Animals using toxic peptides and proteins for predation or defense typically depend on specialized morphological structures, like fangs, spines, or a stinger, for effective intoxication. Here we show that amphibian poisons instead incorporate their own molecular system for toxin delivery to attacking predators. Skin-secreted peptides, generally considered part of the amphibian immune system, permeabilize oral epithelial tissue and enable fast access of cosecreted toxins to the predator’s bloodstream and organs. This absorption-enhancing system exists in at least three distantly related frog lineages and is likely to be a widespread adaptation, determining the outcome of predator–prey encounters in hundreds of species. To avoid being eaten, poisonous prey animals must rely on fast passage of toxins across a predator’s oral tissue, a major barrier to large molecules. Here, Raaymakers et al. show that antimicrobial peptides co secreted with frog toxins enhance intoxication of a snake predator by permeabilizing oral cell layers. In the absence of structures that create a wound to inject toxins, fast delivery is typically only possible for small molecules (<300 Da) that are easily absorbed through oral epithelia, like cyanides, alkaloids, or steroids2. Many frog species however, secrete much larger molecules with systemic targets, like peptides  and in some cases even small proteins (4–8 kDa)7. These toxins generally resemble vertebrate hormones or neuropeptides and undergo posttranslational modifications that increase their lifespan in a predator’s bloodstream or enhance their affinity to a target receptor10. Upon receptor binding, these toxins essentially simulate an overdose of a predator’s hormone or neuropeptide, resulting in a range of adverse effects, including nausea, hypotension, and hyperalgesia10.When a poisonous or venomous animal is attacked by a predator, it usually has little time to deploy its toxins and induce a response in the aggressor to avoid being killed Fig. . To achi13. The poisons of many frogs thereby seem to defy the constraints of passive toxin uptake, and hint at a mechanism that allows systemic delivery fast enough to evade predation. Besides toxins, many frogs secrete antimicrobial peptides (AMPs), capable of killing a broad range of microorganisms through cell lysis15. Since their first characterization in the late 1980s, AMPs have been generally considered a component of the amphibian innate immune system, and investigated primarily in light of their potency against clinically important pathogens18. Recently however, an alternative role in antipredator defense has been suggested, and it was predicted that AMPs, through a similar cytolytic activity, could permeabilize a predator’s epithelial tissue to facilitate toxin delivery7. Proof for this hypothesis would highlight a long-overlooked function of amphibian AMPs besides innate immunity, and drastically alter our perception of how they contribute to an amphibian’s survival. Here we report on a series of in vitro and in vivo experiments that provide compelling evidence for an antipredatory role of AMPs. Our results complete the picture of a toxin delivery system as sophisticated as that of many venomous animals, be it molecular rather that morphological in nature.The large size and physicochemical properties of peptides renders them unsuitable for fast epithelial absorptionXenopus laevis20  of the intestinal wall. Because caerulein is a widespread toxin in frogs9 with a long half-life in the bloodstream (up to 45 min)23, this peptide makes a suitable model to investigate mechanisms of toxin delivery.As the main model for our study, we used a peptide pair composed of the systemic toxin caerulein, and the AMP caerulein precursor fragment-3 (CPF), both of which are key constituents of the skin secretion of the frog 20 Fig. . CaeruleThe absence of lactate dehydrogenese (LDH) leakage after applying caerulein (100 μM) to epithelial cell cultures indicate that the toxin is incapable to damage epithelial cells by itself Fig. . However25. Despite the long half-life of caerulein, radioactivity measurements across such period may increasingly overestimate the amount of intact toxin in a predator’s body. Nevertheless, the radiolabeled peptide provides a useful tool to quantify and compare the efficiency of epithelial absorption as the first step of predator intoxication.Our in vitro experiments provide a strong indication that frog AMPs indeed enhance the transepithelial passage of cosecreted toxins. To test whether this pattern holds in a live model predator, we quantified radiolabeled caerulein 22. Over 30 min, systemic exposure to the toxin (area under the curve (AUC) derived from repeated blood measurements) ranged up to 13 times higher in the presence of CPF . This is a biologically realistic concentration for frog skin secretions according to reported estimates for AMPsCPF Fig. , and aveCPF Fig. , c. The 30. Besides CPF and related peptides in species of the family Pipidae20, AMPs are particularly prominent in the skin secretions of discoglossoid and neobatrachian frogs, in which they, too, co-occur with peptide toxins ; and the toxin dermorphin and the AMP dermaseptin-S1 (Drs) from the skin of the waxy tree frog Phyllomedusa sauvagii (Neobatrachia: Hylidae). The toxin bombesin is a potent ligand of neuromedin receptors expressed on smooth muscle cells surrounding the gut and blood vessels, which causes hypotension and smooth muscle spasms33. Dermorphin causes sedation and shock upon binding its target opioid receptors in the nervous system35. Like caerulein, both toxins require access to the bloodstream to induce these effects, and have structural adaptations to extend their half-lives in the bloodstream and tissues36.AMPs have been characterized in hundreds of frog species, and have been postulated to represent independent evolutionary origins in distantly related frog lineagesins Fig. . To inveins Fig. : The toxSimilar to caerulein, neither bombesin nor dermorphin induced any damage to epithelial tissue by themselves Fig. . In contMost research on amphibian skin-secreted peptides has a predominantly pharmacological scope, aiming to characterize their physiological effects, not their natural mode of delivery. By investigating the process of intoxication “from a frog’s perspective”, our study has two major implications.38. The present evidence however, implies that pharmacological studies seeking to optimize the therapeutic potential of frog AMPs should take their potential damaging effect on epithelial tissue into account.First, we provide evidence for a function of amphibian AMPs outside the immune system. Due to their pore-forming activity and expression in the skin, AMPs are inclined to serve a dual role, in innate immunity as well as antipredator defense. In fact, besides enhancing the absorption of cosecreted toxins, the cytotoxic activity of amphibian AMPs may cause irritation or pain on oral mucosa, thereby providing a secondary and fast-acting effect on some predators. The broad-spectrum antimicrobial activity of many amphibian AMPs continues to fuel the perception that they are promising lead compounds for the development of new antibioticsX. laevis skin secretion found here, quantities ranging up to milligrams have been reported for toxins in the skin secretion of other frog species, including caerulein in Litoria caerulea 6. The question remains whether toxins of this size would also benefit from AMP-enhanced absorption. The degree by which epithelial transfer was enhanced in our in vitro experiments seemed inversely correlated to the toxins’ molecular weights, which may indeed indicate that a peptide’s size affects an AMP’s capacity to mediate its transepithelial uptake.Second, our results show that toxin systems relying on epithelial absorption may be more complex than currently perceived, involving multiple factors that together maximize the effectiveness of intoxication. At least in peptide-secreting frogs, epithelial permeabilization is likely to be a decisive step, reducing the time for toxins to reach an adverse effect in the predator. The secretion of large amounts of toxin additionally increases the probability that effective toxin levels are attained in the predator’s system even if only a small percentage of the toxin is absorbed. Similar to the large amounts of caerulein in lea Fig. , and bom24 and in some cases ranged up to 50 min25. Similarly, amphibians have been documented to survive up to 20 min in the gastrointestinal tract of bullfrogs and fish after being swallowed completely25. If in the latter case sickness is induced before the frog dies, it might be regurgitated and escape. Even in predators whose preying technique precludes survival of the frog , enhanced toxin absorption could still adversely affect the predator and be beneficial to the remaining frog population, possibly aided by predator learning42.Absorption enhancement is likely to be effective against predators whose limited prey-handling skills require considerable time to subdue their prey, and/or swallow their prey whole . In addition, it may act in concert with ingestion-delaying behavior such as struggling or inflating. For various snake species, recorded times between capture and ingestion of amphibians averaged 15–35 min43. Examples involving toxic peptides or proteins with systemic targets may include salamanders44, nemertean worms45, and cone snails releasing insulin-based secretions46. Moreover, the venoms of animals relying on active injection like biting or stinging  often include cytolytic agents, which may further improve toxin spreading49. As such, our findings call for a rethinking of the textbook distinction between poisonous animals (relying on absorption of their toxins) and venomous ones (creating a wound to guide toxins into the victim)51. Well-known toxin delivery systems range from macroscopic structures (like snake fangs or insect stingers) down to cellular adaptations (nematocysts in cnidarians). The molecular delivery system identified here represents an extension down the biological scale, and show that toxin delivery can be mediated at any suborganismal level.We anticipate that other animals administering their toxins through absorption may use similar mechanisms of tissue permeabilization. The defense secretions of several whipscorpion species (order Thelyphonida), for example, contain caprylic acid, which, besides increasing the effective contact area of the sprayed secretion, enhances the penetration of cosecreted acetic acid through arthropod cuticleExperiments involving live animals were conducted in accordance with the European guidelines and the Belgian legislation on animal housing and experimentation, and were approved by the Ethical Committees of Animal Experimentation of the Vrije Universiteit Brussel (Permit nos. EC15-272-7 and EC16-334-1) and by the Ethical Committee of the Faculty of Veterinary Sciences of Ghent University (Permit no. EC2015/26).X. laevis (Pipidae); (2) the toxin bombesin (Bom) and the AMP BLP from the skin of the oriental fire-bellied toad B. orientalis (Discoglossoidea: Bombinatoridae); and (3) the toxin dermorphin (Drm) and the AMP dermaseptin-S1 (Drs) from the skin of the waxy tree frog P. sauvagii (Neobatrachia: Hylidae). Sequences and molecular weights of these peptides are provided in the appropriate figures . The selected AMPs show broad-spectrum antimicrobial activity as confirmed by previous studies53 and are considered part of the frogs’ innate immune system18. All peptides were synthesized via standard Fmoc-based solid-phase peptide synthesis by the authors and by CASLO Laboratory ApS . For radiolabeling, a caerulein analog, modified with a diethylenetriaminepentaacetic acid (CHX-A″-DTPA) at the N-terminal side of the peptide , as a chelator for the isotope indium-111  the toxin caerulein (Cae) and the AMP caerulein precursor factor-3 (CPF) from the skin of the African clawed frog  the toxiipidae);  the toxi2) at a constant temperature of 37 °C. To prepare an experiment, cultured cells were seeded on the appropriate inserts/wells/coverslips 21 days in advance, at a concentration of 2×105 cells/ml and further cultured to develop confluent and fully differentiated monolayers . The progress of confluence and differentiation was checked by light microscopy and measuring electrical resistance across the cell layer, respectively.Two models of gastrointestinal epithelia were selected to represent the mucosal barrier that toxins must cross in order to enter the basolateral tissue and blood of a predator. The Caco-2 cell line  was chosen because of its wide application in in vitro epithelial drug transport studies. EpiOral 3D tissues  were selected to test toxin transport across a more complex epithelial model composed of 8–11 cell layers. Caco-2 cells were cultured in cell medium  in a humidified incubator /(ODP − ODN), where ODX is the corrected OD value of sample x, ODN is the average corrected OD obtained for the nine negative control wells, and ODP is the average corrected OD obtained for the six positive control wells. Note that the resulting % LDH leakage can be higher than 100%, e.g., if a peptide does not lyse all cells , leaving the surviving and damaged cells to continue to produce and release additional LDH. Consequently, our LDH leakage assays should be regarded as measures of cell membrane damage, not cell death.Cell-damaging activity of the peptides was tested using commercial LDH II assay kits  and Caco-2 cell cultures prepared in 96-well plates (see above), with each well containing 200 µl of medium. For each toxin–AMP pair (see above), cell cultures were exposed for 5 min to three different AMP + toxin concentrations  and a single toxin concentration (100 µM). Additionally, we tested the effect of only administering the AMPs at the same three concentrations. Nine wells containing cells and medium but no peptide served as negative controls . Six wells containing 1% triton-X lysis solution were used as positive controls . After completing the colorimetric reaction according to the kit’s manual, optical density (OD) was measured at 450 nm using a multiskan FC microplate photometer/ELISA reader (Thermo Scientific). The cell-damaging effect of a peptide (in % LDH leakage) was calculated as: 100 × (ODn = 9) were monitored: (1) apical administration of the toxin alone, at 100 μM; (2) apical administration of toxin + AMP, both at 100 μM; (3) toxin + AMP, both at 50 μM; (4) toxin + AMP, both at 25 μM; and (5) no peptide administration (negative control). Caco-2 cells were prepared (see above) on collagen-coated polystyrene transwell insert filters with a 0.4 µm pore size . Inserts were placed in 24-well plates (provided with the inserts), creating an apical compartment (the insert) and a basolateral compartment (the well). Monitoring of TEER was done using a STX2 “Chopstick” electrode connected to an EVOM voltohmmeter (World Precision Instruments Inc). The original TEER , was measured 30 min before administration of the peptides. Peptides were administrated by replacing the apical medium by fresh medium in which the peptides were dissolved. As frequent exposure to electrical currents can damage epithelial cells , five treatments (Caco-2 cells were seeded on glass coverslips that were coated with rat tail collagen 1 (Sigma-Aldrich) the day before. The effect of four treatments  on differentiated Caco-2 monolayers was visualized. Chemical fixation was performed in 12-well culture plates 5 min after peptide administration . For fixation, plate wells were filled with 1 ml 2,5% glutaraldehyde in 0.05 M sodium cacodylate buffer  for 20 min, and subsequently washed twice with the same buffer. Postfixation was performed with 1% osmium tetroxide for 30 min, followed by three rinses with double-distilled water. Dehydration was carried out using a graded hexylene glycol series: 30; 50; 70; and 90%, taking 15 min for each step. Cells were rinsed three times for 10 min with 100% hexylene glycol, and then two more times for 10 min with 100% ethanol. Critical-point drying of coverslips was done using a Balzers union CPD020. Dried coverslips were mounted on carbon-coated stubs with double-sided conductive tape. Gold coating happened with a Jeol 1200JFC fine coater, after which coverslips were examined with a Jeol JSM-840 scanning electron microscope operating at 12 kV.n = 9), or after apical administration of 20 nmol toxin + 20 nmol AMP . Toxin passage across EpiOral multilayered cell cultures was investigated for the caerulein–CPF pair in a similar setup (n = 8). Each basolateral sample was stepwise diluted in cell medium and transferred to an ELISA kit plate. Six wells containing negative control samples (only cell medium) and two wells containing positive control samples (supplied with the kits) were added. Assays were performed following the standard protocols provided with the kits’ manuals. Standard curves for each toxin were obtained in duplicate. OD was measured at a wavelength of 450 nm using a multiskan FC microplate photometer/ELISA reader (Thermo Scientific). OD values were converted into toxin concentrations using the four-parameter curve fitting software .Toxin concentrations were quantified by ELISA using commercially available kits that show 100% cross-reactivity with the toxin in question. Caerulein was quantified using the Cholecystokinin Octapeptide (CCK) (26-33) (non-sulfated) ELISA kit , bombesin was quantified using the Bombesin ELISA kit , and dermorphin was quantified using the MaxSignal Dermorphin ELISA kit (Biooscientific.com). For each toxin–AMP pair, AMP-induced increase in the passage of the toxin across Caco-2 monolayers was investigated by comparing basolateral concentrations at 60 min after apical administration of either 20 nmol of the toxin alone . Caerulein concentrations in X. laevis skin secretions were estimated using ELISA. Adult female X. laevis frogs (n = 8) were ordered from a licensed commercial breeder (Xenopus Express France) and housed in plastic containers of 70 × 45 × 45 cm (L×B×H), filled with 25 cm of aged tap water with a filtration system, and kept in a acclimatized animalarium with a 12/12-h day-and-night cycle. To collect skin secretion, each frog was placed in a plastic ziplock bag and manually massaged to simulate the swallowing motion and esophageal peristalsis of a predator (as expected during an attack). The secretion produced during this stimulation tends to stick to the inner surface of the bag and is therefore easy to collect. After 5 min, all secretion on the frog and the bag’s inner surface was collected in a pre-weighed 1.5 ml microcentrifuge tube using a spatula, briefly centrifuged at 10,000 r.p.m. and weighed again to obtain a net weight for the skin secretion. Obtained skin secretion samples ranged between 85.7 and 419.8 mg, roughly corresponding to 85–420 μl. The secretion samples were subsequently diluted to obtain 10 ml of a 9 M urea/0.1% TFA solution, and fully dissolved by brief vortexing. A 100 μl sample of each solution was used to measure caerulein amounts and concentrations via the ELISA procedure described above. Using the ELISA results, we inferred that the individual frogs produced between 0.12 and 4.27 mg caerulein, amounts that are high, but comparable to those estimated for several peptide toxins in other frogs40. Combined with the estimated skin secretion volumes, the estimated amounts correspond to skin secretion concentrations between 1.0 and 7.5 mM, and the lowest limit of this range was used as a biologically realistic concentration for in vivo experiments (see below).Previous studies have estimated that AMP concentrations in the skin secretion of various frog species are in the millimolar rangeThamnophis eques (Colubridae: Natricinae) after oral administration in the presence vs. absence of CPF. Adult female snakes of this species (n = 18) were purchased from J & M Garters , and housed in 70 × 40 × 20 cm plastic boxes (L×B×H), kept in a acclimatized animalarium with a 12/12-h day-and-night cycle at 20–25 °C. A first experiment was designed to test a biologically realistic peptide concentration, and involved a comparison of two treatments (n = 3): (1) oral administration of 50 µl buffer solution, containing 50 nmol caerulein + 50 nmol CPF ; and (2) oral administration of 50 µl buffer solution containing 50 nmol (1 mM) caerulein alone. A sample size of three was chosen because it allows for sufficient statistical power using linear mixed modeling (see below) at a minimum cost of animal lives. A second experiment was designed to test a peptide concentration corresponding to our in vitro tests, which involved the following two treatments (n = 6): (1) oral administration of 50 µl buffer solution containing 5 nmol caerulein + 5 nmol CPF ; and (2) oral administration of 50 µl buffer solution containing 5 nmol (100 µM) caerulein alone. Here a sample size of six was chosen because we suspected a lower effect size than in the previous experiment due to the lower peptide concentrations.To investigate toxin absorption in a live model predator, radiolabeled caerulein was quantified in the blood and organs of snakes of the species 29, yielding values in pmol/gram BW. Note that these values may represent an increasingly larger underestimation of the actual amount of toxin absorbed by a snake’s body, since an increasingly large fraction of the orally administered dose will have been transferred from the blood to other tissues. As an additional measure for systemic toxin exposure, we calculated for each snake the AUC value (expressed in pmol×min/g). The AUC value incorporates multiple successive measurements of the amount of toxin in the blood, and is calculated as the area under the connecting lines between successive data points as defined by the commonly used “trapezoidal rule” method56.For each experiment, snakes were selected for both treatments to obtain identical distributions of BWs. Caerulein-DTPA (65 or 375 nmol) was labeled with 111InCl3 (65–90 MBq)  in 0.2 M NH4OAc  by incubation at 50 °C for 30 min . Radiochemical purity (>95%) was verified by iTLC on silica gel  using 0.1 M sodium citrate buffer (pH 5.0) as mobile phase. Snakes were anesthetized with an intracardial injection of propofol (12 µg/g BW) and subsequently positioned on their back. Peptide solutions were orally administered with standard syringes by carefully dripping the content (50 µl of a 1 mM or 100 µM buffer solution) on the palate of the snakes, after which the remaining radioactivity of the syringe was measured to determine the radioactivity of actual administered oral dose. Blood samples (10–50 µl) were taken from the heart with standard insulin syringes at predetermined time points after the start of the experiment . After 30 min, snakes were euthanized with an intracardial injection of T61 (0.5 µg/g BW) and organs and tissues were collected and weighed. Organ and tissue radioactivity was measured in a gamma counter against a standard of known activity and corrected for the decay in radioactivity. The investigator conducting these measurements was blinded to the oral administration treatments (presence vs. absence of CPF). Before the onset of the experiment, it was decided that samples would be excluded from the analysis only if radioactive contamination during the dissection was deemed highly probable. This was the case for one measurement for the brain (one snake treated with 1 mM caerulein + CPF), where radioactive contamination from the oral cavity during excision of the brain from the skull could not be ruled out. Caerulein levels in the snakes’ blood and organs were expressed in pmol/gram (blood or tissue). Unlike experiments based on intravenous injection, it is impossible to infer absolute absorption efficiencies based on the total amount of toxin in a subject’s body at any time after oral administration. Yet, to allow comparison with doses based on whole BWs, as reported in previous studies on caerulein toxicity, the obtained blood values were corrected for the proportion of blood in a snake’s body (5–8%)https://www.r-project.org; linear mixed models). Differences in TEER among peptide treatments were evaluated using one-way analysis of variance with a Tukey  or Games–Howell  post hoc test. Pairwise differences in transepithelial toxin passage between treatments (ELISA) were evaluated using Student’s t-tests. Linear mixed models considering “time” and “treatment” (different peptide administrations) as fixed factors and “snake individual” as random factor were used to test for differences in caerulein blood levels over time between treatments. Linear mixed models considering “organ” and “treatment” as fixed factors and “snake individual” as random factor were used to evaluate their effects on caerulein levels in organs and blood after 30 min.All data series were tested for normality and equality of variance and subsequently analyzed using SPSS version 23.0  and R version 3.1.1 (The data that support the findings of this study are available from the corresponding author upon reasonable request.Supplementary InformationPeer Review File


Even at these low concentrations, H2O greatly affects the physico-chemical properties of mantle materials, governing planetary dynamics and evolution. The diffusion of hydrogen (H) controls the transport of H2O in the Earth’s upper mantle, but is not fully understood for olivine 2SiO4) the most abundant mineral in this region. Here we present new hydrogen self-diffusion coefficients in natural olivine single crystals that were determined at upper mantle conditions (2 GPa and 750–900 °C). Hydrogen self-diffusion is highly anisotropic, with values at 900 °C of 10−10.9, 10−12.8 and 10−11.9 m2/s along [100], [010] and [001] directions, respectively. Combined with the Nernst-Einstein relation, these diffusion results constrain the contribution of H to the electrical conductivity of olivine to be σH = 102.12S/m·CH2O·exp−187kJ/mol/(RT). Comparisons between the model presented in this study and magnetotelluric measurements suggest that plausible H2O concentrations in the upper mantle (≤250 ppm wt) can account for high electrical conductivity values (10−2–10−1 S/m) observed in the asthenosphere.Nominally anhydrous minerals formed deep in the mantle and transported to the Earth’s surface contain tens to hundreds of ppm wt H Downwelling oceanic lithosphere (at subduction zones), and upwelling magmas  are vehicles for transport of H2O between the surface and the Earth’s deep interior1. Experimental studies have shown that substantial concentrations of H2O may be present in the mantle, hosted as defects in the structure of nominally anhydrous minerals (NAMs). The major mineral component of the uppermost mantle, olivine 2SiO4), can incorporate as much as ~1 wt% H2O at conditions encountered at the lowest portion of the upper mantle4. Minerals found in mantle xenoliths, including olivine, that are brought to surface by alkaline or kimberlitic magma eruptions, contain up to hundreds of ppm wt (parts per million by weight) H2O5, providing direct evidence of H2O incorporated in NAMs from the Earth’s interior. Recently, a crystal of terrestrial ringwoodite (a high-pressure polymorph of olivine that forms below ~410 km depth), discovered as an inclusion in a natural diamond, was found to contain ~1.4 wt% H2O6. This confirms that at least some regions within Earth’s transition zone contain large concentrations of dissolved H2O.Earth’s hydrosphere is a distinctive feature of our planet where massive oceans affect its climate and support its ecosystem. The distribution of H2O, as hydrogen (H) bonded to the crystal structure, can influence the physical and chemical properties of minerals even if only present at ppm wt levels7. For example, H influences the rheological properties of minerals and has been proposed as a possible factor in weakening H-bearing olivine in the convective mantle while relatively dry olivine is thought to stabilize the mantle beneath continents8. Furthermore, H enhances the formation of fluids/melts within the Earth by lowering the melting temperature of mantle minerals9, affecting the chemical differentiation of the planet. Therefore, constraining the presence and transport of H in the Earth’s mantle is crucial to understanding planetary evolution and geodynamics.H12, relative to other cations and oxygen, resulting in a substantial contribution to its electrical conductivity13. Due to the high sensitivity to H content, electrical conductivity can thus be envisaged as a tool to investigate the H2O contents throughout the mantle.Knowledge of the electrical conductivity of mantle minerals is critical for interpreting magnetotelluric sounding measurements used to interrogate the structure and composition of the Earth’s interior. The diffusion of H in olivine is very fast2O on the electrical conductivity of olivine has been measured in high pressure (P) and high temperature (T) experiments. Two recent studies agreed that H enhances olivine’s electrical conductivity, but reached contrasting conclusions regarding the implications of this enhancement with respect to measurements of mantle electrical conductivity15. High electrical conductivity anomalies (10−2–10−1 S/m) are observed in the asthenosphere18, the region directly beneath the lithosphere where the upper mantle becomes relatively ductile. Yoshino et al.14 concluded that hydrous olivine is not capable of explaining these high electrical conductivity values, while Wang et al.15 concluded that limited amounts of H (~80 ppm wt H2O) dissolved in olivine are sufficient to match these geophysical measurements. The use of different analytical techniques and calibrations to determine sample H2O contents complicates direct comparisons of the measurements from various groups19. Moreover, the use of different experimental procedures may have affected the electrical conductivity measurements and also could be to blame for the apparent disagreements between datasets22. Despite substantial follow-on work devoted to determining the influence of H on olivine and mantle conductivity26, the disparate interpretations remain unresolved.The influence of Hin-situ electrical conductivity measurements. The Nernst-Einstein relation can be used to relate generalized mobility of H to its electrical mobility13. In the absence of a melt or fluid phase, the diffusion of H in mantle minerals, and along their grain boundaries, controls its distribution in the Earth’s interior. Chemical diffusion of H in olivine, i.e. the diffusion due to a concentration gradient, has been investigated by a number of groups27. Hydrogen self-diffusion  rate-limits chemical diffusion and is poorly constrained, limiting our knowledge of hydrogen mobility in the Earth’s mantle. Two studies have investigated H self-diffusion coefficients in enstatite28 and ringwoodite29. One recent study reports H self-diffusion coefficients in natural olivine along the fast [100] direction, but was unable to resolve profiles in the slow [010] and [001] directions30. The lack of experimental data is mainly due to (i) the challenges in successfully performing consecutive, multi-step self-diffusion experiments at elevated P-T conditions30 and (ii) the spatial resolution required to analyze diffusion profiles along both the fast and slow diffusion directions. Hydrogen diffusion profiles can be hundreds or tens of microns in length within the same sample of highly anisotropic olivine30.Hydrogen self-diffusion in olivine provides an alternative method for determining electrical conductivity that circumvents some of the experimental difficulties associated with previous H) in single crystal olivine are reported for all three principal crystallographic orientations. Results were obtained at 2 GPa and between 750–900 °C via H-D (hydrogen-deuterium or 1H-2H) exchange experiments followed by high spatial resolution analysis of the resulting isotopic profiles utilizing Secondary Ion Mass Spectrometry (NanoSIMS). The new results better constrain the effect of H on olivine electrical conductivity for all orientations, and reconcile some of the discrepancies in previously reported results. A diffusion-based model of electrical conductivity is used to infer that olivine H2O contents comparable to those expected for mid-ocean ridge basalt (MORB), and below ocean island basalt (OIB), mantle sources can explain electrical conductivity anomalies observed in the asthenosphere18.Here, for the first time, H self-diffusion coefficients (D2H (D) were successfully measured along [100], [010] and [001] directions of samples cut from a single crystal of olivine. The samples were prepared using a three-step procedure consisting of a ‘dry’ anneal at high T and controlled fO2, a ‘wet’ anneal to saturate the samples with hydrogen at high P and T, and a final ‘exchange’ anneal for the isotopic exchange at high P and T (see Methods and Du Frane and Tyburczy)30.Diffusion profiles for 3+) accommodated by existing metal vacancy sites, or (2) slower concurrent diffusion of negatively charged metal vacancies into the crystal (i.e. creation of new sites)11. The ‘dry’ anneal step, >16 hrs at 1300 °C and fO2 close to that of a Ni-NiO buffer  is used to establish a homogeneous, equilibrium concentration of small polarons and metal vacancies throughout the crystal lattice11. During the wet anneal at 2 GPa and 750–900 °C, hydrogen is then expected to enter the crystal via the more rapid mechanism of redox exchange with polarons on metal vacancy sites that were established during the prior dry anneal step11. Then in the final step, deuterium is exchanged with hydrogen established in the wet anneal. The center of the single-crystal sample PC28  using the same capsule assembly32. H2O concentrations of samples PC25 and PC33 are expected to be similar because their dry anneal steps were performed at the exact same conditions, and their wet anneal steps were performed at only slightly higher or lower temperatures.Two mechanisms have been proposed for incorporation of hydrogen into Fe-bearing olivine: 1) redox exchange with positively charged small polarons , DH,[hkl] is H self-diffusion coefficient along [hkl] direction (m2/s), Ha,[hkl] the activation enthalpy (in kJ/mol), T is the absolute temperature (K) and R is the gas constant (8.314 J/mol·K). The subscript in equation , CH is the concentration of H (1/m3), q is the charge (C), k is the Boltzmann constant (1.381 × 10−23 J/K) and T is temperature (K). The electrical conductivity of mantle olivine as a function of temperature  as a function of orientation . Du Frane and Tyburczy30 proposed a similar model, employing more appropriate H self-diffusion coefficients for the [100] direction, but only estimates for the [010] and [001], and concluded that H2O contents required to satisfy the observed conductivity anomalies in the asthenosphere likely exceeded the olivine storage capacity (up to 1 wt%). This recent effort30 is substantially improved in this study by the inclusion of experimentally determined H self-diffusion coefficients for DH,[010] and DH,[001], and higher precision coefficients for DH,[100] 30, which is implausible based on the relative mass, ionic radius, and substantially higher site density of polarons in olivine.Use of the Nernst-Einstein relation and H diffusivity to estimate electrical conductivity in mantle olivine has been recently challenged30 and presented in this study, it is likely that mixed vacancy-interstitial diffusion in olivine is controlled by a dissociative mechanism. In this case, an activated H dissociates from a metal vacancy and migrates as an interstitial particle until it is trapped again at another thermal vacancy47. This mechanism explains the relatively high hydrogen self-diffusion coefficients that are closer in magnitude to those of small polarons than metal vacancies within a crystal structure that predominantly contains sites that are metal vacancies. For the dissociative mechanism, the mean free terminal drift velocity of H would be the same in response to a chemical potential gradient or an electrical potential gradient (i.e. mobility), justifying the use of Nernst-Einstein relation for H diffusion and H diffusion data to model electrical conductivity.To be consistent with chemical- and self-diffusion results for H in olivine published previously25 for H2O contents at or below 250 ppm wt H2O, which is the storage capacity of olivine at ~240 km depth in the asthenosphere37  become significantly higher (2.14–2.25 eV or 206–217 kJ/mol)23 and consistent with values presented here for hydrogen self-diffusion (172–229 kJ/mol). For 1380 ppm wt, the model is in poor agreement with studies that included samples having very high H2O contents26 that would only be possible in the lowermost portion of the upper mantle  to diffusively exchange with hydrogen (1H). The method imposes a hydrogen isotopic gradient in the absence of a hydrogen concentration gradient. 2H is predicted to diffuse similarly to 1H but with some diffusive fractionation (with D(1H) = D(2H)/√2). Samples were all cut from one single, large crystal that was oriented by Laue back-scattered x-ray diffraction. From this procedure, olivine cuboids whose edges of ~1 mm length were aligned along the three principal orientations [100], [010] and [001]. Further details regarding the samples and experimental setup are reported previously30.Oriented single crystals of San Carlos olivine were pre-annealed via the redox exchange mechanism16O2H- normalized to 28Si- using a CAMECA NanoSIMS 50 housed at Lawrence Livermore National Laboratory (LLNL). The unique features of the NanoSIMS allow for both the high spatial resolution and detection sensitivity that are required to resolve the extremely short diffusion profiles of trace elements, such as H, along [010] and [001] directions.Hydrogen self-diffusion along all three principal orientations in olivine were measured as a gradient in + beam of ~10 pA that was focused on the sample surface resulting in the ejection of secondary ions. A normal incidence electron flood gun (~100 nA e− in ~60 µm diameter area) was used for charge compensation during the analyses. For each analysis, the surface was sputtered prior to data collection for 3 minutes over a 4 × 4 µm2 square, which was followed by ~7 minutes of collecting time from a 2 × 2 µm2 area at the center of the previously sputtered region . 16O1H−, 16O2H−, 28Si− and 12C2− were simultaneously collected on electron multipliers in pulse counting mode. Despite these efforts, 16O2H− profiles are used to fit diffusion coefficients, because 16O1H− measurements are more challenging and generally have higher errors associated with tuning and alignment of the ion beam, and interference from background levels and surface contamination of 1H. The 12C2− count rate was used to identify analysis locations that included cracks or impurities  analytical profiles, quantified as 16O2H− /28Si−, begin at the sample edge and progress toward the center of the crystal, on a path perpendicular to the edge. To avoid interference with diffusion from other orientations, profiles were positioned near the center of each edge .The dataset presented here is limited to three experiments at three different temperatures, but further work will be needed to increase the robustness of knowledge of hydrogen self-diffusion in olivine. Additional experiments over a wider spread and repeat temperatures would reduce uncertainties in activation enthalpies, and therefore extrapolations to higher temperatures. It is possible for there to be slight crystal to crystal variations in hydrogen diffusion coefficients measured in NAMsSEM images were collected to investigate the array of analyses forming the different profiles. This procedure is essential to determine the exact distance from the edge of the crystal of the craters formed by the NanoSIMS analyses, and to identify the presence of sources of impurities such as cracks or fractures in the analyzed pits that could invalidate a measurement. The images were collected with an FEI INSPECT F SEM at LLNL, which was operated with an accelerating voltage of 15 kV. Both secondary electron and backscatter electron images were collected to evaluate topographical and compositional details of the analyzed areas  wavelength dispersive x-ray analyses were also performed to accurately determine the composition of olivine and exclude from analyses any areas of chemical zonation in the investigated samples. No significant crystal growth was observed at the boundaries of samples under electron microprobe. The analyses were conducted by means of a JEOL JXA 8200 electron microprobe at LLNL, which is equipped with 5 separate spectrometers. Spot analyses (~1 µm diameter) were conducted on the crystals at 15 kV accelerating voltage and 15 nA beam current. Counting time was 20 s on peak position and 10 s on each side for background, and quantitative analyses were made possible by means of MgO, FeSupplementary information


Ribosomal RNA modifications are important in optimizing ribosome function. Sugar 2′-O-methylation performed by fibrillarin-associated box C/D antisense guide snoRNAs impacts all steps of translation, playing a role in disease etiology (cancer). As it renders adjacent phosphodiester bonds resistant to alkaline treatment, 2′-O-methylation can be monitored qualitatively and quantitatively by applying next-generation sequencing to fragments of randomly cleaved RNA. We remapped all sites of 2′-O-methylation in human rRNAs in two isogenic diploid cell lines, one producing and one not producing the antitumor protein p53. We identified sites naturally modified only partially  and sites whose 2′-O-methylation is sensitive to p53. We mapped sites particularly vulnerable to a reduced level of the methyltransferase fibrillarin. The remarkable fact that these are largely sites of natural hypomodification provides initial insights into the mechanism of partial RNA modification. Sites where methylation appeared vulnerable lie peripherally on the 3-D structure of the ribosomal subunits, whereas the numerous modifications present at the core of the subunits, where the functional centers lie, appeared robustly made. We suggest that vulnerable sites of 2′-O-methylation are highly likely to undergo specific regulation during normal and pathological processes. Ribosomal RNA is extensively modified by base isomerization and by covalent addition of chemical groups to nucleotides2. RNA modifications extend the topological repertoire of RNAs, stabilizing rRNA structural elements at the core of the ribosomal subunits3. Covalent modifications include ribose and base methylation, base acetylation, and base aminocarboxypropylation4. Base isomerization consists in the conversion of uridines into pseudouridines. With around fifty of each on yeast ribosomes and 100 of each on human ribosomes, 2′-O-methylations and pseudouridines are by far the most abundant rRNA modifications2.Ribosomes are essential nanomachines which, in all living cells, convert the information encoded in messenger RNAs into proteins. At the functional core of the ribosome is ribosomal RNA (rRNA), a ribozyme catalyzing the critical steps of decoding and amino acid polymerization5. Ribosomopathies are cancer predisposition syndromes caused by ribosome biogenesis dysfunction resulting from a mutation in either a ribosome assembly factor or a ribosomal protein6. There are ribosomes in every cell of our body, but the effects of reduced ribosome production or altered ribosome quality depend on the tissue and cell type7. Understanding how this cell/tissue specificity originates is a major challenge in the field. There is increasing evidence that cells produce heterogeneous populations of ribosomes that may differ in composition and translational capacity8. Specialized ribosomes may play important roles in normal processes such as embryonic development and in diseases such as cancer10. The best demonstration of the coexistence of distinct ribosomes in cells is partial modification, i.e. modification at specific positions on some but not all ribosomes , reading frame maintenance, and stop codon recognitionRecently, deep-sequencing-based strategies have been developed to map RNA modifications at the transcriptome-wide level. These strategies rely on the ability of RNA modifications to be detected by antibodies, to promote specific reverse transcriptase ‘drop off’, to confer specific resistance to RNase digestion, or to induce specific cleavage of the RNA chain when subjected to particular chemical treatments  and one that does not (HCT116 p53 −/−)15. Altogether, our RiboMethSeq analysis identified 106 positions on mature rRNAs: 39 sites on 18S rRNA, 65 sites on 28S rRNA, and 2 sites on 5.8S rRNA  by unassembled ribosomal components see refs  and 40. 35. Expecting this to be important in interpreting the results of systematic 2′-O-methylation site mapping after fibrillarin depletion (see below), we examined the extent and kinetics of the impact of fibrillarin depletion on the formation of mature rRNAs in the cell lines used here.Fibrillarin homologs in budding yeast  and other eukaryotes, including in HeLa cells, have been shown to be important for ribosome biogenesis, and in particular for the initial pre-rRNA processing steps leading to synthesis of the small-ribosomal-subunit rRNA (18S rRNA)Total RNA was extracted at different time points during fibrillarin depletion  from both HCT116 p53 +/+ and HCT116 p53 −/− and resolved by denaturing agarose gel electrophoresis. The large mature rRNAs (18S and 28S) were visualized by ethidium bromide staining Fig. . This reTo gain precise information on the inhibited step(s) of ribosome biogenesis, pre-rRNA processing was analyzed in detail by high-resolution quantitative northern blotting. Specific probes were used to detect all the major known pre-rRNA intermediates  involves extensive processing of long primary transcripts (47S pre-rRNA) synthesized by RNA polymerase I. In the primary transcripts, the sequences of the mature rRNAs are interspersed with the 5′ and 3′ external transcribed spacers (5′ and 3′ ETS) and the internal transcribed spacers 1 and 2 (ITS1 and 2) . All of the modified positions described above were identified, a methylation score was computed for each, and the methylation scores were clustered with the dedicated software package ‘R’ Fig.  and 4. AAt this point we can see how important it was to establish precisely the levels of the various pre-rRNA intermediates over the time course of fibrillarin depletion: it appears that the reduced methylation observed at specific sites cannot be explained solely by reduced production of the precursors harboring them. Most of the positions where methylation appeared particularly sensitive to fibrillarin depletion  were grouped in six categories . We predict that in cells, these snoRNAs compete particularly efficiently for fibrillarin binding during snoRNP assembly.Lastly, we mapped the sites of methylation vulnerability identified in this work to 3-D models of ribosomal subunits, using a specific color code for sites of putative hypomodification, sites sensitive to p53, and sites particularly sensitive to fibrillarin depletion Fig. . Sites o9. This makes it important both to establish the complete repertoire of ribosomal RNA modifications and to identify positions likely to be modified on only a subset of ribosomes, as modification is liable to undergo considerable regulation at these sites.Ribosomal RNA modifications have been shown in multiple cases to optimize ribosome function . The cell lines used in this work were obtained directly from ATCC and passaged in the laboratory for fewer than 6 months after receipt. All cell lines were diagnosed by ATCC by short tandem repeat (STR) profiling. In HCT116 p53 −/−, the endogenous alleles of p53 were disrupted sequentially by homologous recombination37.Human cells (HCT116 p53 +/+ and HCT116 p53 −/−) were grown at 37 °C under 5% COThe total number of viable cells and the percentage of viable cells in each sample analyzed were established with a Muse® cell analyser (Merck-Millipore) and the Cell Count and Viability Reagent (REF MCH100102), according to the manufacturers’ instructions.5 cells (for 72 hours), 3 × 105 (for 48 h) 6 × 105 (for 24 h) resuspended in 2.5 ml antibiotic-free medium were seeded into each well. Inactivation was carried out for 24, 48, and 72 hours. The silencers used are listed in Supplementary Table HCT116+/+ and HCT116−/− cells were reverse transfected as follows: 1.5 µl of 20 µM DsiRNA (Integrated DNA Technologies) or 20 µM siRNA (Life Technologies) and 4 µl Lipofectamine RNAiMAX (Life Technologies) were mixed with 500 µl Opti-MEM (Life Technologies) in each well of a 6-well plate. After a 20-min incubation at room temperature, 1.5 × 1046. The NGS-based method of 2′-O-methylation detection was as described for yeast in ref. Total RNA was extracted as previously described6 HCT116 cells. Cells were washed once with ice-cold 1x phosphate buffered saline (PBS) and lysed in lysis buffer  plus protease inhibitors  for 20 min on ice. Lysates were cleared by centrifugation at 20,000 g and 4 °C for 20 min. Protein concentrations were estimated by the Bradford protein assay (Biorad). 30 µg total human protein extract was loaded on a 12% SDS PAGE gel and transferred to a PVDF membrane . Antibodies against the following proteins were used at the indicated dilutions: fibrillarin ; p53 . The secondary antibody used was either a horseradish-peroxidase-conjugated anti-mouse IgG  or a horseradish-peroxidase-conjugated anti-rabbit IgG . Probed membranes were incubated for 10 min with the Super Signal West Pico Chemiluminescent Substrate (Pierce), and the luminescence signal was acquired and processed with a ChemiDoc MP (Bio-Rad).Total protein was extracted from ∼1 × 1046. The membranes were hybridized with 32P-labelled oligonucleotides and the signals acquired with a Phosphorimager .Total RNA was extracted from HCT116+/+ and HCT116−/− cells in Tri-reagent solution (Life Technologies) according to the manufacturer’s recommendations, except that the RNA pellets were washed with absolute ethanol and then with 75% v/v ethanol. The concentration and quality of the RNA were assessed with a Nano Drop 1000 spectrophotometer (Thermo Scientific). RNA electrophoresis on denaturing agarose gels and northern blotting were conducted as described previouslyThe RiboMethSeq reads were aligned to the reference rDNA sequence NR_046235. Clustering of methylation scores and snoRNA levels was performed with “R” with hclust function using Euclidean distances. To perform statistical analysis of snoRNA abundances after fibrillarin depletion, we used DESeq. 2.Supplementary InformationSupplementary Dataset Number 1


These LumC13 derivatives were tested both in vitro and in vivo to evaluate their ability to promote corneal epithelial cell migration and corneal wound healing, respectively. These validations add to the therapeutic value of LumC13 (Lumikine) and aid its clinical relevance of promoting the healing of corneal epithelium debridement. Moreover, our data validates the efficacy of our computational approach to design active peptides based on interactions of receptor and chemokine/ligand.Lumican, a small leucine rich proteoglycan (SLRP), is a component of extracellular matrix which also functions as a matrikine regulating multiple cell activities. In the cornea, lumican maintains corneal transparency by regulating collagen fibrillogenesis, promoting corneal epithelial wound healing, regulating gene expression and maintaining corneal homeostasis. We have recently shown that a peptide designed from the 13 C-terminal amino acids of lumican (LumC13) binds to ALK5/TGFBR1 (type1 receptor of TGFβ) to promote wound healing. Herein we evaluate the mechanism by which this synthetic C-terminal amphiphilic peptide (LumC13), binds to ALK5. These studies clearly reveal that LumC13-ALK5 form a stable complex. In order to determine the minimal amino acids required for the formation of a stable lumican/ALK5 complex derivatives of LumC13 were designed and their binding to ALK5 investigated  Proteoglycans (PGs) are complex polymers composed of a core protein to which one or more glycosaminoglycan (GAGs) chains are covalently attached. They play a central role in physiology and disease123459111315171219in vivo add to the therapeutic value of Lumikine in promoting healing of damaged corneal epithelium.Herein, we exploited the C-terminal Lumican peptide surroundings, followed by further exploration of ALK5 as a potential target and further developed suitable peptidomimetics of LumC-peptides. Molecular docking and MD simulation studies were carried out to study the interaction of the LumC-peptides and ALK5 complex, which provides insights into vital binding interactions. These interaction studies not only revealed the relatively stable interaction of LumC peptide with ALK5 but also highlighted the significance of these interactions in activating ALK5. We were also able to establish the minimal amino acid sequence required for the formation of the lumican/ALK5 complex. Validations of the peptides functions in wound healing et al.We used a modified HADDOCK benchmark for an efficient protein-peptide docking protocol, combining the conformational selection with induced fitΔC20 score is −77.3 ± 11.4, Lumikine (LumC13C-A) and LumikineY-F show a score of −83.8 ± 7.1 and −84.8 ± 7.0, respectively  and Lumikine complexes have again a similar HADDOCK score as the LumC13 complex, illustrating again a good affinity between the biological partners. LumC18ΔC5 and the score of the hybrid peptides  amount to similar score values of −79.5 ± 1.7, −55.8 ± 4.0, −76.0 ± 1.6 and −82.5 ± 0.5, respectively. The BSA is used to quantify the protein surfaces, which are not exposed to water. The LumC13 complex shows a BSA value of 1490.8 ± 109.1, while the BSA values of the LumC33ΔC20, Lumikine, LumikineY-F are 1375.6 ± 140.2, 1384.9 ± 79.8 and 1574.4 ± 138.3, respectively, and the LumC18ΔC5 and the score of the hybrid peptides exhibit a BSA score of 1450.3 ± 71.0, 1266.2 ± 138.5, 1441.9 ± 35.8 and 1284.2 ± 24.2, respectively , van der Waals (vdw), desolvation (Dsolv) and AIR energies, and a buried surface area (BSA) was obtained, allowing for a comparison of values. While the LumC33ectively . The lesvely see . A higheMolecular dynamics (MD) calculations have been previously used in understanding and characterizing the structural, dynamical, and functional properties of biomolecular systems. Our group has recently developed its application for the rational design of lumican peptides capable of binding ALK5ΔC20 monomer essentially cluster within mixed conformations, specifically around the C-terminus, leading to a large backbone root-mean-square deviation (rmsd) of 2.3 Å. We next clustered the similar conformations of hybrid peptides in comparison with LumC18ΔC5 using the cluster plugin in VMDC-A and LumikineY-F, while the mutation of a Tyr1 to phenylalanine in peptide LumikineY-F does not affect binding significantly. Those residues bind specifically to the GS-domain region  of the peptides are involved in ALK5 binding, while significant participation of the polar , and hydrophobic  residues is also observed. These results imply that the LumC33C-A is consistent with their frequent interaction with the GS domains of ALK5. Given the more heterogeneous nature of the hybrid surface, it is likely easier for the hybrid N-terminus tyrosine to find a complementary binding pocket that can satisfy all or majority of the possible charge-charge interactions. Thus, the C-terminus  appears to remain in contact with GS domain more frequently compared to the N-terminus.The higher contact probability of the mainly hydrophilic LumC13Further analysis reveals that this difference is caused by the different flexibility of hybrid 2/3 conformations sampling, reflecting its entropy. As shown in α atoms were analyzed to determine if these systems reached equilibrium.LumC13, a peptide that binds to ALK5 and peptides derivate from rational design were evaluated regarding their binding/interacting potency623In The probability of occurrence of hydrogen bonds was calculated by using the H-bonds plugin v1.2 in vmdΔC20-ALK5 complex system, one pair of hydrogen bonds exist between the side chain of Lys2 and the carboxyl of Glu247 and Glu250. The carbonyl oxygen atom of Glu247 also interacts with Ile1 through hydrogen bond. The side chains of Arg5 and Leu6 tend to form hydrogen bond with Ser191. In addition, the hydroxyl of Thr251 establishes a hydrogen bond with the N-gama nitrogen of His3 of LumC13.In order to further validate the binding modes, we carried out molecular dynamics simulations to gain detailed information about the key residues of the active site in each complex, the electrostatic, van der Waals, solvation and total contribution of the residues to the binding free energy of Lumikine–ALK5 and LumC-peptides-ALK5 interaction were calculated with the MM-PBSA method. The contributions from the residues around the GS-domain are shown in molecular mechanics Poisson–Boltzmann surface area (MMPBSA), and molecular mechanics generalized Born surface area (MMGBSA) techniquesSeveral techniques exist to predict binding free energies based on MD simulations, including free energy perturbation, thermodynamic integration, MMPB(GB)SA approaches have become standard for estimation binding free energies since they offer a good balance between speed and accuracy. MMPB(GB)SA combines force field based molecular mechanics (MM) with a continuum model, based on the Poisson–Boltzmann (PB) or generalized Born (GB) equation to handle electrostatic interactions, and a nonpolar solvation energy correlated with solvent accessible surface area (SA). This method can be applied to any aqueous binding process: it is not dependent on assumptions of small (perturbative) differences between two similar systems. The configurational entropy due to ligand–protein binding can be calculated by a normal mode (NMODE) method. The MMPB(GB)SA methodology has been applied in various small molecule–protein systems, including drugs binding to their receptors2627ele , and are correlated to the formation of several hydrogen bonds with LumC13 qualitatively, as shown in The contribution of selected residues surrounding the GS domain active site can be clearly seen from ele  implies that Ser187 might make a special contribution for active site of GS-domain. Strong electrostatic interactions result in the formation of hydrogen bonds between ALK5 and Asp7 and Glu2 in LumC13, LumC13C-A and LumC13AY-F.The strongest electrostatic contribution comes from the GS domains and the helix 8 located on its opposite site, which promotes one strong H-bond in complex. The lowest averaged value of ΔEele  implies that this residue might make a special contribution for peptide binding. Peptides outside EVTLN domain region reduced the binding by 10,000- fold , which were −218.0 kcal mol−1 in LumC-ALK5 complexes and −446.3 kcal mol−1 in Hybrid 2/3–ALK5 complex  shows negative contributions at 134.9 and 378.3 kcal mol−1 for LumC13 and Hybrid 2/3Y-F, respectively, which largely cut down the total contribution. Thus, the values of ΔGtotal for LumC13 and Hybrid 2/3Y-F are −83.1 and −68.0 kcal mol−1, respectively, which indicates that ALK5 has a stronger binding interaction to this hybrid than the LumC13/Lumikine peptide. We qualitatively studied the difference in terms of binding energy between the peptide and ALK5 domains; which indicated that the LumC13 has a stronger ability than the N-terminal peptides to bind to ALK5, which is in good agreement with our conclusion. In addition, the relative affinities of the C-terminal active sites of Lumican were very similar; thus, the difference in total binding energy between each domain and ALK5 might just be consistent with that C-terminal region.When the decomposition of the binding energy of the residues in both domains were compared, we found that the similar van der Waals’ interactions are found in peptides containing the EVTLN region. Nonetheless, the substitution of Y9 to F mutation in Hybrid2/3 complex . Howeverin vivo to determine capability to promote wound healing using a debridement wound healing model. As our computational studies predicted, peptides Lum13/LumC-A and Hybrid 2/3 exhibit an increased ability to promote wound healing. Hybrid 2/3 possesses the highest affinity in all designed peptides (Kd = 28.7 ± 3.9 lM), and thus can be considered as a promising lead to develop peptide therapeutics targeting wound healing.In short, computational methods identified peptide Hybrid 2/3 as capable of binding ALK5 domain with the lowest interactions energies, and that binding depends on the EVTLN domain. The computational studies predicted that although LumC peptide derivatives bind to ALK5 binding and pose as a potential new target for promoting wound healing. In order to further improve the peptides affinity, we subsequently attempted to modify and optimize the primary sequence of the peptide. The designed peptides were assayed in vivo, mice were subjected to a 2 mm with corneal epithelium debridement. The injured corneas were treated with 10 μl eye drops lumican peptides (0.3 μM) every 15 min for 5 h as previously describedC-A) treated corneas, the epithelium sheet moved ∼144 μm. The control peptide, LumC30ΔC20, did not promote epithelium wound healing, whereas peptide hybrid 2/3 promotes wound migration of about 107 μm. The average epithelium migration (μm) and standard deviation were calculated from n corneas of each of two separate experiments.To confirm peptide action promoting corneal epithelium wound healing Lum−/−) mice were also used to examine the efficacy of LumC peptides in promoting epithelium sheet migration following debridement wound , preventing further canonical TGFβ signaling via Smad cascade following polymerization of TGFBR2 and ALK5/TGFBR137384041in silico methodology, peptides were successfully tested experimentally for their propensity to promote in vivo wound healing after binding TGFBRI. The most potent of these peptides exhibited activity in an in vivo model of corneal epithelium debridement. With the in vitro evidence for modeling experiments, our analysis suggests that lumikine binding is targeted to the GS-domain of ALK5. Conclusions based on these simulations should take into account the effect of peptide initial conformations; although multiple copies of trajectories on the 100-ns timescale (enhanced sampling) may still affect the simulation results. The exploration of the contribution of binding free energy and its decomposition into per-residue analysis indicate that the conformational changes of the region around the GS-domain play an important role in understanding of the binding affinity to the LumC peptides.Here, we evaluated the binding free energy after decomposition into per-atom contributions, which could be summed over atom groups to obtain different energy contributions arising from residues backbone and side-chain by using the MMGBSA method, which is particularly useful to explore the variability of these peptides. After applying Our data also demonstrates great pharmacological applications of using LumC13 derivatives for promoting the rate of wound healing. This could be of particular interest in conditions where patients display delayed wound healing, such as diabetes, which culminates in an increased risk of inflammation and scarring. The significance of synthesizing lumican peptides that mediate TGFbRI signaling also goes beyond promoting skin and corneal wound healing. We have previously demonstrated that lumican inhibits prostate cancer cell migration, and thus is mediated through the TGFβ signaling pathway1Our approach represents a fast and straightforward approach that can be applied as a general method to determine binding modes of protein−peptide complexes and also to design TGBβRI inhibitors.in silico approach combining molecular docking and molecular dynamics was successfully used to predict minimal lumican amino acid sequence necessary for forming a stable complex with ALK5/TGFbRI. In vitro and in vivo wound healing assays demonstrated that LumC13, markedly the EVTL motif, is essential to promote epithelial cell migration to the same extent as lumican, thereby confirming the in silico data.An Lum−/− mice in C57BL/6 genetic background aged 7–8 weeks were used in this study. Mice were anesthetized with intraperitoneal injections of a combination of ketamine  and xylazine  prior to the surgery, and a drop of proparacaine hydrochloride 0.5% (Alcon) was applied to the cornea to deliver local anesthesia before injury. Briefly, a 2 mm diameter trephine was used to demarcate the central epithelial region of the eye and the epithelium within the demarcated region was mechanically removed using an Algerbrush II  while viewing the cornea under a stereomicroscope . Eight mice per group were treated and the experiments were repeated at least two times. Each mouse was topically treated with one drop peptide solution (10 μl) at concentration of 0.3 μM or 10 μL PBS every 15 min for 5 h after wounding. After 5 h, mice were sacrificed. The eyes were excised and fixed in 4% paraformaldehyde in PB . The migration of epithelium sheet was determined as previously described5−/− mice were used as negative controls, in which the epithelium sheet migrated less than 50 μm in 5 h  interactions were switched off within a distance of 10–12 Å, and the list of non-bonded interactions was truncated at 13.5 Å. A time step of 2 fs was used, and the trajectory data were written at 1 ps intervals. Intermolecular contacts (hydrogen bonds and non-bonded contacts) were analyzed with the LIGPLOT softwareEach complex structure solvated with 17,216 water molecules and 0.1 M KCl. After pressure coupling, individual protein-peptides complexes were gradually relaxed in 50-ns MD simulations, where convergence was monitored using the RMSD of the backbone atoms. MD simulations were performed using the NAMD programbind) for each complex was estimated by using the MM/PBSA approach previously based on the snapshots extracted from the single trajectory of the complex (single trajectory method)252853MM/PBSA binding free energy calculations: To determine the most stable complexes predicted by the molecular docking, the binding free energy  and the electrostatic (∆Eele); ∆GGB and ∆GSA are the relative polar and nonpolar contributions to desolvation free energy, respectively, and [−T∆S] represents the conformational entropic contribution at temperature T. In this work, ΔGpolar and ΔGnonpolar were calculated with APBSwhere ΔEGB) was calculated using the generalized Born (GB) approximation model developed by Onufriev et al.SA) part was computed based on the SASA determined with the ICOSA algorithmTo properly evaluate individual residue influence in the interaction between ALK5 and LumC peptides, a free energy decomposition process was applied to the mm_pbsa analysis. During the decomposition process, the polar contribution of desolvation free energy .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The molecular crowding effect on cytoplasmic mechanics was selectively studied by preparing simple in vitro models of cytoplasm from which both the metabolism and cytoskeletons were removed. We obtained direct evidence of the cytoplasmic glass transition; a dramatic increase in viscosity upon crowding quantitatively conformed to the super-Arrhenius formula, which is typical for fragile colloidal suspensions close to jamming. Furthermore, the glass-forming behaviors were found to be universally conserved in all the cytoplasm samples that originated from different species and developmental stages; they showed the same tendency for diverging at the macromolecule concentrations relevant for living cells. Notably, such fragile behavior disappeared in metabolically active living cells whose viscosity showed a genuine Arrhenius increase as in typical strong glass formers. Being actively driven by metabolism, the living cytoplasm forms glass that is fundamentally different from that of its non-living counterpart.Physiological processes in cells are performed efficiently without getting jammed although cytoplasm is highly crowded with various macromolecules. Elucidating the physical machinery is challenging because the interior of a cell is so complex and driven far from equilibrium by metabolic activities. Here, we studied the mechanics of  Active biomolecules such as enzymes and reactants must be transported via the cytoplasm and supplied at appropriate occasions and locations in cells. Physical agitation in the cytoplasm leads to jiggling of intramolecular conformations of proteins that are crucial to initiate biochemical reactions and facilitate subsequent signaling cascades. Cells that do not undergo metabolic activation in their cytoplasm are either not viable or sometimes enter a dormant state4.Elucidating the mechanics of the cytoplasm is essential for understanding cell behaviors because cytoplasm governs the dynamics of core functional molecules essential for living systems7 and slow glassy behaviors11 have been observed. Apart from cytoskeletons, living cytoplasm is highly condensed, containing a much greater amount [~30% weight fraction12] of macromolecules  that are not tethered to cytoskeletal structures. Although less noticed than the cytoskeleton, this macromolecular crowding also drastically affects cell mechanics13 and therefore regulates metabolism15. Physical activation by molecular motors directly induces out-of-equilibrium fluctuations and further alter the mechanics of a living cell’s interior16. Glass transition in such activity-driven systems19 was implied with the anomalous fluctuations observed in living cells20. In contrast to thermal fluctuations, however, little is known how to relate such activity-driven fluctuations to the glass formation of the system. The mechanics of living cytoplasm might thus be affected by cytoskeletal networks, metabolic activity, and molecular crowding in a complex manner21. It is therefore necessary to design an experimental study that dissects out each factor affecting cytoplasmic mechanics and selectively evaluates its consequences.Nonetheless, the physical nature of the cytoplasm is poorly understood. The mechanics of reconstituted cytoskeletons purified from the cytoplasm have been studied intensively; strikingly nonlinear responsesliving cells by comparing them with several simple in vitro models of the cytoplasm that lack cytoskeletons and metabolic activity: a globular biomacromolecule  solution and three types of cell extracts . The mechanics of these models and living cells were studied at the subcellular scale, using the optical-trap-based microrheology (MR) technique29. Without metabolic activity and cytoskeletons, we found direct evidence that glass transition occurs in these in vitro cytoplasm. The viscosity of these materials rapidly increased with the macromolecule concentration. This behavior in a manner of super-Arrhenius31 is similar to that of fragile colloidal suspensions close to their glass transition. All cell extracts from evolutionary and developmentally distinct cells  showed diverging viscosity at similar critical concentrations c* (~0.34 g/mL) that were surprisingly close to the physiological concentration in living cells (~0.3 g/mL).In this study, we investigated the mechanics in c*, metabolically active living cells (both eukaryotes and prokaryotes) show decent fluidity; however, it is lost in spontaneously inactivated cells. The fluidity of living cells was lost also when macromolecule concentrations were increased by osmotic compression13. The manner of the viscosity increase upon crowding was completely different from that in metabolism-deficient cytoplasm. Viscosity of a living cell’s interior retains the Arrhenius-like exponential increase over a wide range of concentrations, that is typical for strong glass formers8. These results indicate that living cells are not simply fluidized by their metabolic activity; they still maintain glass-forming property but are driven toward the qualitatively altered state that can be attained in activity-driven systems. We discuss the possible mechanism for the loss of fragility, in relation to the homogenization/relaxation with active stirring in living cells.These observations indicate that the fluidity of metabolism-deficient cytoplasm is “frozen” at physiological concentrations. Regardless of being similar to 32. Across this range of concentrations, aggregates or inhomogeneities34 were rarely observed under high-magnification and high-contrast microscopy.Before exploring the cytoplasmic mechanics, we studied a simple model for the crowding of biomacromolecules: a single-component solution of the globular protein BSA which was dissolved in aqueous buffer (HEPES NaCl) homogeneously up to 600 mg/mLu(t) of probe particles (diameter 2a = 1 μm) embedded in samples were measured using laser interferometry36. After application of the fluctuation-dissipation theorem (FDT)37 to the power spectral density P(ω) = u(t)u(0)〉eiωtdt of the observed fluctuations, the shear viscoelastic properties were obtained: G(ω)  of the surrounding material. For details, see the protocol in Supplementary Note S1 and our prior study25. As detailed later, we found that the BSA solution surrounding the probe did not elastically support the applied stress even at high concentrations. The viscosity η of the samples is then given as η = G″(ω)/ω. This estimate of viscosity should be equal to the static viscosity at a low shear rate limit38.We performed high-bandwidth passive microrheology (PMR). Thermal fluctuations η of the BSA solutions were measured over a wide range of concentrations (0.003–0.60 g/mL). A rapid increase in viscosity was observed above ~0.45 g/mL; this effect does not follow the simple power law or Arrhenius-type exponential behavior. Rather, the Doolittle equation39 fit the data well:A = 1.9 and the critical concentration c* = 0.78 g/mL (solid line). Here, ηw denotes the viscosity of water. Extrapolation of the fit to higher concentrations predicted that η would diverge at c* = 0.78 g/mL. The corresponding volume fraction, φ* was estimated to be ~57 % using a partial specific volume of BSA ~7.33 × 10−3 mL/g40. In the case of hard-sphere colloids, Equation  that lack metabolism by removing small metabolites using a centrifugation filter. With this protocol, most proteins and organelles involved in cell mechanics remained in the sample. In the IVCE, the aging-associated increase in viscosity was greatly reduced 29. A sinusoidal force F(t) = F(ω) exp(iωt) was applied to the probe particles, and the displacement responses u(t) = u(ω) exp(iωt) were measured. Using the compliant response function, α(ω) ≡ u(ω)/F(ω), we obtained the linear shear modulus of the surrounding medium: G(ω) [ = G′ (ω) + iG″ (ω) = 1/[6πaα(ω)], where a is the radius of probe particles] as detailed in Supplementary Note S1 and in our prior study25. Because this protocol does not assume FDT, complete agreement between AMR and PMR confirmed that our samples were in thermodynamic equilibrium , Xenopus eggs21 (blue squares), and HeLa cells (black diamonds), yielded almost the same results, as shown in Fig. η of the E. coli extracts fits well Equation 12 in living cells.After these control studies, we measured 25. The resulting high-bandwidth spectrum (0.1 Hz < ω < 100 kHz) also supported the concept of the glassy cytoplasm. For predominantly elastic materials, G′ (ω) is constant in frequency and greater than G″ (ω), whereas purely viscous materials have G″ (ω) which is proportional to frequency [G″ (ω) ≈ ωη] and G′ (ω) which is negligible. For all BSA solutions as well as IVCEs below 0.2 g/mL, G″ (ω) was much greater than G′ (ω) over the entire frequency range measured. This result indicates that the samples behave as a simple fluid, whereas their kinetics gradually slow down upon crowding , allowing the overall system to gradually flow. Because of the shortage of the bandwidth in our experiments toward smaller frequencies, the viscosity determined by PMR may be underestimated at concentrations close to c*.Similar phenomena have been observed for many colloidal suspensions close to the glass transitionx from the center of the probe . Using the effective spring constant k of the optical trapping potential (therefore referred to as trap stiffness), the applied force is obtained as F = kx. After measurement of the resulting constant velocity v of the probe, Stokes’ law provides the viscosity of the surrounding material asF. If a material is solidified because of gelation, for instance, it may flow only after “yielding” induced by the failure of stress-bearing structures. For example, probes in crosslinked actin gel stay at rest below yield forces and show occasional jumps and hops because of intermittent yielding when they move  in F  showed shear thickening (increased viscosity during shear) at greater F (>0.4 pN), Newtonian behavior was also observed at a smaller F  although ηpull was slightly greater than η, particularly when the sample concentration was close to c*.Accordingly, we measured the viscosity of the sample in an alternative way. Optical trapping forces ranging from 0.09 to 1.1 pN were applied to the probe particles, by the feedback control of the laser focus that maintains the constant distance ory Fig. . All datn F Fig. . Such liφ* ≈ 57% was also reported for eye lens α-crystallin41. It is reasonable that tightly folded globular proteins  show jamming at the volume fraction similar to hard spheres (φ* ~ 64%)49. A slight difference in φ* as compared to hard spheres was expected because φ* can weakly depend on the shape and polydispersity of colloids51. In contrast, it remains unclear why IVCEs showed much smaller c* values than did tightly folded proteins or hard spheres. In the cytoplasm, macromolecules assemble into higher-order structures which contain hydrated water. The situation is similar to suspension of dilated hydrogel particles that vitrify at extremely small concentrations of macromolecules52. The effective volume fraction φ in IVCE must be higher than the estimate from the dried mass fraction. This phenomena may explain the apparent discrepancy in c* between BSA and IVCE.The jamming volume fraction 53  as an Angell plotη of IVCE that is higher than 108 [Pa ⋅ s]. Here, the concern was whether the cells were viable with the virtually solidified cytoplasm where efficient transport of molecules was stopped. We therefore investigated “apparent” diffusion of fluorescently labelled macromolecules (GFP) in living cells. We first chose giant protoplasts of E. coli, which were artificially grown to spherical shape by digesting the cell walls with lysozymes  ceased growing after the cell wall was digested. Such minor spheroplasts showed irregularly heterogeneous textures under an optical microscope  were on the verge of glass transition; simple extrapolation of the fit in Fig. η measured by microrheology . For the small protein GFP, cytoplasm in the surrounding medium is not regarded as a continuum. Hence, the Stokes-Einstein relation η ∝ 1/D may not hold. Furthermore, the unavoidable errors enter the estimate of concentrations when cytoplasm is encapsulated in liposomes. We could still say that diffusion of both BSA solution and IVCE showed trends consistent with those obtained by microrheology; diffusion of GFP decreases rapidly toward the glass transition point.As a control, we encapsulated IVCEs into spherical bags (liposomes and emulsions) together with GFP and measured its diffusion by FRAP. We compared the inverse of the diffusion of GFP  of molecules is not sufficient to study their relation to glassy dynamics because fluctuations in such activity-driven glasses have been rarely explored and remain elusive in the field of glass studies to date.In contrast, the “apparent” diffusion of GFP in normal living spheroplasts  incorporated into the cell interior . The power spectral density of probe fluctuations P(ω) at high frequencies is converted to the imaginary part α″ of the response function α(ω) based on the FDT [P(ω) = 2kBT α″/ω]  and living cells in confluent epithelium  share a similarity in their mechanics; both showed the same power-law form of frequency dependence at high frequencies, i.e., G ~ (i ω)0.5 or equivalently α ~ 1/G ~ (i ω)−0.5 0.75 rather than the cytoplasm G ~ (i ω)0.5.We found that 8. The volume fraction of intracellular macromolecules was estimated from the ionic strength of the media according to Ponder’s law66. Notably, fluctuations of the probe particle responded immediately to the change of osmotic pressure , the osmotic pressure was changed by adding sucrose or water to the culture medialiving cells ηcell@4kHz/ηw is plotted in Fig. in vitro cytoplasm (ηext@4kHz/ηw). We found another typical glass-forming behavior that ηcell@4kHz/ηw increased exponentially with the macromolecule concentration. This result indicates that the living cell interior can be categorized as a strong glass former50. Angell plots of ηcell@4kHz revealed Arrhenius-like behavior, which is evidently different from in vitro cytoplasm . Since the power-law exponents β of the form α ∝ ωβ− in NIH3T3 cells are distinct from 0.5 at low concentrations  toward higher concentrations overlaps with that of living cells, ηcell@4kHz/ηw, as shown with the eye guide given by the thick pink line.The same tendency in static “elasticity” of cell cortex was reported in ref.living cells are quantitatively universal, which were not expected prior to this study  as well as in germ cells (Xenopus egg)69. Constituents of macromolecules in living cells dynamically change during the cell cycle70 and at different stages of embryonic development71. If cytoplasm was solidified because of gelation with specific cross-linking , its mechanics would largely depend on the slight difference of these chemical constituents. On the other hand, repulsive interactions that govern colloidal glass transitions are generally non-specific, and are affected by physical properties of colloidal constituents such as their shape, size, and stiffness, rather than their chemical details38 . The FDT violation and super diffusion seen in Supplementary Fig. 19. Note that not only the IVCEs but inactive cells are glassy (or less fluidized at least) at biologically-relevant concentrations ]/[activation energy] as a function of colloidal concentration; a greater slope leads to fragile glass formers50. Activation is solely thermal in fragile IVCEs whereas non-thermal activations in living cells make the slope (dE/dc) smaller. Without non-thermal activation, colloidal glasses are strong only when constituent colloids can be compressed more than 10-fold in their volume during glass formation74. Such extreme deformability is unlikely for a large part of cell constituents such as ribosomes. Therefore, the softness of cell constituents alone does not explain the non-fragile behavior of living cells; the metabolic activity is essential.Non-thermal activation is the key to understand the difference of fragility of cell extracts and in living cells Fig. . Being s76. When dE/dc is large, colloids in suspension cannot relax the frustration in local arrangements independently. This leads to inhomogeneous distributions of residual forces and stresses in the sample which can relax only through rare cooperative rearrangements77. On the other hand, soft gels, emulsions and foams form strong glasses50. Even if they are packed, they can still locally relax that the heterogeneous dynamics might be less likely to be observed. In our experiments, heterogeneity of viscosity in cell extracts were not large . The critical concentration c* for jamming was found to be nearly identical to the physiological concentration of metabolically active cells. In contrast, microrheology performed in living cells revealed finite fluidity though it is reduced in spontaneously deactivated cells. Viscous modulus in living cells was also increased by the increased macromolecular concentration via osmotic compression, however, more gently in a manner of complete Arrhenius typical of strong glass formers. All these observations support that the non-living cytoplasm at the physiological concentration is on the verge of glass transition. The living cytoplasm gains some fluidity via metabolic activity by changing its glass-forming characters from that of fragile to that of strong glass formers.Direct evidence was obtained for the glass-forming behavior of 36  of the quadrant photodiode (QPD), which is proportional to probe displacements u(t), was used for the PID feedback of the piezo-actuated stage that maintains the particle in the center of the laser focus. The total displacement of the probe particle in the sample was obtained as the sum of the piezo displacement ustage and the displacement of the probe particle from the laser focus uQPD. PMR in living cells and the particle pulling were both performed under the feedback.Details of the MR using the optical trap and laser interferometry technique are given elsewhere47. The schematic of this pulling method is shown in Fig. u = uQPD + ustage.In order to stably apply an extremely small force (~0.1 pN) to the probe particle, we used another feedback technique known as a force clamp36  of the QPD must be appropriately calibrated to probe displacements u(t) and optical-trapping forces F(t). The conventional method of calibration is to measure the Brownian motion of the same probe particle in a pure solvent35. Comparing the observation and theoretical prediction based on FDT, calibrations for both displacements and trapping forces were attained simultaneously. This protocol is valid when the difference in the refractive index between sample solutions and a pure solvent is negligible, i.e., for dilute samples.For microrheology experiments using the optical-trap and laser-interferometry techniques, diffraction of the laser due to the displacement of a trapped particle was detected using a quadrant photodiode36 Fig. . The outCal ≡ u(t)/V(t) directly in situ for every particles. A probe particle was trapped using a drive laser and oscillated slowly at 0.5 Hz. Cal for the probe laser was obtained by comparing the output V(t) of the QPD for the fixed probe laser and u(t) obtained from the video of the probe particle  and u(t) were compared in the same manner.The calibration method described above is not valid for the concentrated samples used in this study. We therefore obtained the calibration factors for probe displacements k is determined as F/x, where F is the trapping force and x is the distance between the probe center and the laser focus. We describe here the response function of the probe that is in the trap with stiffness k as Ak(ω) and the response in the absence of the optical trap as α(ω). The response in trap Ak(ω) was suppressed as compared to α(ω), as shown in Figs Akn(ω) by increasing the laser strength by n-times , Akn(ω), and α(ω) is given as follows25:A new calibration method (for trap stiffness) that is applicable to concentrated cytoplasmic models was employed in this study. A particle in an optical trap is confined within a harmonic potential created by the trapping laser. Therefore, optical trapping affects both the thermal motion and the response of the particle to the applied force. Trap stiffness mes Fig. . As showThe trap stiffness was obtained asEquation .For BSA solutions, 100 mg of BSA  was dissolved in 1 mL of NH buffer . The samples were washed with a 10-fold volume of NH buffer using a 50-kDa filter unit  at 4 °C. After four washes, the samples were concentrated to ~600 mg/mL by centrifugation with a 3-kDa filter at 4 °C.Escherichia coli, HeLa cells, and Xenopus eggs were extracted as given in Supplementary Note Escherichia coli, HeLa cells, and Xenopus eggs. Before experiments, cell extracts (IVCE) were diluted with a 10-fold volume of NH buffer and washed by centrifugal filtration at 4 °C using a filter with a 3-kDa molecular cut-off . After washing 4 times, the samples were concentrated by further centrifugal filtration. The concentration of the resulting IVCE was determined using a Bradford assay. We assumed that the efficiency of the assay and the ratio of proteins to total macromolecules were constant for different concentrations of IVCEs taken from each cell species. The results of the assay were calibrated to the actual concentrations of macromolecules by measuring the dry weight of the stocks. In IVCE obtained from E. coli, for instance, approximately 1.5-fold more biomacromolecules than proteins  were present in the final solutions. The ratio of contents obtained in this study was consistent with that in other studies on the ratio of proteins to RNA in E. coli12. All experiments using Xenopus were approved by the Institutioonal Animal Care and Use Committee of Kyushu University  and carried out in accordance with the guidelines set by this committee.The cytoplasm of E. coli were prepared as described elsewhere43 . The probe particles  were introduced into the cells using a Gene gun 78. The dishes were then incubated at least for several hours until the cell membranes recovered. Excess beads were removed by washing the dishes with phosphate-buffered saline. Finally, a CO2-independent culture medium without serum was added to perform the measurements.Giant spheroplasts of 80 with 1 mM eggPC in mineral oil . Initial macromolecule concentrations of BSA and IVCE were 0.14 and 0.11 g/mL in NH buffer (220 mosM), respectively. After formation, liposome solutions were mixed with equal volumes of 220, 660, and 1100 mM aqueous sucrose in order to osmotically compress and concentrate the contents. Macromolecule concentrations inside the liposomes were estimated from the fluorescence intensity of GFP (0.2 mg/mL) measured by confocal microscopy . After photobleaching with a circular spot of a radius r ~ 1 μm for 1 s at full laser power, the fluorescence recovery was recorded. For convenience, the recovery intensity as a function of time was fitted to the following conventionally used equation:I0 and I1 indicate modified Bessel functions, and τ is the characteristic time scale of diffusion81. This formula is merely valid for 2D diffusion, typically when photobleaching was performed with an unfocused laser in bulk solution. The formula r2/4 τ then gives the diffusion coefficient of GFP. In this study, however, FRAP was performed with a focused laser in samples with limited dimensions (spheroplasts and liposomes). Because the measured value of r2/4 τ merely reflects the scale of the diffusion in this case, it is crucial to normalize it to the value measured for pure solvents with all other conditions unchanged.Liposomes containing concentrated BSA and IVCE were prepared using the droplet-transfer methodSupplementary Information