL) toad discovered while searching pondside burrows for one of the tracked male toads. However, this female was missing a leg . As this could have biased her movements, we describe these movements in Fig. We captured 34 adult toads across the four sites, sexed, and weighed them. Sex was determined from morphology  and the male-specific “release call”19, so we noted whether or not it had rained in the 24 h preceding each capture event. Toad positions in the absence and presence of recent rain were not available at Gosselin, as it did not rain at Gosselin during the tracking period, but it did rain at all other sites .After equipping them with radio-transmitters, we located each animal daily , at which time we took a GPS reading each day for 5 days. During that period, the toads were also located on 3 nights . Our estimates of how far toads travelled from their diurnal shelter during their nocturnal expeditions are therefore conservative, as they are based on a single point per night. Sunset occurred at 1840 h during the tracking period, and toads typically began to emerge at 1900 h. The nocturnal locations of each toad were then taken between 2030 and 0130 h. At the end of the 5-day period, the toads were recaptured, and their radio-transmitters were removed. To determine whether body size had influenced toad movements we also measured SVL at this time. Rain can also affect toad movements43. To calculate the body condition of the tracked toads we obtained body mass (g) and SVL (mm) data from 142 male and 98 female toads from across northern French Guiana40. Using these data we calculated sex-specific relationships between mass and SVL by plotting ln(mass) against ln(SVL). For males this relationship was y = 2.868x − 8.739 (Pearson r = 0.9692), and for females it was y = 2.985x − 9.292 (Pearson r = 0.9696). We then used these values to calculate the scaled mass index for each tracked toad44. This index uses the formula Mi * (L0/Li)^bSMA, to compute the mass each toad would have if they all had the same body length (L0). Here, Mi and Li are the mass and SVL of the individual, L0 was taken to be the mean SVL of the tracked toads (120 mm), and bSMA is the slope of the sex-specific ln–ln regression of mass by SVL divided by the Pearson correlation coefficient .Behaviour can also be state-dependent; for example, body condition can influence foraging decisions(i)Distance between days . The distance between a toad’s position and its position on the previous day .(ii)Probability of changing shelters . Whether or not a toad had moved > 5 m from the shelter it used the day before .(iii)Distance between shelters . If a toad had left its previous diurnal shelter, the distance between the old shelter and the new one .(iv)Number of diurnal shelters used (integer between 1 and 5). Toads sometimes left a diurnal shelter for a new one, only to return at a later date; we therefore also quantified the total number of diurnal shelters used by each toad over the 5-day period, using a 5 m cut-off to determine whether the toad had returned to the same shelter .(v)Total dispersal distance . The distance between the toad’s original shelter site on Day 1 and its diurnal shelter on Day 5 .(vi)Path straightness (proportion). Calculated as the ‘total dispersal distance’ divided by the sum of the distances between days ; this proportion was logit transformed prior to analysis.(vii)Shelter type . Each shelter site was categorized according to whether or not standing water was present; at shelter sites where water was classified as present, the toad was either sheltering in the water or within 5 m of it . This variable was created to account for the possibility that the importance of standing water could vary in response to rain or between coastal and rainforest sites. For example, potential effects of rain on toad movements could have been obscured by the fact that, if a toad moved away from a shelter adjacent to standing water during rain, it might subsequently move back after the rain stopped Distance from diurnal shelter . The distance between the toad’s diurnal refugium and its nocturnal location; where the location of the refugia used before and after that night were both known, we took the mean of these two distances .(ii)Probability of foraging . Whether or not the toad had emerged from its diurnal shelter to forage .(iii)Foraging distance . If a toad had emerged from its diurnal shelter, the distance between the diurnal shelter and where the animal was found foraging .Note that we refer to nocturnal movements as “foraging movements” for simplicity. However, breeding activity occurs year-round in cane toads . Therefore, breeding activity could also have contributed to these movements. Although tadpoles were present and breeding occurs at all of the study sites, we never observed breeding or new egg clutches during the tracking period .We used data on the nocturnal position of each toad to calculate:Two toads at Montjoly lost their transmitters before the end of the tracking period; a female M10; 115 g) that dropped her transmitter on the final day, such that we obtained 5 days and 2 nights of data, and a female  for which we only obtained 2 days and 2 nights of data. This latter female (M4) was excluded from analyses of the number of diurnal shelters used, total dispersal distance, and path straightness. Another female toad entered private property on the final day of the tracking period, and we were only able to estimate her position within 10 m for Day 5 .We first used data from the coastal toads to determine whether initially capturing toads at night (rather than during the day) biased our movement estimates. We therefore used separate models to analyse all of the movement parameters outlined above as responses to the fixed effect of initial capture method. To account for variation between sites and individuals, site and individual ID were included in these models as nested random effects. Upon finding that locating toads at night did not have pronounced effects on our movement estimates, we excluded this factor from all subsequent models.We then used data from all of the sites  to test for site-specific differences in toad movement or shelter selection. For these models, each parameter outlined above was analysed as a response to the fixed effect of site. Because toads are nocturnally active, their nocturnal position might vary depending on when they were captured. To control for this factor, we also included time in analyses of nocturnal position . Rain could also affect toad movements, or have effects that vary between sites. We therefore re-ran all of these models, but with the added predictor of whether or not it had rained in the previous 24 h , as well as the interaction between site and rainfall.To determine whether variation between individuals affected toad movements as well as whether the effects of individual variation differed between sites, we retained the fixed effect of site in each model  and added individual-level predictors to each model. These individual-level predictors included SVL (continuous: mm), body condition , and sex . We also included all first-order interactions. Because all of the toads tracked in the rainforest were males, we first excluded the rainforest sites from all models and looked for an effect of these individual-level predictors on toad movement within coastal sites alone. For models where a significant effect of sex was found, we retained sex as a predictor in subsequent analyses in which data from all four sites were included. However, if no effect of sex was found within coastal sites, we omitted sex as a predictor in subsequent analyses of the full dataset. This was done to avoid confounding any effects of sex with those of site. .46. In all models, distances were ln-transformed (ln[y + 1]) prior to analysis to meet homoscedasticity assumptions. Any continuous response variables for which multiple measures were taken per toad were analysed as mixed-effect models (package nlme:lme47). Individual ID was included in these models as a random effect to account for variation between individual toads. Continuous measures for which there was only one final value per toad were instead analysed via linear regression using the lm procedure. Binomial responses were analysed via logistic regression (package lme4:glmer48), with individual ID included as a random effect. The number of diurnal shelters used per toad was analysed using a quasipoisson model . For models that included multiple predictors , we used a stepwise removal procedure to eliminate non-significant predictors from the model, starting with non-significant interactions. We used p = 0.1 as the threshold for elimination; therefore, the final models only included variables for which p < 0.1 (though non-significant main effects were retained if they were involved in a significant interaction). All models were initially assessed using the anova function . Where significant effects were identified we used Tukey post-hoc tests (package emmeans50) to conduct pairwise comparisons and identify significant differences.All analyses were conducted in RAt all four sites our radio-tracked toads typically remained within shelter-sites by day, and moved at night. The spatial ecology of these toads varied among sites Figs. ,  2. Thep = 0.172), nor did night-captured toads move significantly farther from their diurnal shelters during the night than day-captured toads . Indeed, the only significant difference between toads captured during the day and those captured at night was that those captured at night moved farther between diurnal shelters . As toads that sheltered in the sand moved more between diurnal shelters . Montjoly Beach toads were also marginally less likely to change shelters than were toads at Regina Wash (p = 0.0528). There was also an effect of site on the distance between shelter sites  or Regina Wash (p = 0.0002). As a result, the distances moved between days also varied by site ; toads at Gosselin moved less than toads at Regina Wash (p = 0.0369) and tended to move less than those at Montjoly (p = 0.0940). The number of independent shelter sites a toad used over the 5-day period also varied between sites  and toads from Montjoly using fewer shelters than those from Regina Wash (p = 0.0474). However, the odds that a toad would shelter near water did not vary significantly among sites . Total dispersal distance by the conclusion of the study period  also differed among sites ; toads at Gosselin did not disperse as far as those at Regina Wash (p = 0.010) and tended to disperse less than those at Kaw (p = 0.0559). However, path straightness did not vary significantly among sites .The probability that a toad would change shelter sites varied among sites  or distance from the diurnal shelter , foraging distances did vary between sites  and tended to travel farther than toads at Gosselin (p = 0.095). There was also an effect of time on foraging position; toads located later in the night were less likely to be outside of their diurnal shelter , were closer to their diurnal shelters than were toads located earlier in the night , and had reduced foraging distances .Foraging movements also varied by site. Although there was no significant effect of site on the probability that toads would emerge to forage . At Montjoly, toads were less likely to shelter near water following rain, such that the relative odds of sheltering near water when it had not rained were 86.6 times those when it rained . At Regina Wash, rain did not affect shelter preference . We excluded Kaw from this analysis due to the low sample size combined with a lack of variation in toad position, which prevented a good model fit.  In contrast, rain did not significantly affect the distance moved between days, the probability of changing diurnal shelters, or the distance between consecutive shelter sites, nor were there significant interactions between site and rainfall for these variables . Rain did not significantly affect nocturnal positions at Regina Wash .The type of diurnal shelter site a toad selected  was affected by a significant interaction between site and rainfall . There were no significant effects of site, SVL, or body condition on these odds. Once coastal toads had emerged, the distance they travelled was negatively associated with their body condition , with toads in poor body condition travelling farther during their foraging expeditions ; larger toads travelled farther from their diurnal shelters to forage at Montjoly, but not at Gosselin . There was also a significant interaction between time and site in this model ; toads were found farther from their diurnal shelters earlier in the night at Montjoly (p = 0.031), but there was no effect of time on nocturnal position at Gosselin (p = 0.829). In contrast to these nocturnal movements, which were apparently affected by variation among individuals, the only factor that significantly influenced coastal movements between days and the probability of changing shelters was site (see previous section for model outputs), although coastal toads in poorer body condition tended to travel farther when they moved to a new diurnal shelter .Because we did not detect females at our rainforest sites during the tracking period, we first analysed the effects of individual-level predictors within the coastal sites alone. This allowed us to determine whether sex affected movement by comparing toads of different sexes from the same site. The only parameter significantly affected by sex was the probability of emerging to forage; within coastal sites, the odds that a male toad would emerge were 7.37 times those of a female . To avoid confounding the influences of site and sex, we omitted sex from all models in which no sex effect had been detected at coastal sites . Across sites, the distance travelled by toads that had emerged to forage was affected by a significant interaction between site and SVL , as well as site and body condition . Unlike at coastal sites (see above), there were no significant effects of size or body condition on foraging distance at rainforest sites . Distance from the diurnal shelter was not related to any of the individual-level characteristics measured .When potential effects of individual variation on the probability of emerging to forage were considered across all four sites, sex was the only significant predictor; overall, the odds that a male would emerge to forage were 5.48 times those of a female  and site . Larger toads were less likely to move to a new shelter , toads at Gosselin were less likely to change shelters than were toads at any other site (p < 0.05), and toads at Montjoly were less likely to change shelters than were toads at Regina Wash (p = 0.0029). However, no individual-level predictors were significantly associated with the distance between consecutive diurnal shelters, leaving only the site effect in the final model  and site , with smaller toads travelling farther between days  and toads at Gosselin staying closer to their previous shelter than did toads at any other site . Path straightness was also associated with SVL ; the movements of larger toads were straighter  and site . Toads in better body condition dispersed farther . When body condition was included in the model, dispersal was significantly lower at both of the coastal sites than either rainforest site , but there was no significant difference between sites of the same habitat type . However, note that we lacked data on body size for the toad that dispersed the farthest at Montjoly , so this toad was excluded from models that included body condition.The probability that a toad would change diurnal shelters was affected by both SVL  indicate that diurnal foraging sometimes occurs within the native range as well as invaded regions, though such behaviour is relatively rare. Overall, movements varied among sites as well as among individuals, indicating that toads exhibit substantial behavioural flexibility in their native range; these toads not only adjusted their movement patterns in response to environmental conditions, but also exhibited environment-dependent flexibility in response to individual-level variation in morphology or state.Broadly, activity patterns of cane toads in their native range resemble those in the areas to which this species has been translocated. Adult toads are largely inactive by day, although occasional individuals forage during daylight hours55). For example, a trend for males to move further at night was seen in toads spool-tracked in Hawai’i27, where males travelled farther than females at certain sites in order to reach their breeding ponds. . Ultimately, these nocturnal foraging excursions took the animals up to 200 m from their diurnal retreats, with foraging toads showing a mean displacement of 56.4 m . These nocturnal movements are similar to those recorded in their invasive range in Hawai’i, where toads were found to have a mean displacement from their diurnal shelter of 27.6 m 27.Although our toads were generally sedentary by day, they regularly emerged after nightfall and moved to places where they could access resources such as food, water, or mates. Male toads were more likely to emerge, which may partially reflect differences in breeding behaviour; adult males can spend long periods in waterside habitats (requiring migration to and from the pond) whereas females visit ponds less often , when larger individuals moved, they did so in straighter paths—that is, a more consistent direction from one day to the next. Interestingly, studies on invasive toads in Australia have revealed inter-population (geographic) differences in path straightness, and significant heritability of this trait13. Hence, a trait that is linked to body size within the native range has evolved to differ geographically (but not related to body size) within the species’ invasive range. That evolved shift contributes to the faster dispersal of invasive toads within Australia, a target of selection61, suggesting that evolution has taken a dispersal-relevant trait (path straightness) that showed pre-existing variation, and changed the distribution of that trait among rather than within populations. Such a decoupling of pre-existing variation to morphology may provide a rapid way for selection to generate novel trait distributions.Because toads often move extensively at night, studies that rely upon locating toads during daylight hours only, when the animals are ensconced in their retreats, can substantially underestimate the total distances moved by individuals on a daily or weekly basis. However, daily checks of shelter sites are well-suited to measuring overall rates of displacement, a critical variable for determining rates of geographic spread in invasive populations45. This shift is associated with increased desiccation risk during the dry season, coupled with greater predation risk and lower prey availability, all of which increase the risk of moving at this time45. Similarly, toads in the Australian semi-desert sheltering away from water move rapidly through the landscape along relatively straight paths following rain, likely to increase their probability of encountering standing water . In contrast, semi-desert toads sheltering near water bodies are more mobile during dry periods, but move along meandering paths . Although seasonality is more extreme in the Australian savannah than in French Guiana12, rainfall may also affect toad movements at our coastal sites. Coastal toads were more likely to reuse shelters than were toads in the rainforest, and at Montjoly, where the preferred foraging area was > 100 m from the rock pool typically used for rehydration, rain had a pronounced effect on where toads sheltered . In addition, although breeding occurs year-round, influxes of toads to French Guiana breeding sites occurred in December, indicating that toads may be more mobile during the wet season. This reliance on limited freshwater resources during the drier months may also explain why toads lived at such high densities at coastal sites, despite the fact that these dense populations suffered from high levels of parasitism as a result40. Limited rainfall during the tracking period may therefore have restricted toad movements, especially at coastal sites, where seasonality is more pronounced and water availability was limited.Weather can also have pronounced effects on amphibian movements. In Australia, cane toads exhibit nomadic behaviour during the wet season, and greater fidelity to both their diurnal shelters and their site during the dry season63, we never observed toads moving along roads in French Guiana.) As a result, cane toads in our study only displaced 37.4 m (SE = 14.1 m) on average over the 5-day period, with a mean displacement between consecutive shelters of only 68.9 m (SE = 9.6 m). Although greater than the 11 m movements between consecutive shelters recorded in invasive Hawai’ian populations27 and similar to mean values recorded in established Australian populations , this value is substantially lower than the very high dispersal rates of invasion front toads in tropical Australia, where toads move ~ 270 m between consecutive diurnal shelters (and up to 1.8 km)20. This discrepancy indicates that these high dispersal rates along the invasion front are indeed derived traits for the species, whereas movements in Hawai’ian toads may actually be somewhat reduced relative to those in the native range.The variation in toad spatial ecology among sites, and its links to toad size, body condition, and weather, make it difficult to draw simple comparisons with studies of movement ecology in the species’ invasive range. Nonetheless, our study supports a basic (but hitherto untested) premise of previous interpretations of evolved shifts in movement ecology within invasive populations of cane toads. Within their native range , toads tend to be sedentary, with little net displacement over a 5-day period. Indeed, the net displacement of toads at the Gosselin beach site (~ 1.5 m over 5 days) is lower than reported in most other studies. In the course of our study, most toad movements were more meandering than directional, and only one toad actually left a study site; a female toad that dispersed from the Montjoly pool to a suburban ditch 420 m away . Toads from both coastal and rainforest habitats moved in meandering paths, such that path straightness did not differ significantly between habitat types . Whereas coastal toad dispersal was likely restricted by a lack of water during the study period, dispersal by rainforest toads may have been restricted by their nocturnal preference for open habitats for breeding and foraging activities. The availability of such open areas is limited in dense rainforest environments, and rainforest toads frequently returned to the same forest gaps at night. . Additionally, some strategies thought to be novel behavioural adaptations to the arid environments of Australia were also observed at coastal sites; for example, whereas toads generally spend the day in terrestrial shelters, moving to water to rehydrate nocturnally as necessary, toads in semi-arid environments have also been found to rehydrate in standing water during the day . We observed similar behaviours at our coastal sites, where a high proportion of toads diurnally sheltered partially immersed in standing water, rather than in terrestrial shelters  xeric environments, such as those they would subsequently encounter in Hawai’i and Australia. Given the higher densities and greater observability of toads in beach relative to forested habitats, as well as their proximity to the sailing ships used to export these toads from Cayenne, the individuals originally collected from French Guiana for translocation to sugarcane plantations in other countries were almost certainly from coastal habitats.  In the course of their invasion across tropical Australia, these pre-existing abilities have been fine-tuned by evolutionary processes; e.g., the capacity for long-distance dispersal exhibited facultatively by coastal native-range toads is now expressed constitutively in invasion-front individuals within Australia71. Future research may usefully explore the role of local adaptation and behavioural plasticity within the native range in preparing these toads to successfully invade and exploit such extreme habitats in their introduced range.Our study of native-range toads has shown great flexibility in movement patterns in response to local conditions. Toads in the native range exploit a wide range of habitats, including beach and rainforest sites otherwise typified by profoundly disparate faunal groups, exhibiting a level of habitat generalism that likely facilitated this toad’s success and subsequent colonization of a wide range of habitats in their introduced rangeIn summary, cane toads within their native range exploit a wide range of environments, and modify their behaviours to match the risks and opportunities presented by these different habitats. This highly adaptable species may have benefited from this broad ancestral niche, enabling the cane toad to become one of the most successful invasive species in the world.Supplementary Information.


Ice shelf fracture is responsible for roughly half of Antarctic ice mass loss in the form of calving and can weaken buttressing of upstream ice flow. Large uncertainties associated with the ice sheet response to climate variations are due to a poor understanding of these fracture processes and how to model them. Here, we address these problems by implementing an anisotropic, nonlocal integral formulation of creep damage within a large‐scale shallow‐shelf ice flow model. This model can be used to study the full evolution of fracture from initiation of crevassing to rifting that eventually causes tabular calving. While previous ice shelf fracture models have largely relied on simple expressions to estimate crevasse depths, our model parameterizes fracture as a progressive damage evolution process in three‐dimensions (3‐D). We also implement an efficient numerical framework based on the material point method, which avoids advection errors. Using an idealized marine ice sheet, we test the creep damage model and a crevasse‐depth based damage model, including a modified version of the latter that accounts for damage evolution due to necking and mass balance. We demonstrate that the creep damage model is best suited for capturing weakening and rifting over shorter (monthly/yearly) timescales, and that anisotropic damage reproduces typically observed fracture patterns better than isotropic damage. Because necking and mass balance can significantly influence damage on longer  timescales, we discuss the potential for a combined approach between models to best represent mechanical weakening and tabular calving within long‐term simulations. Our shallow‐shelf creep damage model can represent the full evolution of ice shelf fracture from crevasse initiation to tabular calvingStrongly anisotropic creep damage produces sharp, arcuate rift patterns more consistent with observations than isotropic creep damageThe zero‐stress damage model poorly captures rifting, but a modified form accounts for damage evolution due to mass balance and necking Approximately half of ice mass loss is attributed to fracture‐induced calving, while the other half is attributed to ocean‐driven basal melting , a two‐dimensional (2‐D) vertically integrated form of the incompressible Stokes equations  models. Crevasse/rift propagation in LEFM models is based on the stress intensity factor and fracture toughness of ice, which are inherently related to strain energy release rate (where rate indicates per unit crack length) and fracture energy , respectively. In the literature, analytical LEFM models were applied to estimate the penetration depth of crevasses  by weakening the material response =, i are the spatial indices and e^i are the orthonormal basis vectors. Second‐order tensors are similarly denoted as A=Aije^i⊗e^j, where ⊗ is the dyadic product of the Cartesian base vectors. We adopt Einstein's convention of summation that repeated indices imply summation. Principal values of A are written as Ai, where in this case, index i indicates principal components rather than Cartesian directions. Variables at time step m are indicated using the superscript Am. Constants used for the damage model are listed in Table We begin this section by briefly reviewing the SSA equations. Then, we present the creep damage model and its parameterization for the SSA. We use a mix of tensorial and indicial notation as needed for conciseness and clarity. Vectors are donated as  system x,x2,x3=, the seawater pressure is applied using a depth‐integrated Neumann boundary condition asσ is the Cauchy stress, n^ is the unit (outward) normal to the ice front, ρw is the seawater density, and b is the elevation of the ice draft  of the ice shelf/stream by reformulating Equations ε˙ij=12(∂vi∂xj+∂vj∂xi). Along with momentum balance equations in Equation a˙ (m a−1) is the sum of the basal and surface accumulation rates  and orthotropic damage . The other two principal axis lie in the horizontal x1−x2 plane, where we always ensure D1≥D2. Because vertical shear stress components are neglected in the SSA, the orthotropic damage tensor has only four non‐zero components D11,D22, D33, and D12 that need to be determined.We implement the anisotropic creep damage model originally proposed by Murakami , Murakamσij , as for its undamaged state under the effective stress, σ∼ij . A linear transformation between the two stress spaces that ensures the symmetry of the effective stress tensor can be written as , and f is the dynamic damage evolution function given as , the effective von Mises stress (weighted by β), and the trace of the effective stress (weighted by λ = 1−α−β), which is three times the volumetric stress. The Hayhurst weights must satisfy the conditionIn Equation α = 0.21, β = 0.63, and λ = 0.16 as previously calibrated from laboratory data  is the eigenvector corresponding to the maximum effective principal stress, σ∼1, which we always assume lies within the horizontal x1−x2 plane to be consistent with crevasse formation along vertical planes. Operator Tr[⋅] denotes the trace. Although the exponent k∗ has been calibrated based on laboratory experimental data to be a function of the Cauchy stress  and one  direction). According to Equation D and σ∼ are always coaxial, the relationship between the principal components of the damage rate is controlled by the anisotropy parameter asIn the second and third terms of Equation D and σ∼ will cause damage accumulation to become more weighted toward D2 at the expense of D1. Misalignment can occur, for example, as a rift develops and causes the orientations of principal stresses to change downstream. Note that growth of D3 is probably not physically motivated because it corresponds to development of horizontal crevassing, rather than vertical crevassing. From Equations x1−x2 plane, it is evident that damage will accumulate on D3 if γ<1, but not if damage is fully anisotropic (γ=1). We test sensitivity to γ in Section Any misalignment between 2.4z according to the deviatoric stresses, a parameterized pressure, and the existing damage. The deviatoric stresses are obtained at vertical coordinate z using the nonlinearly viscous constitutive relation for ice flow ]ε˙e1−nn is the depth‐dependent form of Equation B, is dependent on temperature, T∗(z), which may be depth‐varying. The ice pressure used to derive the SSA is approximated using the hydrostatic assumption σD=σijD(z)−peff(z)δij. It may appear that this stress could then be converted to the needed effective stress using Equation σ∼ij=σ∼ijD−peffδij, where we define the effective deviatoric stress as . Lastly, we detail incorporation of the depth‐averaged damage variable into the SSA solution.3.11 continuous shape functions to ensure smooth transfers of stiffness as material points move between grid cells, and in the GIMPM, such shape functions are assembled by convolving linear grid functions with characteristic functions associated with each material point  due to frequent remeshing that may be required when modeling large‐deformation processes like ice shelf flow. While our creep damage model may be adopted for any flow‐modeling framework, we implement it here within our GIMPM‐SSA code to avoid these diffusion errors  term in Equation Within the GIMPM‐SSA framework, we track damage and any other 3‐D fields, such as temperature, upon a series of vertical layers assigned to each material point. For mesh‐based methods, the vertical layers could be assigned to nodes or quadrature points instead. For the simulations in this study, we always maintain an even distribution of layers between the local ice base and surface elevations, which is possible because we do not incorporate mass balance processes such as surface and basal melt, or infill of crevasses with snow at the surface or marine ice at the base. Therefore, the only vertical motion of layers here is driven by changes in ice thickness from extension or compression, owing to the 3.2ΔDm is the damage increment over a time step and may be expressed in local or nonlocal form. For each material point layer, the local damage increment, ΔlocDm, is found by integrating the damage evolution rate, D˙m, over the length of the time step Δt using the Runge‐Kutta‐Merson (RKM) method as detailed in Ling et al. ] can vary with depth, it must be included in Equation D(z) to properly capture the combined effect of damage and thermal softening on the depth‐averaged viscosity of ice  indicates the maximum time step that satisfies the Courant‐Friedrichs‐Lewy (CFL) condition with constant δ3≤1. Here, the time step is almost always restricted by damage rather than the CFL condition, and consequently, dD¯max≈δ2 each computational cycle. The typical time increment varies based on the chosen damage parameters, but in all the simulations in this study, it is on the order of days for subcritical damage accumulation to hours during rapid rift propagation.If 3.4Implementing nonlocal damage is motivated by both physical and numerical considerations. Physically, the progressive accumulation of microcracks that damage mechanics describes is distributed over a characteristic length scale in quasi‐brittle materials like glacier ice Bazant, . NumericΔDm(xm), is calculated asN is the number of material points, x^jm, positioned within a characteristic length, lc, of xm at time step m. The weight function, ϕ is a Gaussian curve given asHere, we apply the nonlocal scheme within each layer of neighboring material points. For example, local damage of the second layer of a material point is only reweighted according to the local damage of the second layer from surrounding material points, but not the layer above or below it. The nonlocal damage increment, lc, should reflect the size of the fracture process zone and should be set so that the number of neighboring material points, j, is large enough to alleviate grid dependence 〈Dmax〉, unless this update reduces their values. This rupture criterion accounts for the transition from subcritical damage, associated with the gradual nucleation and growth of microcracks and microvoids, to rapid critical crack growth, or rupture of a macrocrack, once crack density reaches a critical value. The value for the critical damage, Dcr, can be estimated from uniaxial tension data  and GIMPM (SJp) shape functions at a material point, where ϕIp=ϕI(xp) and SJp=SJ(xp). After the SSA is solved, the computational cycle for the GIMPM then continues as described in Part I ) to perform the velocity and position updates, an algorithm that eliminates potential noise or overdamping associated with simpler update schemes , the bed has a retrograde slope. The higher sidewalls of the bedrock trough result in thin, lateral protrusions of grounded ice that define the maximum longitudinal extent of the grounding line at x1∼537 km. All floating ice upstream of this point constitutes a laterally supported shelf ice, whereas all ice downstream constitutes an unsupported floating ice tongue. The trajectories overlaying Figure D¯1 direction.The MISMIP+ geometry is rectangular. In the longitudinal direction, the domain spans from an ice divide at Starting from a thin slab of ice defined over the domain, we grew the system to steady state using the given MISMIP+ ice flow parameters and accumulation rate and a modified Coulomb law for friction  0.06 years and (b) 0.2 years. Nearly identical early damage patterns develop for the other values of γ tested  can be considered symmetry boundaries because the normal velocities are set to zero, so that the rifts can be considered to have initiated from the center of small ice shelves. While rifts typically initiate at grounded margins, rift initiation from the center of ice shelves has occurred, for example, at Pine Island Glacier in Antarctica . However, the jump in damage induced by setting D¯cr lower than D¯max yields more visually distinct rifting, and is likely physically justified because highly damaged shelf ice may experience vertical shear stresses not accounted for in the SSA , where B is calculated from temperature according to the standard Arrhenius relation as given in Zwinger et al. . However, unlike in the creep damage case, this region does not develop into a sharp rift that propagates across the shelf to result in a calving event. Over time, the zero‐stress damage field mostly evolves from its initial configuration through advection, as evident following the black tracer particle in Figures In the first experiment, we run the zero‐stress damage model as given for 30 years. No critical rupture scheme is enforced. Note that this test was performed previously using the isotropic form over a longer time scale using the MISMIP+ geometry with the finite volume ice flow model Berkeley Ice Sheet Initiative for CLimate ExtremeS; Sun et al., D^cr=0.7 and D^cr=0.6 for isotropic and anisotropic damage, respectively. While the choice of these values is rather ad hoc, they allow for the formation of sharp rifts. The critical rupture criterion is enforced after each combined zero‐stress damage and SSA solution. At the first time step, rupture occurs near the shear margins where D^1>D^cr, and the resulting high stresses allow rifts to propagate across the domain to calve tabular icebergs. The final maximum principal zero‐stress damage fields are given in Figures In the second zero‐stress damage experiment, we rerun the MISMIP+ experiment, but encourage rifting to initiate by setting critical damage values of δ2=0.05  It is formulated on the basis of estimating depths of closely spaced crevasses, which is not appropriate for modeling rifting or ice shelf crevasses, which tend to be widely spaced; (b) the zero‐stress damage evolution exhibits grid dependence, which especially affects both the path and rate of rift propagation; and (c) deviatoric stresses are assumed to be constant with depth, so that variations in temperature and damage with depth are not accounted for, which typically results in an overestimation of basal crevassing. We further caution that there may be additional numerical challenges when working with the zero‐stress damage model concerning stability and time‐stepping. To ensure stability in the zero‐stress damage response, we implement a similar adaptive time‐stepping scheme as in the creep damage model, where time steps are adjusted with the goal of achieving a maximum change in 2‐D damage between time steps of  Section . This sc4.4A drawback of both the creep and zero‐stress damage models as tested above is that they do not account for the potential impact that processes associated with necking and mass balance may have on damage evolution. In Section S0 in the necking model (see Section S0>1), so that damage healing occurs. Due to this trend of accelerated damage in some areas, with healing in the immediately surrounding areas, rifting in the modified isotropic case develops into sharper patterns than in the standard isotropic case. However, rifts still do not propagate across the center of the shelf. Sharper damage patterns also occur in the modified anisotropic case as compared to the standard anisotropic case, especially near the lateral boundaries, though rifting is similarly minimal in both cases. Interestingly, the configuration of fully damaged material points in the isotropic modified zero stress simulation , and interstitial liquid water content. Pralong and Funk  in the Hayhurst criterion, assuming the weight associated with effective pressure (λ) is small. Aside from this, we recently developed a two‐parameter phase field damage model , and improved representation of rift boundary conditions, in our future work we intend to combine the necking/mass‐balance and creep damage models for an ideal representation of ice‐shelf fracture on decadal timescales.By testing the creep damage model on an idealized marine ice sheet, we find that large‐scale damage of ice should be treated as fully anisotropic. Anisotropic creep damage yields sharper, more arcuate rifting and crevasse patterns that are more consistent with observations. In addition, anisotropic nonlocal damage is thermodynamically consistent (Pralong et al., Supporting Information S1Click here for additional data file.


They serve a myriad of functions ranging from energy and metabolite exchange to highly debatable roles in apoptosis. Their role in molecular transport puts them on the center stage as communicators between cytoplasmic and mitochondrial signaling events. Beyond their general role as interchangeable pores, members of this family may exhibit specific functions. Even after nearly five decades of their discovery, their role in plant systems is still a new and rapidly emerging field. The information on biochemical regulation of VDACs is limited. Various interacting proteins and post-translational modifications (PTMs) modulate VDAC functions, amongst these, phosphorylation is quite noticeable. In this review, we have tried to give a glimpse of the recent advancements in the biochemical/interactional regulation of plant VDACs. We also cover a critical analysis on the importance of PTMs in the functional regulation of VDACs. Besides, the review also encompasses numerous studies which can identify VDACs as a connecting link between Ca Oryza sativa and Nicotiana tabacum have three VDAC isoforms , first reported by isoforms , Arabidoisoforms , and Medve VDACs . Their vve VDACs , plant V2+) and reactive oxygen species (ROS) signaling may crosstalk in the cell. This information should help us understand the functional significance of VDACs and explore the possibility that VDACs might have a role in inter-organellar communication for major cellular processes in plants.Voltage-dependent anion channel, being one of the major membrane protein in the OMM, can be modulated by factors like chemical compounds and other proteins or cellular metabolites. In this review, we provide an update on the biochemical regulation of VDACs and their impact on the physiological function of VDACs, with a particular emphasis on the plant system. Post-translational modifications (PTMs) are essential mechanisms that diversify protein functions . SeveralIn the following sections, we discuss the various factors that influence the biochemical, structural, and functional aspects of VDACs.Curcumin longa, is known for its anti-cancer properties  has been shown to modulate rat brain-VDAC1 through an electrophysiological approach. The conductance of VDAC was shown to increase upon H2O2 treatment, and its activity was restored by curcumin. The binding site of H2O2 on the channel identified through in silico molecular docking indicates a possibility of direct interaction between VDAC and H2O2 (Centella asiatica (CA) quench ROS during ischemia-reperfusion injury by protecting N2a cells due to antioxidant property. However, leaf extracts from CA failed to prevent IR injury in VDAC1 knocked down-N2a cells, which indicates the involvement of VDACs in the protective effect of CA. This was followed by reconstitution of hVDAC1 in the lipid bilayer membrane, CA treatment resulted in its increased single-channel conductance and stabilized the open state of hVDAC1  and five neighboring pore residues . Steric interaction and hydrogen bonds stabilized the binding, with Thr9, Asp12, and His184 contributing most highest in the binding , resulting in healthy cells and leaves in plants. The expression ratio of VDAC–hexokinase and their interactions are essential in cell death pathways in plants , and ubiquitination. These modifications on VDACs will be discussed in the following sections.In vitro and in silico analyses revealed many phosphorylation sites in isoforms of VDAC, which can play an important role in studying the function of VDAC in the near future. If we look into the studies performed till date to understand the phosphorylation events of VDAC, we can divide them into three main classes: (a) studies that proved VDAC can be phosphorylated which alters the channel properties, (b) studies where the whole proteome was examined to understand the PTM and (c) where the specific study on VDAC phosphorylation site was performed, followed by identification and validation.Voltage-dependent anion channels undergo phosphorylation for modulation of their channel properties. To understand the biological significance of the phosphorylation event(s) in VDAC, it is necessary to identify the phosphorylation sites in VDACs, the protein mediating the phosphorylation, the physiological stimulus mediating the phosphorylation and the resultant change in VDAC (in particular) and physiology in general. in vitro kinase assays and followed it up with investigation of the changes in channel dynamics of VDAC due to phosphorylation in real time, using reconstitution in lipid membrane bilayer (in vitro phosphorylation of mouse VDAC1 by protein kinase C (PKC). These studies gave some critical shreds of evidence on the PKC and VDAC interaction and probable regulation of mitochondrial permeability transition pore by PKC, but in terms of phosphorylation, the information did not yield more knowledge beyond just the event.The first group majorly consists of reports from the Ghosh laboratory . They ha bilayer . Their pThe second group of studies provided an improvement from the first group by indicating the exact site of phosphorylation in VDAC  under a certain stimulus (or a disease). Tyr phosphorylation of pig VDAC1 and VDAC2 were reported using immunoblotting followed by mass spectrometry (MS) under hypoxia, with no specific site identified . Using ain vitro kinase assay showed that this particular variant was not phosphorylated by GSK-3 (in vitro kinase assay. This particular study used MS only to identify the VDAC isoform and not the phosphorylation site(s). But there are more Thr residues available  that could be phosphorylated and need further validation. Nevertheless, Thr51 in human VDAC1 (or VDAC3) is a target of GSK-3 for modulating the channel properties. in vitro kinase assays. The Nek1 is regulated by TKL1 (Tousled-like kinase), which is also involved in chromatin assembly and DNA repair mechanism  the phosphorylation of VDAC by glycogen synthase kinase-3 (GSK-3), (2) the phosphorylation of VDAC during endostatin treatment, and (3) phosphorylation of VDAC by NIMA-related protein kinase 1 (Nek1). VDAC can be phosphorylated by Akt and GSK-3  . Pastoriby GSK-3 . By analechanism . On inveechanism .in vivo and in vitro experiments)  .in vivo research. Although VDACs have similar sequences, there are abundant proofs that phosphorylation sites can differ based on species and kinase involved. We believe that the best approach to deal with this problem is -after the identification of a kinase partner of VDAC, an in vitro kinase assay is to be followed to first answer the question of phosphorylation-based regulation. This should be then followed by identification of sites by MS following either perturbing the organism with the stimulus that is being investigated or alternatively, by MS after an in vitro kinase assay. The results obtained from the MS analysis should be used to create either phospho-mimic or phosphor-mutant, and then one can proceed to analyze the channel conductance of these variants. These approaches should give us better results for the identification of actual site(s). Distressfully, the data on plant VDAC phosphorylation is still lacking.The phosphorylation sites detailed in the above paragraphs  (in vitro nitrosation using PAPA NON-Oate (PPN) at 25 μM showed that the conductance is reduced and the dwell time of rat VDAC is increased in the closed state. Also, at PPN100, the conductance was reported to be half of that is present in wild type (wt)  . Moreoveype (wt) . The modype (wt) . VDACs sype (wt) . Interfeype (wt) . It direype (wt) . The modype (wt) . It eithype (wt) . Howeverype (wt) . Ox-PTMsype (wt) . Cysteinype (wt) . There aype (wt) . A conseype (wt) . Howeverype (wt) . In the ype (wt) . hVDAC3 ype (wt) . Functioan VDAC3 . Cys 2, henotype . Cys modhenotype . OverexpR injury . VDACs eR injury . It is fR injury . After m2+ is an important second messenger and plays a crucial role in signaling in all eukaryotes , and endoplasmic reticulum (ER). However, organelles such as mitochondria can also regulate Ca2+ signals , from the suspended medium during electron transport  to Inositol 1,4,5-triphosphate receptor (IP3R: on ER) and is a determining factor for the interaction as its absence abolished the stimulatory effect. This direct enhancement of Ca2+ accumulation in mitochondria forms a molecular bridge , generating ROS signals under physiological conditions depending on tissue specificity. This signaling aspect depends on [Ca2+] threshold, which, when overcome, results in detrimental mROS levels, compromising mitochondrial bioenergetics and cellular functioning , thus, blocking oxidative stress damage responsible for cardiac dysfunction (2+-induced mitochondrial permeability transition, causing the induced opening of permeability transition pores of high conductance in IMM due to excessive Ca2+ uptake (2+ but we still lag in understanding the initiation of this loop. It is still emerging if mitochondrial Ca2+ signal generates a ROS signaling event or flux of ROS via mitochondrial stimulation of Ca2+ signaling in cytosol/mitochondria. It is still unclear whether the process is only facilitated by VDACs majorly and independently or does it need a complex to regulate the signaling. We expect studies in the future to depict the cytosolic and mitochondrial Ca2+ dynamics in the absence of VDAC in plants that can clarify the involvement of VDAC in the process. The simultaneous imaging of ROS and Ca2+ in reference to VDACs and their patterns in real-time also demand attention. Nevertheless, at present, it is an open question that needs to be investigated in plants. We also expect research in the future that can solve the mode of regulation in VDACs in these signaling events. It is a challenging opportunity to elucidate the entire signaling mechanism, including Ca2+-ROS homeostasis, in the context of cell biology. The interconnection between pathways and the role of VDACs as the central node as shown in Antioxidant systems in mitochondria regulate the maintenance of ROS at physiological levels through mitochondrial metabolism. ROS imbalances in mitochondria, either through overproduction or impaired antioxidant system, lead to mitochondrial dysfunction and induction of the apoptotic cascade. Such ROS imbalances in pathological conditions exhibit a Cachondria . Since mchondria . Mitochoctioning . The recoverload . VDAC mahaperone , indicat in vivo . In lungoduction . The genfunction . The clo+ uptake . The not2+ and ROS is also mediated by VDACs, and hence, they can be a Ca2+-ROS connecting link. However, many interesting questions remain to be addressed. At present, the ongoing research on plant VDACs faces many challenges, with burning questions and controversies. Its structure, isoforms in plants, role in apoptosis, relation with PCD and the VDAC interactome are gray areas. The active components extracted from plants can modulate animal VDACs. Their functional significance on plant VDACs will be a great help to further our understanding. Fortunately, the once lagging plant VDACs have emerged as a new arena of research. Based on the studies so far, VDACs have immense potential in therapeutics and the improvement of crops. The ongoing research will further unmask its functional significance, particularly, in plants.Significant knowledge on VDAC has accumulated in recent years. The functional range of VDACs as gating proteins has now expanded. We know the localization of VDACs, their modulation, biochemical regulation and functional implications in animals and, to some extent, in plants. Multi-localization of VDAC on different membranes, yet the difference in their functions, is an example of how nature increases diversity with existing tools in the cell. The multi-localization of VDAC can be predicted as a connecting route for better intra-cellular communication. However, its localization on PM is highly debatable. Different modes of regulation of VDACs make them versatile and hence, explains their ability to regulate and participate in multiple signaling events. The research on biochemical regulation is still limited and extensive intervention is the need of the hour. These channels are well known for molecular transport, and this function also puts them under the spotlight as communicators between cytoplasmic and mitochondrial signaling events. A mutual interplay between CaGP conceptualized the review. BR and GP wrote the manuscript. BR, PK, SS, MB, and GP reviewed and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Metabolic dysfunction is a major driver of tumorigenesis. The serine/threonine kinase mechanistic target of rapamycin (mTOR) constitutes a key central regulator of metabolic pathways promoting cancer cell proliferation and survival. mTOR activity is regulated by metabolic sensors as well as by numerous factors comprising the phosphatase and tensin homolog/PI3K/AKT canonical pathway, which are often mutated in cancer. However, some cancers displaying constitutively active mTOR do not carry alterations within this canonical pathway, suggesting alternative modes of mTOR regulation. Since DEPTOR, an endogenous inhibitor of mTOR, was previously found to modulate both mTOR complexes 1 and 2, we investigated the different post-translational modification that could affect its inhibitory function. We found that tyrosine (Tyr) 289 phosphorylation of DEPTOR impairs its interaction with mTOR, leading to increased mTOR activation. Using proximity biotinylation assays, we identified SYK (spleen tyrosine kinase) as a kinase involved in DEPTOR Tyr 289 phosphorylation in an ephrin (erythropoietin-producing hepatocellular carcinoma) receptor–dependent manner. Altogether, our work reveals that phosphorylation of Tyr 289 of DEPTOR represents a novel molecular switch involved in the regulation of both mTOR complex 1 and mTOR complex 2. The serine/threonine kinase mechanistic target of rapamycin (mTOR) is a central regulator of cellular metabolism, and its dysfunction is frequently observed in cancer. Indeed, constitutive activation of mTOR-regulated pathways is now considered as a potent inducer of tumor growth and cancer cell survival , 4, 5, 6mTOR functions as a central metabolic regulator involved in the modulation of cellular processes, such as autophagy, macromolecule biosynthesis, lysosome biogenesis, cell mobility, cell survival, and cytoskeletal rearrangement . The invR-2-hydroxyglutarate (R-2HG), an oncometabolite, instead of their normal product, α-ketoglutarate  by mTOR and CK1α under growth factor stimulation, leading to its degradation by the ubiquitin/proteasome system /PI3K/AKT canonical activation pathway , 16. Howlutarate , 21. Bec enzymes , resulti enzymes . R-2HG-it manner . Moreovee system , 25, 26.Erythropoietin-producing hepatocellular carcinoma (EPH) receptors are the largest family of receptor protein tyrosine kinase. They are involved in a vast variety of cellular processes, such as proliferation, differentiation, cell shape, and mobility . Their dWe identified a novel phosphorylation event targeting DEPTOR tyrosine (Tyr) 289, which rapidly affects its inhibitory function on both mTORC1 and mTORC2. Phosphorylation of DEPTOR on Tyr 289 weakens its association with mTOR, thus allowing a rapid and sustained activation of both mTORC1 and mTORC2. Proximity biotinylation experiments identified SYK (spleen tyrosine kinase) as a kinase implicated on DEPTOR Tyr phosphorylation. Using combinatory methods, we found that the EPHB2 receptor signaling pathway also promotes DEPTOR Tyr 289 phosphorylation through SYK activation.Taken together, our findings identified a novel molecular mechanism involved in modulating mTOR activity and a previously unidentified potential pathway contributing to PTEN/PI3K/AKT-independent increase of mTOR activity observed in a subset of cancers.A).Increasing evidence is pointing toward multiple PTMs playing a disruptive role on DEPTOR function as an endogenous inhibitor of mTOR. While serine phosphorylation was shown to trigger DEPTOR proteasomal degradation , 25, 26,Fig. 1B), we found Tyr phosphorylation on the C-terminal half of the protein (amino acids 214–409) . We further divided the C-terminal fragment in two smaller fragments (D3 and D4 fragments) and found that Tyr phosphorylation was restricted to the D4 fragment (amino acids 276–409) .Since Tyr phosphorylation is known to modulate protein interactions and activity in a rapid and reversible manner , we sougFig. 1C and D). For both of them, the upper band seems to be the one harboring most of the Tyr phosphorylation. We then investigated the nature of this bands pattern by using a DEPTOR D4 fragment tagged on both its N terminus (FLAG) and C terminus (hemagglutinin [HA]). Detection of both bands of DEPTOR fragment D4 using FLAG and HA antibodies suggested that these forms of DEPTOR are not resulting from proteolytic cleavage but rather resulting of PTMs (A). Only the higher molecular weight band of DEPTOR fragment D4 was Tyr phosphorylated and recognized using 4G10 antibody (B). In addition, we validated that the ∼18 kDa band was not solely the result of DEPTOR Tyr phosphorylation, since it was still observable using anti-FLAG in Tyr phosphatase-treated immunoprecipitates (B). Sequence alignment revealed that three tyrosines are present within D4 fragment and that only two are evolutionarily conserved, namely Tyr 289 and Tyr 326 (C). By generating point mutation of each conserved tyrosine within the D4 fragment into nonphosphorylatable phenylalanine (Y to F), we determined that only Tyr 289 can be phosphorylated (E).We observed multiple bands above the major band for DEPTOR C-terminal and DEPTOR D4 fragment , C and D of PTMs A. Only tantibody B. In addipitates B. Sequen Tyr 326 C. By genorylated E.A). We then infected these cells with shRNA-resistant WT DEPTOR or mutants harboring Tyr 289 phosphomimetic Y to E substitution (DEPTORY289E) or a nonphosphorylatable Y to F substitution (DEPTORY289F). In starved cells, nonphosphorylatable DEPTORY289F showed increased association to mTOR when compared with DEPTORWT, whereas the phosphomimetic version of DEPTOR (DEPTORY289E) showed a slight decreased binding when compared with DEPTORWT  and FAT (mTOR) domain . We inveFig. S2DEPTORWT . This di to mTOR . These rS240/244) and mTORC2 (phospho-AKTS473) activation. To monitor the regulation of mTOR activity, cells were first serum starved for 16 h, then stimulated with complete media for 30 min, and then replaced with starving media for 12 h in order to follow kinetics of the regulation of mTOR activity. We observed a faster decrease in phosphorylation of S6 in DEPTORY289F-expressing cells, compared with cells expressing DEPTORWT, whereas those expressing DEPTORY289E showed a sustained mTORC1 activation until 4 h poststimulation (A), suggesting that phosphorylation on Tyr 289 impairs DEPTOR ability to suppress mTORC1 activity. All DEPTOR mutants showed similar behavior regarding phospho-AKTS473, a readout for mTORC2 activation, after serum stimulation, followed by rapid decrease of phosphorylated AKT within the first hour (A). Because of the faster regulation of mTORC2 following stimulation, phospho-AKTS473 levels in mutant DEPTOR-expressing cells was rather determined within the first hour after serum stimulation. Like mTORC1 activity, cells expressing DEPTORY289F mutant showed a faster decrease of phospho-AKTS473 compared with cells expressing DEPTORWT, whereas DEPTORY289E-expressing cells displayed sustained mTORC2 activation following serum removal (B). These results confirmed that DEPTOR phosphorylation on Tyr 289 decreases its inhibitory functions on mTOR activity.Growth factor–induced DEPTOR dissociation from mTOR was shown to positively modulate mTOR activity , 25, 26.mulation A, suggesrst hour A. Becaus removal B. These WT, DEPTORY289F, or DEPTORY289E at different time points for 18 h after growth factor stimulation (A). Cell area rapidly increased 1 to 3 h following stimulation and reached maximum size at 12 h poststimulation. The increase in cell area was even more noticeable in cells expressing DEPTORY289E comparatively to cells expressing either DEPTORWT or nonphosphorylatable DEPTORY289F (A). These results are consistent with our assessment of mTORC1 and mTORC2 activation  and Torin 1 (inhibitor of both mTOR complexes) on growth factor–induced cytoskeletal modulation of cells expressing DEPTORY289E. We observed a significant decrease in cell area upon treatment with Torin 1. When only mTORC1 was inhibited, the decrease in cell area was less marked, confirming the impact of mTORC2 on cell area (B). These results confirm that DEPTOR phosphorylation on Tyr 289 sustained mTORC2 activity leading to increased cytoskeletal reorganization.To further investigate the cellular impact of DEPTOR Tyr 289 phosphorylation on mTOR-dependent mechanisms, we assessed its effect on cytoskeletal reorganization by measuring cell size area upon stimulation. We measured cell size area of HeLa cells depleted of endogenous DEPTOR expressing DEPTORmulation A. Cell aTORY289F A. These tivation . Moreoveell area B. These Y289E was more stable through time than DEPTORWT and the unphosphorylated version DEPTORY289F (A). To assess if DEPTOR Tyr 289 phosphorylation was preventing its degradation, we immunoprecipitated each construct and measured their respective ubiquitination following insulin stimulation. We observed similar ubiquitination patterns for DEPTORWT and DEPTORY289F, whereas ubiquitination of DEPTORY289E was significantly decreased (B). To further confirm the effect of Tyr 289 phosphorylation on DEPTOR ubiquitination, we assessed its effect on its ability to associate with β-transducin repeat–containing protein 1 (β-TRCP1), an E3 ubiquitin ligase previously identified to be involved in DEPTOR degradation . Taken together, these results suggest that while Tyr 289 impairs the repressive function of DEPTOR, it also prevents it from β-TRCP1-induced ubiquitin-mediated degradation. Hence, this also suggests that this phosphorylation event could act as a molecular switch allowing a rapid activation/inactivation of DEPTOR repressive function on mTOR.Ubiquitin-mediated degradation of DEPTOR is an important molecular process allowing the release of mTOR from DEPTOR-mediated inhibition , 25, 26.TORY289F A. To assecreased B. To furradation , 25, 26. β-TRCP1 B. Taken A and B). DEPTOR Tyr 289 lies closely to serine residues phosphorylated by mTOR (Ser 293 and 299) (A). While Torin 1 showed no effect, PP242 provoked a robust reduction in DEPTOR Tyr 289 phosphorylation. As PP242 is known to lose specificity at higher concentration, we tested a range of PP242 concentrations and observed inhibition of DEPTOR Tyr phosphorylation only at higher concentrations of 5 to 10 μM (A). These results suggested that the observed effect was not caused by mTOR inhibition but by another kinase inhibited at higher PP242 concentrations.We demonstrated that DEPTOR Tyr 289 phosphorylation allows a rapid relief of DEPTOR-mediated inhibition of mTOR , A and Band 299) , 25, 26.and 299) A. While to 10 μM A. These B). While both kinases are known to participate in DEPTOR regulation, PI3K through the canonical pathway of mTOR . Finally, we tested inhibitors against the SRC Tyr kinase family  and observed a significant decrease of DEPTOR Tyr phosphorylation following PP2 treatment but did not detect any significant response with other inhibitors of this family . The PP2-mediated decrease in DEPTOR Tyr phosphorylation was more noticeable at concentrations over 1 μM (B). Similarly, to results obtained using PP242, the requirement of higher doses of PP2 suggested that the effect was most probably not because of inhibition of SRC kinase family members but from other kinases potentially sensitive to PP2-mediated inhibition at higher concentration , 3736, 3Fig. 6 of mTOR  and CK1αf DEPTOR , neitherver 1 μM B. Similantration  . Conversely, EPHB2 depletion (A) led to a decrease in Tyr phosphorylation . In addition, we validated that EPHB2 overexpression increased Tyr phosphorylation on full-length DEPTOR (B), and that Tyr 289 was the residue targeted by EPHB2 (C). As EPHB2 seems to be a major regulator of DEPTOR Tyr phosphorylation, we observed that a kinase-dead EPHB2 mutant (K653A) failed to phosphorylate DEPTOR when overexpressed . Moreover, depletion of EPHB2 or overexpression of the kinase-dead version decreased the cellular levels of DEPTOR (B and C). Hence, we proceeded to confirm that preventing DEPTOR Tyr phosphorylation, through impairing the EPHB2 pathway, decreased its protein stability. As expected, we observed a significant decrease in DEPTOR half-life in the presence of kinase dead EPHB2, when compared with WT, following growth factor stimulation (D).Of the possible secondary targets of PP242 and PP2 inhibition, only EPH Tyr kinase receptors were previously reported to be inhibited at high concentrations of the two drugs , 42, 43.Fig. 7epletion A led to h DEPTOR B, and thby EPHB2 C. As EPHmulation D.Figure D) and EPHB2 (E) Tyr phosphorylation, while we observed a synergistic reduction of the phosphorylation in cells expressing EPHB2-targeting shRNAs (A and B). These results suggest that other Tyr kinase(s) can at least partially compensate for the loss of EPHB2 involved in DEPTOR Tyr phosphorylation . To assess the effect of EPHB2 on the activation levels of both mTOR complexes, we either overexpressed or depleted EPHB2WT in HeLa cells (E). EPHB2WT-overexpressing cells showed increased mTORC1 and mTORC2 activation when compared with both shNT and shEPHB2 cells, confirming that EPHB2 levels can affect mTOR activation thorough DEPTOR phosphorylation.Using the EPHB2 overexpressing or depleted cells, we assessed PP242 and PP2 ability to repress EPH receptor–induced DEPTOR Tyr phosphorylation. In EPHB2-overexpressing cells, both inhibitors drastically decreased DEPTOR D and EPHnd EPHB2 E Tyr phog shRNAs , A and BLa cells E. EPHB2WA) and by revealing their autobiotinylation following streptavidin pull down (B). We then proceeded with duplicate biotinylated assays and identified, among all the potential interactors (A), two Tyr kinases: Fer  and SYK. Since SYK was already shown to interact with EPH receptors (B). We then assessed if SYK could phosphorylate DEPTOR Tyr 289 by either overexpressing or abrogating SYK expression, and then assessed the level of phosphorylation of DEPTOR. As expected, SYK overexpression greatly increased the level of Tyr phosphorylation on DEPTOR (C), whereas decreasing SYK expression, using a validated shRNA (A), showed the opposite effect (D). We further confirmed these observations using DEPTOR full length (B) and the specificity of the phosphorylation site by using DEPTOR D4Y289F fragment (C).As we were unable to confirm direct interaction between EPHB2 and DEPTOR (data not shown), we reasoned that DEPTOR Tyr phosphorylation was mediated through another Tyr kinase activated by EPHB2 receptor. To identify the potential kinase able to phosphorylate the Tyr 289 of DEPTOR, we performed a miniturboID experiment using DEPTOR D4 fragment as bait. We first validated our constructs by evaluating their capacity to biotinylate partners A and by ull down B. We theeractors A, two Tyeceptors , as welleceptors , we firseceptors B. We then DEPTOR C, whereaed shRNA A, showede effect D. We furl length B and thefragment C.Figure Taken together, our results exposed a new mTORC1 and mTORC2 regulatory mechanism mediated by the activation of EPHB2 receptors and SYK, which promote the phosphorylation of DEPTOR. This phosphorylation of DEPTOR promotes its dissociation from mTOR complexes, thus allowing the rapid and sustained activation of mTORC1/mTORC2 .Figure 9mTOR is frequently hyperactivated in cancer, conferring cancer cells with proliferative and survival advantages over normal cells . While t289 phosphorylation can decrease its affinity toward both mTOR complexes (mTORC1 and mTORC2), which lead to increased mTOR activation, while increasing DEPTOR stability. This represents a novel mode of DEPTOR regulation contrasting with the reported mechanism where DEPTOR dissociation to mTOR was initiated by serine phosphorylation, which subsequently leads to its proteasomal degradation , it also seems possible that mTORC2 activation was regulated by the DEPTOR Tyr 289 phosphorylation. This is further supported by our observation that DEPTOR Tyr 289-mediated regulation of mTOR bypasses the PI3K/PTEN pathway. While this remains to be validated, DEPTOR Tyr 289 phosphorylation could have been oversighted in their assessment of mTORC2 activation, thus corroborating the existence of two modes of regulation by DEPTOR, one going through the retroactivation of PI3K and a transient one going through the phosphorylation of DEPTOR Tyr 289.Recent advances on DEPTOR regulation mechanisms have led to a model of retroactive feedback effect of DEPTOR on mTOR where DEPTOR can directly inhibit mTORC1, whereas its effect on mTORC2 was indirectly caused by PI3K activation . HoweverWhile studies already revealed a possible link between EPH receptors and mTOR pathways, we observed that the effect of EPH receptors on mTOR activation or inhibition was highly specific. Some previous studies indicated that EPHA receptor can inhibit the mTOR pathway , 51, whee.g., DEPTOR), explaining how SYK mediates mTOR activation independently of the PI3K/AKT axis  supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Wisent).Cells were transfected with 8 μg of DNA using 30 μg of linear polyethylenimine. The following vectors were used: pCDNA3.1 FLAG-eYFP, pRK5 FLAG-DEPTOR, pLJM1 FLAG-DEPTOR-D1/D2, pLJM1 FLAG-DEPTOR-D3/D4, pLJM1 FLAG-DEPTOR-D4 , pLJM1 FLAG-DEPTOR-D4-HA , pLJM1 HA-GFP, pLJM1 EPHA1-GFP, pLJM1 EPHA2-GFP, pLJM1 EPHA3-GFP, pLJM1 EPHB1-GFP, pLJM1 EPHB2-GFP, pLJM1 EPHB3-GFP, pLJM1 EPHB4-GFP, pCDNA5 EPHB2 Wt, pCDNA EPHB2 KD, and pLJM1 HA-SYK.http://n2t.net/addgene:8453; RRID: Addgene_8453)  , and 2 μg pMD2.G envelope plasmid . Media were changed 24 h after transfection, and lentiviral particles were harvested 24 h later. Viral supernatant was filtered through 0.45 μm filters and supplemented with 8 μg/ml polybrene and 10% fetal bovine serum. The following plasmids were used: pLKO.1-sh_NT, pLKO.1-shGFP, pLJM1 FLAG-DEPTOR , pLJM1 FLAG-GFP, pLKO shDEPTOR_3, pLKO shDEPTOR_5, pLKO shEPHB2_GFP_3, pLKO shEPHB2_GFP_5, pLKO shSYK_1, pLKO shSYK_2, pLJM1_FLAG_GFP_miniturbo, pLJM1_FLAG-DEPTOR_D4-miniturbo.Lentiviral transductions were performed similarly as previously described , 59, 60.ne_8453)  or expre_19319]) , 6 μg ofAGAAAGGTGTTGAAATGCTTCTCTATCAAGAGTAGAGAAGCATTTCAACACCTTTCTTTTTTTG-3′), DEPTOR shRNA_5 (5′-CCGGTGCAGCTATTTAATAGGGAATCTAGATCAAGAGTCTAGATTCCCTATTAAATAGCTGCTTTTTTG-3′), EPHB2 shRNA_3 (5′-CCGGTTTAAAGAGGATTCTCATAAGCTCGAGCTTATGAGAATCCTCTTTAAATTTTTTG-3′), EPHB2 shRNA_5 (5′-CCGGGCTAGACAAGATGATCCGCAACTCGAGTTGCGGATCATCTTGTCTAGCTTTTT-3′), SYK shRNA_1 (5′-CCGGTCAGAGGAATTTGGCTGCTTCTTCAAGAGAGAAGCAGCCAAATTCCTCTGTTTTTTG-3′), SYK shRNA_2 (5′-CCGGTGTGATTGCACTTGGACATCAGTCCATCAAGAGTGGACTGATGTCCAAGTGCAATCACTTTTTTG-3′), and shRNA nontarget (5′-CCGGGCGCGATAGCGCTAATAATTTCTCGAGAAATTATTAGCGCTATCGCGCTTTTTG-3′).DEPTOR shRNA_3 , anti-pan-AKT , antiphospho-AKTS473 , anti-ACTIN  and anti-GAPDH , anti-4G10 , anti-GFP , antiubiquitin P4D1 , anti-EPHB2 D2X2I , anti-EPHB2 , and anti-SYK D3ZE1 .Cells were lysed in Laemmli buffer, proteins were separated on SDS-PAGE, and transferred to polyvinylidene fluoride membranes (Bio-Rad). Primary antibodies used were anti-FLAG , anti-HA , anti-mTOR , anti-DEPTOR , anti-S6 , anti-phospho-S6g at 4 °C. The supernatant was incubated for 1 h at 4 °C with 15 μl of FLAG M2 agarose beads (Sigma). Immunoprecipitates were washed three times with lysis buffer containing before elution in Laemmli buffer. For denaturing IP, after specific treatment, cells were lysed in 1 ml of denaturing buffer , boiled for 5 min, and sonicated with FISHER SONIC dismembrator model 150 at level 6 for 20 s. All lysates were complemented with 9 ml of nondenaturing buffer  and 15 μl of FLAG M2 agarose beads (Sigma) and incubated for 1 h at 4 °C. Immunoprecipitates were washed three times with nondenaturing buffer before elution in Laemmli buffer.For nondenaturing IP, after specific treatment, cells were lysed in 1 ml of lysis buffer  and incubated on ice for 10 min. After vortex, lysate was centrifuged for 10 min at 17,000After washes of denaturing IP, beads were washed twice in PBS and separated in two. One part was treated 30 min at 37 °C with only calf intestinal alkaline phosphatase (CIP) buffer , and the other one was treated with CIP buffer and CIP phosphatase .Cells were plated on Ibidi plates  and starved for 16 h. Stimulation treatments with complete media were done, and cells were fixed with 4% formaldehyde at different time points  after three washes of PBS 1×. After fixation, cells were washed five times with PBS 1× and permeabilized with PBS 0.5% Triton for 20 min. Five washes were made, and cells were incubated at room temperature for 1 h with PBS–Tween bovine serum albumin 0.2%. Cells were washed five times with PBS–Tween and incubated for 1 h at room temperature with an anti-FLAG M2  diluted in PBS 1×. After five washes with PBS–Tween, cells were incubated 1 h with an anti-Alexa-Fluor 488 , phalloidin , and 4′,6-diamidino-2-phenylindole . About ten washes were made (five washes with PBS–Tween and five washes with PBS 1×) and kept in PBS 1× until imaging. Total internal reflection fluorescence microscope was used to image the experiment. Large images were taken (8 × 8) with a 15% stitching between each image at 20×. Images were then analyzed using NIS Element Analysis software. The mean area of the cells was calculated for each condition .Cells were starved for 16 h and stimulated 30 min with complete media. After the stimulation, they were starved again during 1, 2, 3, 4, 6, 8, and 12 h or during 5, 15, 30, 45, 60, 120, and 180 min. Cell extracts were also made for starve and 30 min stimulation condition.18-StageTips, dried with a SpeedVac, and stored at −80 °C until MS analysis.Infected 293T cells with FLAG-GFP-MiniTurbo and FLAG-DEPTOR_D4-MiniTurbo were treated 1 h with 50 μM of biotin. Cell lysates were cleared of free biotin using a 3K Microsep advance centrifugal device , and the supernatant was used for a denaturing streptavidin pull down. To prepare for MS analysis, the pulled-down biotin-labeled proteins were washed three times with 50 mM of ammonium bicarbonate, resuspended in 100 μl of ammonium bicarbonate with 1 μg of trypsin, and incubated overnight at 37 °C with gentle lateral mixing. About 16 h later, each sample was supplemented with 1 μg of trypsin and incubated for another 4 h. The supernatants were collected, beads were washed twice with 200 μl of MS-grade acetonitrile, and the washes were pooled with the collected supernatant. Digestion was stopped with a concentration of 2% of formic acid. Peptides were desalted with homemade C18 column, 0.3 mm id × 5 mm; Dionex Corporation) and eluted onto an analytical C18 column . Peptides were eluted over a 2-h gradient of acetonitrile (5–40%) in 0.1% formic acid at 400 nl/min while being injected into a TimsTOF Pro mass spectrometer equipped with a Captive Spray nano electrospray source . Data were acquired using data-dependent auto-MS/MS with a 100 to 1700 m/z mass range, with parallel accumulation serial fragmentation enabled with a number of parallel accumulation serial fragmentation scans set at 10 (1.27 s duty cycle) and a dynamic exclusion of 0.4 min, m/z-dependent isolation window, and collision energy of 42.0 eV. The target intensity was set to 20,000, with an intensity threshold of 2500. The resulting files were analyzed using MaxQuant  using a mass tolerance of 20 ppm for precursor ions and a tolerance of 50 ppm for fragment ions and searched against the UniProt human reference genome  containing 75,776 entries. Two miscleavages were allowed, while enzymes were set to trypsin (K/R not before P). Carbamidomethylation of cysteine was set as a fixed modification. Phosphorylation of serine, threonine, and Tyr; oxidation of methionine; and acetylation of N-terminal protein were allowed as variable modifications. Peptide-spectrum matches and protein false discovery rates were both applied at 1%. Please note that values of parameters in MaxQuant have not been changed from their default values unless explicitly stated. The complete protein list identified by MaxQuant can be found in https://reprint-apms.org/) portal using default parameters. The results of this analysis can be found in Peptide samples were analyzed at the Sherbrooke mass spectrometry core facility. Briefly, samples were injected into an HPLC  and loaded onto a trap column with a constant flow of 4 μl/min (Acclaim PepMap100 Cmassive.ucsd.edu) and assigned the identified MSV000087055. They can be downloaded at ftp://MSV000087055@massive.ucsd.edu.All MS files were deposited at MassIVE (This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Saccharin is a commonly used artificial sweetener that exhibits both sweetening and sweet inhibition activities. The species‐dependent response towards saccharin and the interaction between saccharin and the sweet taste receptor T1R2/T1R3 remain elusive. In this study we used mismatched chimeras of T1R2 and T1R3 and calcium mobilization functional analysis to reveal a detailed species‐dependent response towards saccharin of human, squirrel monkey, and mouse sweet taste receptors. Our findings, combined with previous results by others, suggest multiple and complex interaction modes between saccharin and the sweet taste receptor, which are helpful guidelines for effective modulation of the sweet taste by sweetener/sweet inhibitors. Saccharin exhibits both sweetening and sweet inhibition activities. Besides the orthosteric binding site in T1R2 for activating the human sweet taste receptor T1R2/T1R3, it appears that there are at least two allosteric inhibitory sites located in T1R2 and T1R3 respectively to inhibit the receptor activity, suggesting multiple and complex interaction modes between saccharin and the heterodimeric sweet taste receptor T1R2/T1R3. CRDcysteine‐rich domainGPCRG protein‐coupled receptorHDheptahelical domainIDintracellular domainSEMstandard error of meanTMtransmembraneVFTMVenus flytrap moduleIt has been well‐known that the sweet taste perception in mammalians is broadly mediated by the sweet taste receptor, which belongs to the family C G protein‐coupled receptors (GPCRs) . The recThe heterodimeric sweet taste receptor T1R2/T1R3 is composed of three domains: a large extracellular domain, which includes a Venus flytrap module (VFTM) and a cysteine‐rich domain (CRD), a heptahelical domain (HD), which includes seven transmembrane helices (TMs 1–7), and an intracellular domain (ID), which interacts with the G protein  and sweet inhibitory (> 3 mm) responses, depending on the concentration of stimulation. Based on the species‐dependent responses towards Na‐saccharin and functional analysis of the human/rat chimeras of sweet taste receptor T1R2/T1R3, it has been suggested that the human T1R3 is the allosteric binding site for inhibition by Na‐saccharin [et al. reported that the extracellular VFTM region of human T1R2 is an orthosteric binding site for the sweetness of saccharin [The artificial sweetener saccharin has been shown to elicit both human sweet sensation  was purchased from Sigma‐Aldrich . Sucralose  was obtained from Tongyuan Sweetener Co. Ltd . Stevioside (purity ≈90%) was obtained from NuSci Institute & Corp . Saccharin  was obtained from Supelco . All other chemicals and reagents were analytical grade and were from Invitrogen .The human, squirrel monkey, and mouse T1R2s and T1R3s (mouse C57BL/6 strain) constructs were generated with the expression vector pcDNA3.1 (Invitrogen) as described previously . The Gα1http://swissmodel.expasy.org/) with the heterodimeric human metabotropic GABA(B) receptor (PDB: 6UO8) as the template. A sequence alignment of the human T1R2 and T1R3 and template sequences was carried out with the ClustalW program [The homology model of full‐length human T1R2/T1R3 was constructed using the Swiss‐Model program , but the responses were inhibited when the concentration of saccharin was above 3 mm. However, mouse receptor showed a dose‐dependent increase of response to saccharin and no inhibition was found at the concentrations of saccharin tested up to 60 mm. These results demonstrated the species‐dependent responses to saccharin for human and mouse but not squirrel monkey sweet taste receptors, while species‐specific inhibition of the response by saccharin at high concentrations (> 3 mm) for human but not mouse receptors.The constructs of sweet taste receptors and Gα16‐gust44 were transfected into the HEK293E cells, and their expression has been demonstrated in our and other previous studies , 13, 14.m saccharin. We selected three sweeteners: sucralose, stevioside, and d‐tryptophan, which can be perceived by all three species. As shown in Fig. m saccharin, whereas the response of mouse T1R2/T1R3 could not be inhibited. The dose–response experiment showed the inhibitory efficiency of the response towards sucralose by saccharin in human, squirrel monkey, and mouse  can inhibit the response of human but not mouse sweet taste receptor T1R2/T1R3 towards sucralose, stevioside, and D‐tryptophan, while the response of mismatched human T1R2 and mouse T1R3 (replacement of mouse T1R2 by human T1R2) towards these sweeteners can be inhibited by saccharin (> 3 mm). These results indicate that saccharin at high concentrations can bind at one or more allosteric sites located in human T1R2 to inhibit the response of human sweet taste receptor towards various sweeteners. However, it was previously described that the human T1R3 is an allosteric binding site of saccharin for inhibiting sweet taste [In this study we reported the detailed species‐dependent responses towards saccharin of human, squirrel monkey, and mouse sweet taste receptors both for sweetening and inhibitory activities, and revealed that the sweet taste receptor T1R2 is a novel allosteric binding site for inhibition by saccharin. As shown in Figs. et taste . Moreoveet taste , 8. Therm), Na‐saccharin preferentially binds into the high‐affinity orthosteric site in T1R2, activating the receptor. When the concentrations increase (>3 mm), the excessive saccharin molecules can bind to allosteric inhibitory sites located in human T1R2, and when more saccharin is added, they can probably bind with both T1R2 and T1R3, giving rise to the inhibited state of the receptor. Interestingly, we have reported that the HD of human T1R2 is an allosteric binding site for the sweet inhibitor amiloride [A two‐state allosteric model was proposed to explain the sweet “water‐taste” that removing the sweet taste inhibitors such as saccharin, acesulfame‐K, and lactisole located at the allosteric binding sites shifts the receptor from a high‐energy inhibited state to the constitutively active state, thus stimulating the sweetness upon water rinsing . Our premiloride . It is mThe authors declare no conflict of interest.The data are available from the corresponding author upon reasonable request.MC and BL conceived and designed the project, XZ and ML acquired the data, MC and BL analyzed and interpreted the data, and BL and MC wrote the article.