4E-BP2–dependent translation in parvalbumin neurons controls epileptic seizure threshold

The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) integrates multiple signals to regulate critical cellular processes such as mRNA translation, lipid biogenesis, and autophagy. Germline and somatic mutations in mTOR and genes upstream of mTORC1, such as PTEN, TSC1/2, AKT3, PIK3CA, and components of GATOR1 and KICSTOR complexes, are associated with various epileptic disorders. Increased mTORC1 activity is linked to the pathophysiology of epilepsy in both humans and animal models, and mTORC1 inhibition suppresses epileptogenesis in humans with tuberous sclerosis and animal models with elevated mTORC1 activity. However, the role of mTORC1-dependent translation and the neuronal cell types mediating the effect of enhanced mTORC1 activity in seizures remain unknown. The eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and 2 (4E-BP2) are translational repressors downstream of mTORC1. Here we show that the ablation of 4E-BP2, but not 4E-BP1, in mice increases the sensitivity to pentylenetetrazole (PTZ)- and kainic acid (KA)–induced seizures. We demonstrate that the deletion of 4E-BP2 in inhibitory, but not excitatory neurons, causes an increase in the susceptibility to PTZ-induced seizures. Moreover, mice lacking 4E-BP2 in parvalbumin, but not somatostatin or VIP inhibitory neurons exhibit a lowered threshold for seizure induction and reduced number of parvalbumin neurons. A mouse model harboring a human PIK3CA mutation that enhances the activity of the PI3K-AKT pathway (Pik3ca^H1047R-Pvalb) selectively in parvalbumin neurons shows susceptibility to PTZ-induced seizures. Our data identify 4E-BP2 as a regulator of epileptogenesis and highlight the central role of increased mTORC1-dependent translation in parvalbumin neurons in the pathophysiology of epilepsy.

Epilepsy is a prevalent (0.5 to 1% of the general population) (1) heterogeneous neurological disorder affecting all age groups and is characterized by seizures and associated psychological and social stigmas (2–4). Hyperactivation of the mechanistic/mammalian target of rapamycin (mTOR) pathway has been reported in brain lesions of epileptic patients with neurodevelopmental disorders (5, 6), and human genetic studies have shown that mutations in mTOR (7, 8) and other components of its pathway are linked to epileptogenesis (6, 9–13). mTOR is a highly conserved serine/threonine protein kinase that forms two distinct complexes: mTORC1 and mTORC2. mTORC1 integrates multiple environmental and intracellular signals to modulate brain functions by controlling key cellular processes such as mRNA translation, nucleotide, lipid and mitochondrial biogenesis, and autophagy (14, 15). Germline or somatic mutations, which result in enhanced mTORC1 activity, including in PIK3CA, PTEN, AKT3, TSC1/2, RHEB, and MTOR, are associated with neurodevelopmental disorders with epilepsy (16–23). Recent studies have also identified mutations in mTORC1 upstream amino acid–sensing GATOR1-KICSTOR-Rag GTPase pathways as a common cause of epilepsy (24), revealing that mutations in GATOR1 (DEPDC5, NPRL2, and NPRL3) (25, 26) and KICSTOR (ITFG2, KPTN, SZT2, and C12ORF66) (6, 7, 27) genes are often found in epileptic pathologies. The link between the mTORC1 and epilepsy has been recapitulated in animal models with enhanced mTORC1 activity (e.g., Pten^+/−, TSC1/2^+/−, and activating mutations in Pik3ca Nestin-Cre knockin (KI), Akt3 KI, MTOR, and Rheb KI) (18, 20, 23, 28–31) while inhibition of mTORC1 reversed epileptogenesis in TSC1^GFAP-Cre and Pten^+/− mice (20, 31). Notably, the mTORC1 rapalog, everolimus, has been approved by the US Food and Drug Administration (FDA) for the treatment of epilepsy in tuberous sclerosis complex (TSC) patients (32, 33).mTORC1 is a master regulator of mRNA translation. Upon activation, mTORC1 phosphorylates S6 protein kinases 1 and 2 (S6K1/2) and 4E-binding proteins (4E-BPs) (34, 35). In the hypophosphorylated form, 4E-BPs bind and prevent the association of the cap-binding protein eIF4E with the large scaffolding protein eIF4G, thereby inhibiting the formation of the eIF4F complex (composed of eIF4E, eIF4G, and an mRNA helicase eIF4A), which is essential for the initiation of cap-dependent translation. Phosphorylation of 4E-BPs by mTORC1 results in the release of eIF4E from 4E-BPs, allowing eIF4F complex formation and initiation of translation (36, 37). Among the three 4E-BP family members (4E-BP1, 4E-BP2, and 4E-BP3), 4E-BP2 is the most abundant paralog in the mammalian brain (38, 39). A recent study (5) has identified aberrant activation of eIF4E as a major mechanism for translational changes in focal malformations of cortical development (FMCD), a condition that is often caused by brain somatic activating mutations in MTOR and presents with intractable epilepsy in children, accompanied by developmental abnormalities and autism spectrum disorder (ASD) (5, 40–42). Increased eIF4E activity has a pathogenic role in inducing epileptic seizures in FMCD as eIF4E knockdown prevented spontaneous seizures in mTOR Cys1483Tyr and Leu2427Pro mutant mice (5), which show mTORC1 hyperactivation.Despite the progress in understanding the causal link between enhanced mTORC1 activity and epilepsy, the mTORC1-downstream molecular mechanisms promoting epileptogenesis and the cell types mediating the effect on seizure threshold and severity remain poorly understood. In this work, we investigated the role of two main mTORC1-downstream effectors, 4E-BP1 and 4E-BP2, in regulating seizure susceptibility and studied the neuronal cell types mediating epileptogenic effects. We report that mice with whole-body or parvalbumin neuron–specific deletion of 4E-BP2 exhibit reduced threshold and increased severity of epileptic seizures. Moreover, we show that Pik3ca^H1047R-Pvalb mutant mice harboring a conditional parvalbumin neuron–specific KI gain-of-function mutation (H1047R) in the PIK3CA kinase domain are prone to seizures. Collectively, these findings demonstrate a central role of 4E-BP2 and parvalbumin neurons in mediating mTORC1-dependent epileptogenesis, thus expanding our understanding of cell type–specific molecular mechanisms of translation dysregulation in epilepsy and other neurodevelopmental disorders.     

Genome-wide CRISPR screens reveal multitiered mechanisms through which mTORC1 senses mitochondrial dysfunction

In mammalian cells, nutrients and growth factors signal through an array of upstream proteins to regulate the mTORC1 growth control pathway. Because the full complement of these proteins has not been systematically identified, we developed a FACS-based CRISPR-Cas9 genetic screening strategy to pinpoint genes that regulate mTORC1 activity. Along with almost all known positive components of the mTORC1 pathway, we identified many genes that impact mTORC1 activity, including DCAF7, CSNK2B, SRSF2, IRS4, CCDC43, and HSD17B10. Using the genome-wide screening data, we generated a focused sublibrary containing single guide RNAs (sgRNAs) targeting hundreds of genes and carried out epistasis screens in cells lacking nutrient- and stress-responsive mTORC1 modulators, including GATOR1, AMPK, GCN2, and ATF4. From these data, we pinpointed mitochondrial function as a particularly important input into mTORC1 signaling. While it is well appreciated that mitochondria signal to mTORC1, the mechanisms are not completely clear. We find that the kinases AMPK and HRI signal, with varying kinetics, mitochondrial distress to mTORC1, and that HRI acts through the ATF4-dependent up-regulation of both Sestrin2 and Redd1. Loss of both AMPK and HRI is sufficient to render mTORC1 signaling largely resistant to mitochondrial dysfunction induced by the ATP synthase inhibitor oligomycin as well as the electron transport chain inhibitors piericidin and antimycin. Taken together, our data reveal a catalog of genes that impact the mTORC1 pathway and clarify the multifaceted ways in which mTORC1 senses mitochondrial dysfunction.

The mechanistic target of rapamycin complex 1 (mTORC1) is a eukaryotic cell growth regulator that responds to nutrient and growth factor availability. Under nutrient-replete conditions, mTORC1 licenses anabolic processes while inhibiting catabolic ones. Given the myriad of stimuli that mTORC1 responds to, it is no surprise that a diverse set of proteins, many as part of large complexes, act in a coordinated manner to regulate mTORC1 activity.The heterodimeric Rag GTPases (RagA/B and RagC/D) play a central role in the control of mTORC1 by nutrients. In response to amino acids, as well as glucose and cholesterol, GTP-bound RagA/B and GDP-bound RagC/D mediate the recruitment of mTORC1 to the lysosomal surface (1–5). Once at the lysosome, GTP-bound Rheb, which is under the control of growth factors through the TSC complex pathway, binds to mTORC1 and stimulates its kinase activity (6–13). Together, Rheb and the Rags form a GTPase-based coincidence detector at the lysosomal surface that ensures that mTORC1 becomes activated only when nutrient and growth factor conditions are optimal. Given that the Rag and Rheb GTPases are central arbiters of mTORC1 activation, the regulation of their respective nucleotide states is of great interest.Dozens of proteins have been shown to modulate mTORC1 activity, many acting indirectly through one of several key effectors. However, the relative contributions of these proteins to the regulation of mTORC1 activity has not been systematically interrogated. Additionally, the majority of the proteins that regulate mTORC1 were identified using proteomic approaches. While fruitful, these studies leave open the possibility that proteins that play a role in mTORC1 regulation through transient or indirect interactions with pathway components, or are not easily detected with mass spectrometry-based proteomics, have not been identified.Advances in CRISPR-Cas9-based screening have generated large catalogs of gene essentiality data in numerous cell lines, which can be leveraged to identify genes that are coessential with those encoding components of the mTORC1 pathway (14, 15). Though this type of analysis reveals many established mTORC1 regulators, a caveat is that it relies on cell fitness rather than mTORC1 activity as a readout. A recent study utilized a gene-trap approach to identify mTORC1 regulators in haploid cells and define new relationships among established components (16). The CRISPR-screening strategy we present here expands this toolbox by enabling screening in a large set of genetically diverse cell lines of different lineages and allows for the identification of genes that regulate mTORC1 signaling but whose loss is not tolerated long term. We carried out a genome-wide CRISPR-Cas9 screen and a series of focused sublibrary screens to identify positive regulators of mTORC1. The hits from these screens ultimately led us to study how mTORC1 senses mitochondrial dysfunction. We find that two kinases (AMPK and HRI) act in a coordinated fashion to mediate the inhibition of mTORC1 caused by mitochondrial stress.     

Placental trophoblast syncytialization potentiates macropinocytosis via mTOR signaling to adapt to reduced amino acid supply

During pregnancy, the appropriate allocation of nutrients between the mother and the fetus is dominated by maternal–fetal interactions, which is primarily governed by the placenta. The syncytiotrophoblast (STB) lining at the outer surface of the placental villi is directly bathed in maternal blood and controls feto–maternal exchange. The STB is the largest multinucleated cell type in the human body, and is formed through syncytialization of the mononucleated cytotrophoblast. However, the physiological advantage of forming such an extensively multinucleated cellular structure remains poorly understood. Here, we discover that the STB uniquely adapts to nutrient stress by inducing the macropinocytosis machinery through repression of mammalian target of rapamycin (mTOR) signaling. In primary human trophoblasts and in trophoblast cell lines, differentiation toward a syncytium triggers macropinocytosis, which is greatly enhanced during amino acid shortage, induced by inhibiting mTOR signaling. Moreover, inhibiting mTOR in pregnant mice markedly stimulates macropinocytosis in the syncytium. Blocking macropinocytosis worsens the phenotypes of fetal growth restriction caused by mTOR-inhibition. Consistently, placentas derived from fetal growth restriction patients display: 1) Repressed mTOR signaling, 2) increased syncytialization, and 3) enhanced macropinocytosis. Together, our findings suggest that the unique ability of STB to undergo macropinocytosis serves as an essential adaptation to the cellular nutrient status, and support fetal survival and growth under nutrient deprivation.

During pregnancy, the health of the mother and the fetus is dominated by the appropriate allocation of nutrients between the two individuals. Maternal–fetal material exchange predominantly depends on the placenta, which is responsible for transferring the bulk of nutrients between maternal and fetal circulations. The placenta plays a critical role in sensing fetal nutritional demands, modulating maternal supply, and adapting its nutrient transport capacity. Limited maternal nutrient availability can lead to adaptive changes in the placental endocrine function, which is thought to attenuate the potential conflict between fetal growth demands and maternal health (1).Fetal growth restriction (FGR) represents a pregnancy complication whereby the fetus fails to attain its genetically determined growth potential due to insufficient delivery of maternal nutrition, especially amino acids, by the placenta (1–3). Annually, ∼30 million newborns, mainly in developing countries, suffer from FGR (4), which leads to increased perinatal morbidity and mortality and multiple lifelong health problems (5). The mechanisms deployed by the placenta to compensate nutrient-deprivation injury and support fetal survival remain unknown.At the outermost surface of the placenta, the syncytial layer lines the placental villi, with a continuous surface measuring 12 to 14 m² at term (6). This layer is directly bathed in maternal blood, and thus positioned to regulate feto–maternal exchanges of gases, nutrients, and waste. The syncytial layer comprises the multinucleated syncytiotrophoblast (STB), the largest multinucleated epithelial surface in the body, which is formed through fusion of the mononucleated cytotrophoblast (CTB). Yet, the physiological advantages of forming such an extensive multinucleated cellular structure and the regulatory mechanisms underlying this process remain to be explored.Macropinocytosis constitutes a specialized route for cellular nutrient uptake from the fluid phase. It is functional in certain cell types, including immature dendritic cells, macrophages, podocytes, and tumor cells (7–10). The process involves the formation of large vesicles of 0.2 to 5 μm in diameter at the sites of membrane ruffling (11). Macropinocytosis promotes the uptake of fluid phase-derived molecules by at least 10-fold (12), especially with respect to internalization of large-sized molecules (>70 kDa) (7). In tumor cells, deprivation of amino acid supply inhibits mammalian target of rapamycin (mTOR) and enhances macropinocytosis and lysosomal catabolism of extracellular proteins to sustain cell survival and growth (13, 14), indicating macropinocytosis as an efficient way to attenuate nutrient shortage in the high-demanding cells.Based on the above evidence, we hypothesized that trophoblasts, particularly STB, may utilize macropinocytosis to facilitate nutrient absorption from the maternal environment, thereby negotiating fetal demands in the face of diminished maternal supply. We tested our hypothesis by using cultures of primary human trophoblast (PHT) cells, human trophoblast cell lines, and rapamycin-treated pregnant mice, and demonstrated the physiological significance of macropinocytosis in STB to adapt to maternal undernutrition stress during pregnancy.     

Photosynthesis tunes quantum-mechanical mixing of electronic and vibrational states to steer exciton energy transfer

Photosynthetic species evolved to protect their light-harvesting apparatus from photoxidative damage driven by intracellular redox conditions or environmental conditions. The Fenna–Matthews–Olson (FMO) pigment–protein complex from green sulfur bacteria exhibits redox-dependent quenching behavior partially due to two internal cysteine residues. Here, we show evidence that a photosynthetic complex exploits the quantum mechanics of vibronic mixing to activate an oxidative photoprotective mechanism. We use two-dimensional electronic spectroscopy (2DES) to capture energy transfer dynamics in wild-type and cysteine-deficient FMO mutant proteins under both reducing and oxidizing conditions. Under reducing conditions, we find equal energy transfer through the exciton 4–1 and 4–2-1 pathways because the exciton 4–1 energy gap is vibronically coupled with a bacteriochlorophyll-a vibrational mode. Under oxidizing conditions, however, the resonance of the exciton 4–1 energy gap is detuned from the vibrational mode, causing excitons to preferentially steer through the indirect 4–2-1 pathway to increase the likelihood of exciton quenching. We use a Redfield model to show that the complex achieves this effect by tuning the site III energy via the redox state of its internal cysteine residues. This result shows how pigment–protein complexes exploit the quantum mechanics of vibronic coupling to steer energy transfer.

Photosynthetic organisms convert solar photons into chemical energy by taking advantage of the quantum mechanical nature of their molecular systems and the chemistry of their environment (1–4). Antenna complexes, composed of one or more pigment–protein complexes, facilitate the first steps in the photosynthesis process: They absorb photons and determine which proportion of excitations to move to reaction centers, where charge separation occurs (4). In oxic environments, excitations can generate highly reactive singlet oxygen species. These pigment–protein complexes can quench excess excitations in these environments with molecular moieties such as quinones and cysteine residues (1, 5–7).The Fenna–Matthews–Olson (FMO) complex, a trimer of pigment–protein complexes found in the green sulfur bacterium Chlorobaculum tepidum (8), has emerged as a model system to study the photophysical properties of photosynthetic antenna complexes (9–19). Each subunit in the FMO complex contains eight bacteriochlorophyll-a site molecules (Protein Data Bank, ID code: 3ENI) that are coupled to form a basis of eight partially delocalized excited states called excitons (Fig. 1) (20–23). Previous experiments on FMO have observed the presence of long-lived coherences in nonlinear spectroscopic signals at both cryogenic and physiological temperatures (11, 13). The coherent signals are thought to arise from some combination of electronic (24–26), vibrational (16–18), and vibronic (27) coherences in the system (28–30). One previous study reported that the coherent signals in FMO remain unchanged upon mutagenesis of the protein, suggesting that the signals are ground state vibrational coherences (17). Others discuss the role of vibronic coupling, where electronic and nuclear degrees of freedom become coupled (29). Other dimeric model systems have demonstrated the regimes in which these vibronically coupled states produce coherent or incoherent transport and vibronic coherences (31–33). Recent spectroscopic data has suggested that vibronic coupling plays a role in driving efficient energy transfer through photosynthetic complexes (27, 31, 33, 34), but to date there is no direct experimental evidence suggesting that biological systems use vibronic coupling as part of their biological function.Open in a separate windowFig. 1.(Left) Numbered sites and sidechains of cysteines C353 and C49 in the FMO pigment–protein complex (PDB ID code: 3ENI) (20). (Right) Site densities for excitons 4, 2, and 1 in reducing conditions with the energy transfer branching ratios for the WT oxidized and reduced protein. The saturation of pigments in each exciton denotes the relative contribution number to the exciton. The C353 residue is located near excitons 4 and 2, which have most electron density along one side of the complex, and other redox-active residues such as the Trp/Tyr chain. C353 and C49 surround site III, which contains the majority of exciton 1 density. Excitons 2 and 4 are generally delocalized over sites IV, V, and VII.It has been shown that redox conditions affect excited state properties in pigment-protein complexes, yet little is known about the underlying microscopic mechanisms for these effects (1, 9). Many commonly studied light-harvesting complexes—including the FMO complex (20), light-harvesting complex 2 (LH2) (35), the PC645 phycobiliprotein (36), and the cyanobacterial antenna complex isiA (37)—contain redox-active cysteine residues in close proximity to their chromophores. As the natural low light environment of C. tepidum does not necessitate photoprotective responses to light quantity and quality, its primary photoprotective mechanism concerns its response to oxidative stress. C. tepidum is an obligate anaerobe, but the presence of many active anoxygenic genes such as sodB for superoxide dismutase and roo for rubredoxin oxygen oxidoreductase (38) suggests that it is frequently exposed to molecular oxygen (7, 39). Using time-resolved fluorescence measurements, Orf et al. demonstrated that two cysteine residues in the FMO complex, C49 and C353, quench excitons under oxidizing conditions (1), which could protect the excitation from generating reactive oxygen species (7, 40–42). In two-dimensional electronic spectroscopy (2DES) experiments, Allodi et al. showed that redox conditions in both the wild-type and C49A/C353A double-mutant proteins affect the ultrafast dynamics through the FMO complex (9, 43). The recent discovery that many proteins across the evolutionary landscape possess chains of tryptophan and tyrosine residues provides evidence that these redox-active residues may link the internal protein behavior with the chemistry of the surrounding environment (41, 43).In this paper, we present data showing that pigment–protein complexes tune the vibronic coupling of their chromophores and that the absence of this vibronic coupling activates an oxidative photoprotective mechanism. We use 2DES to show that a pair of cysteine residues in FMO, C49 and C353, can steer excitations toward quenching sites in oxic environments. The measured reaction rate constants demonstrate unusual nonmonotonic behavior. We then use a Redfield model to determine how the exciton energy transfer (EET) time constants arise from changing chlorophyll site energies and their system-bath couplings (44, 45). The analysis reveals that the cysteine residues tune the resonance between exciton 4–1 energy gap and an intramolecular chlorophyll vibration in reducing conditions to induce vibronic coupling and detune the resonance in oxidizing conditions. This redox-dependent modulation of the vibronic coupling steers excitations through different pathways in the complex to change the likelihood that they interact with exciton quenchers.     

The evolution of red color vision is linked to coordinated rhodopsin tuning in lycaenid butterflies

Color vision has evolved multiple times in both vertebrates and invertebrates and is largely determined by the number and variation in spectral sensitivities of distinct opsin subclasses. However, because of the difficulty of expressing long-wavelength (LW) invertebrate opsins in vitro, our understanding of the molecular basis of functional shifts in opsin spectral sensitivities has been biased toward research primarily in vertebrates. This has restricted our ability to address whether invertebrate G_q protein-coupled opsins function in a novel or convergent way compared to vertebrate G_t opsins. Here we develop a robust heterologous expression system to purify invertebrate rhodopsins, identify specific amino acid changes responsible for adaptive spectral tuning, and pinpoint how molecular variation in invertebrate opsins underlie wavelength sensitivity shifts that enhance visual perception. By combining functional and optophysiological approaches, we disentangle the relative contributions of lateral filtering pigments from red-shifted LW and blue short-wavelength opsins expressed in distinct photoreceptor cells of individual ommatidia. We use in situ hybridization to visualize six ommatidial classes in the compound eye of a lycaenid butterfly with a four-opsin visual system. We show experimentally that certain key tuning residues underlying green spectral shifts in blue opsin paralogs have evolved repeatedly among short-wavelength opsin lineages. Taken together, our results demonstrate the interplay between regulatory and adaptive evolution at multiple G_q opsin loci, as well as how coordinated spectral shifts in LW and blue opsins can act together to enhance insect spectral sensitivity at blue and red wavelengths for visual performance adaptation.

Opsins belong to a diverse multigene family of G protein-coupled receptors that bind to a small nonprotein retinal moiety to form photosensitive rhodopsins and enable vision across animals (1–4). The tight relationship between opsin genotypes and spectral sensitivity phenotypes offers an ideal framework to analyze how specific molecular changes give rise to adaptations in visual behaviors (5). Notably, independent opsin gene gains and losses (6–13), genetic variation across opsins (14–16), spectral tuning mutations within opsins (17–21), and alterations in visual regulatory networks (22, 23) have contributed to opsin adaptation. Yet, the molecular and structural changes underlying the remarkable diversification of spectral sensitivity phenotypes identified in some invertebrates, including crustaceans and insects (24–27), are far less understood than those in vertebrate lineages (28–32).The diversity of opsin-based photoreceptors observed across animal visual systems is produced by distinct ciliary vertebrate c-opsin and invertebrate rhabdomeric based r-opsin subfamilies that mediate separate phototransduction cascades (31, 33–35). Vertebrate c-opsins function through the G protein transducing (G_t) signaling pathway, which activates cyclic nucleotide phosphodiesterase, ultimately resulting in a hyperpolarization response in photoreceptor cells through the opening of selective K⁺ channels (31, 36). By contrast, insect opsins transmit light stimuli through a G_q-type G protein (33, 37) with phosphoinositol (PLCβ) acting as an effector enzyme to achieve TRP channel depolarization in the invertebrate photoreceptor cell (34, 38).All vertebrate visual cone opsins derive from four gene families: short-wavelength-sensitive opsins SWS1 (or ultraviolet [UV]) with λ_max 344 to 445 nm and SWS2 with λ_max 400 to 470 nm, and longer-wavelength-sensitive opsins that specify the green MWS (or Rh2) pigments with λ_max 480 to 530 nm and red-sensitive LWS pigments with λ_max 500 to 570 nm (5, 30). Most birds and fish have retained the four ancestral opsin genes (39), with notable opsin expansions in cichlid fish opsins (23, 40), whereas SWS1 is extinct in monotremes, and SWS2 and M opsins are lost in marsupials and eutherian mammals (41). In primates, trichromatic vision is conferred through SWS1 (λ_max = 414 nm) and recent duplicate MWS (λ_max = 530 nm) and LWS opsins (λ_max = 560 nm) (42–44). In vertebrates, molecular evolutionary approaches and well-established in vitro opsin purification have identified the complex interplay between opsin duplications, regulatory and protein-coding mutations controlling opsin gene tuning, and spectral phenotypes notably in birds, fish, and mammals (45–47).Insect opsins are phylogenetically distinct but functionally analogous to those of vertebrates, and the ancestral opsin repertoire consists of three types of light-absorbing rhabdomeric G_q-type opsin specifying UV (350 nm), short-wavelength (blue, 440 nm) and long-wavelength pigments (LW, 530 nm) (48). Given the importance of color-guided behaviors and the remarkable photoreceptor spectral diversity observed in insects (26, 27), the dynamic opsin gene diversification found across lineages (Fig. 1) highlights their potentially central role in adaptation (27, 49, 50), yet the molecular basis of opsin functionality of rhabdomeric invertebrate G_q opsins remains understudied.Open in a separate windowFig. 1.Visual opsin gene evolution and spectral tuning mechanisms in insects. Visual opsin genes of the Atala hairstreak (E. atala, Lepidoptera, Lycaenidae) in comparison with those encoded in the genomes of diverse insects. The opsin types are highlighted in gray for UV, in blue for short wavelength (SW), and in green for long wavelength (LW). Numbers indicate multiple opsins, whereas no dot indicates gene loss. Colored circles indicate instances of shifted spectral sensitivities in at least one of the encoded opsins. The direction of shift is inferred from the opsin lambda max that departs from the typical range of absorbance in the opsin subfamily using wavelength boundaries for the various colors: UV <380 nm, violet 380 to 435 nm, blue 435 to 492 nm, green 492 to 530 nm, and red shifted >530 nm. Coleopteran lineages, and some hemipterans, lost the blue opsin locus and compensated for the loss of blue sensitivity via UV and/or LW gene duplications across lineages (11, 12). In butterflies, extended photosensitivity at short wavelengths is observed in Heliconius erato with two UV opsins at λ_max = 355 nm and 398 nm (10) and in P. rapae with two blue opsins with λ_max = 420 and 450 nm (17). A blue opsin duplication occurred independently in lycaenid butterflies (61). LW opsin duplications occurred independently in most major insect lineages (6, 16, 55) and confer a variable range of LW sensitivities with or without additional contributions from lateral filtering. In order to extend spectral sensitivity at longer wavelengths while sharpening blue acuity, some lycaenid butterflies have evolved a new color vision mechanism combining spectral shifts at a duplicate blue opsin and at the LW opsin. Images credit: Christopher Adams (illustrator).The recurrent evolution of red receptors in insects in particular suggests that perception of longer wavelengths can play an important role in the context of foraging, oviposition, and/or conspecific recognition (6, 27, 51–54). In butterflies, several mechanisms are likely to have provided extended spectral sensitivity to longer wavelengths. LW opsin duplications along with the evolution of lateral filtering between ommatidia has been demonstrated in two papilionids, Papilio xuthus (27) and Graphium sarpedon (55), as well as in a riodinid (Apodemia mormo) (6, 54). Lateral filtering pigments are relatively widespread across butterfly lineages, e.g., Heliconius (56), Pieris (57), Colias erate (58), and some moths [Adoxophyes orana (59) and Paysandisia archon (60)]. These pigments absorb short wavelengths and aid in shifting the sensitivity peak of green LW photoreceptors to longer wavelengths (27, 51, 56, 57, 61, 62). Despite creating distinct spectral types that can contribute to color vision, as identified in nymphalid (56), pierid (57), and lycaenid (62) species, all of which lack duplicated LW opsins (61, 63), lateral filtering alone cannot extend photoreceptor sensitivity toward the far red (700 to 750 nm) beyond the exponentially decaying long-wavelength rhodopsin absorbance spectrum (51). Thus, molecular variation of ancestral LW opsin genes is likely to have contributed an as yet underexplored mechanism to the diversification of long-wavelength photoreceptor spectral sensitivity. However, disentangling the relative contributions of lateral filtering and pure LW opsin properties has remained technically challenging using classical electrophysiological approaches (14, 64, although see, e.g., refs. 65, 66, 67) and has been limited by the lack of in vitro expression systems suitable for LW opsins.While opsin duplicates have been identified in numerous organisms, the spectral tuning mechanisms and interplay between new opsin photoreceptors in invertebrate visual system evolution are less well understood. Here we combine physiological, molecular, and heterologous approaches to start closing this gap in our knowledge of invertebrate G_q opsin evolution by investigating the functions, spectral tuning, and implications of evolving new combinations of short- and long-wavelength opsin types in lycaenid species. This butterfly group, comprising the famous blues, coppers, and hairstreaks, is the second largest family with about 5,200 (28%) of the some 18,770 described butterfly species (68). In light of their remarkable behavioral, ecological, and morphological diversity (69, 70), as well as pioneer studies in the Lycaena and Polyommatus genera supporting the rapid evolution of color vision in certain lineages (56, 61, 62), lycaenids provide an ideal candidate system for investigating opsin evolution and visual adaptations. Using the Atala hairstreak, Eumaeus atala, as a molecular and ecological model, we find coordinated spectral shifts at short- and long-wavelength G_q opsin loci and demonstrate that the combination of six ommatidial classes of photoreceptors in the compound eye uniquely extend spectral sensitivity at long wavelengths toward the far-red while concurrently sharpening acuity of multiple blue wavelengths. Together, these findings link the evolution of four-opsin visual systems to adaptation in the context of finely tuned color perception critical to the behavior of these butterflies.     

A solution to a sex ratio puzzle in Melittobia wasps

The puzzling sex ratio behavior of Melittobia wasps has long posed one of the greatest questions in the field of sex allocation. Laboratory experiments have found that, in contrast to the predictions of theory and the behavior of numerous other organisms, Melittobia females do not produce fewer female-biased offspring sex ratios when more females lay eggs on a patch. We solve this puzzle by showing that, in nature, females of Melittobia australica have a sophisticated sex ratio behavior, in which their strategy also depends on whether they have dispersed from the patch where they emerged. When females have not dispersed, they lay eggs with close relatives, which keeps local mate competition high even with multiple females, and therefore, they are selected to produce consistently female-biased sex ratios. Laboratory experiments mimic these conditions. In contrast, when females disperse, they interact with nonrelatives, and thus adjust their sex ratio depending on the number of females laying eggs. Consequently, females appear to use dispersal status as an indirect cue of relatedness and whether they should adjust their sex ratio in response to the number of females laying eggs on the patch.

Sex allocation has produced many of the greatest success stories in the study of social behaviors (1–4). Time and time again, relatively simple theory has explained variation in how individuals allocate resources to male and female reproduction. Hamilton’s local mate competition (LMC) theory predicts that when n diploid females lay eggs on a patch and the offspring mate before the females disperse, the evolutionary stable proportion of male offspring (sex ratio) is (n − 1)/2n (Fig. 1) (5). A female-biased sex ratio is favored to reduce competition between sons (brothers) for mates and to provide more mates (daughters) for those sons (6–8). Consistent with this prediction, females of >40 species produce female-biased sex ratios and reduce this female bias when multiple females lay eggs on the same patch (higher n; Fig. 1) (9). The fit of data to theory is so good that the sex ratio under LMC has been exploited as a “model trait” to study the factors that can constrain “perfect adaptation” (4, 10–13).Open in a separate windowFig. 1.LMC. The sex ratio (proportion of sons) is plotted versus the number of females laying eggs on a patch. The bright green dashed line shows the LMC theory prediction for the haplodiploid species (5, 39). A more female-biased sex ratio is favored in haplodiploids because inbreeding increases the relative relatedness of mothers to their daughters (7, 32). Females of many species adjust their offspring sex ratio as predicted by theory, such as the parasitoid Nasonia vitripennis (green diamonds) (82). In contrast, the females of several Melittobia species, such as M. australica, continue to produce extremely female-biased sex ratios, irrespective of the number of females laying eggs on a patch (blue squares) (15).In stark contrast, the sex ratio behavior of Melittobia wasps has long been seen as one of the greatest problems for the field of sex allocation (3, 4, 14–21). The life cycle of Melittobia wasps matches the assumptions of Hamilton’s LMC theory (5, 15, 19, 21). Females lay eggs in the larvae or pupae of solitary wasps and bees, and then after emergence, female offspring mate with the short-winged males, who do not disperse. However, laboratory experiments on four Melittobia species have found that females lay extremely female-biased sex ratios (1 to 5% males) and that these extremely female-biased sex ratios change little with increasing number of females laying eggs on a patch (higher n; Fig. 1) (15, 17–20, 22). A number of hypotheses to explain this lack of sex ratio adjustment have been investigated and rejected, including sex ratio distorters, sex differential mortality, asymmetrical male competition, and reciprocal cooperation (15–18, 20, 22–26).We tested whether Melittobia’s unusual sex ratio behavior can be explained by females being related to the other females laying eggs on the same patch. After mating, some females disperse to find new patches, while some may stay at the natal patch to lay eggs on previously unexploited hosts (Fig. 2). If females do not disperse, they can be related to the other females laying eggs on the same host (27–31). If females laying eggs on a host are related, this increases the extent to which relatives are competing for mates and so can favor an even more female-biased sex ratio (28, 32–35). Although most parasitoid species appear unable to directly assess relatedness, dispersal behavior could provide an indirect cue of whether females are with close relatives (36–38). Consequently, we predict that when females do not disperse and so are more likely to be with closer relatives, they should maintain extremely female-biased sex ratios, even when multiple females lay eggs on a patch (28, 35).Open in a separate windowFig. 2.Host nest and dispersal manners of Melittobia. (A) Photograph of the prepupae of the leaf-cutter bee C. sculpturalis nested in a bamboo cane and (B) a diagram showing two ways that Melittobia females find new hosts. The mothers of C. sculpturalis build nursing nests with pine resin consisting of individual cells in which their offspring develop. If Melittobia wasps parasitize a host in a cell, female offspring that mate with males inside the cell find a different host on the same patch (bamboo cane) or disperse by flying to other patches.We tested whether the sex ratio of Melittobia australica can be explained by dispersal status in a natural population. We examined how the sex ratio produced by females varies with the number of females laying eggs on a patch and whether or not they have dispersed before laying eggs. To match our data to the predictions of theory, we developed a mathematical model tailored to the unique population structure of Melittobia, where dispersal can be a cue of relatedness. We then conducted a laboratory experiment to test whether Melittobia females are able to directly access the relatedness to other females and adjust their sex ratio behavior accordingly. Our results suggest that females are adjusting their sex ratio in response to both the number of females laying eggs on a patch and their relatedness to the other females. However, relatedness is assessed indirectly by whether or not they have dispersed. Consequently, the solution to the puzzling behavior reflects a more-refined sex ratio strategy.     

From the Cover: Evidence from South Africa for a protracted end-Permian extinction on land

Earth’s largest biotic crisis occurred during the Permo–Triassic Transition (PTT). On land, this event witnessed a turnover from synapsid- to archosauromorph-dominated assemblages and a restructuring of terrestrial ecosystems. However, understanding extinction patterns has been limited by a lack of high-precision fossil occurrence data to resolve events on submillion-year timescales. We analyzed a unique database of 588 fossil tetrapod specimens from South Africa’s Karoo Basin, spanning ∼4 My, and 13 stratigraphic bin intervals averaging 300,000 y each. Using sample-standardized methods, we characterized faunal assemblage dynamics during the PTT. High regional extinction rates occurred through a protracted interval of ∼1 Ma, initially co-occurring with low origination rates. This resulted in declining diversity up to the acme of extinction near the Daptocephalus–Lystrosaurus declivis Assemblage Zone boundary. Regional origination rates increased abruptly above this boundary, co-occurring with high extinction rates to drive rapid turnover and an assemblage of short-lived species symptomatic of ecosystem instability. The “disaster taxon” Lystrosaurus shows a long-term trend of increasing abundance initiated in the latest Permian. Lystrosaurus comprised 54% of all specimens by the onset of mass extinction and 70% in the extinction aftermath. This early Lystrosaurus abundance suggests its expansion was facilitated by environmental changes rather than by ecological opportunity following the extinctions of other species as commonly assumed for disaster taxa. Our findings conservatively place the Karoo extinction interval closer in time, but not coeval with, the more rapid marine event and reveal key differences between the PTT extinctions on land and in the oceans.

Mass extinctions are major perturbations of the biosphere resulting from a wide range of different causes including glaciations and sea level fall (1), large igneous provinces (2), and bolide impacts (3, 4). These events caused permanent changes to Earth’s ecosystems, altering the evolutionary trajectory of life (5). However, links between the broad causal factors of mass extinctions and the biological and ecological disturbances that lead to species extinctions have been difficult to characterize. This is because ecological disturbances unfold on timescales much shorter than the typical resolution of paleontological studies (6), particularly in the terrestrial record (6–8). Coarse-resolution studies have demonstrated key mass extinction phenomena including high extinction rates and lineage turnover (7, 9), changes in species richness (10), ecosystem instability (11), and the occurrence of disaster taxa (12). However, finer time resolutions are central to determining the association and relative timings of these effects, their potential causal factors, and their interrelationships. Achieving these goals represents a key advance in understanding the ecological mechanisms of mass extinctions.The end-Permian mass extinction (ca. 251.9 Ma) was Earth’s largest biotic crisis as measured by taxon last occurrences (13–15). Large outpourings from Siberian Trap volcanism (2) are the likely trigger of calamitous climatic changes, including a runaway greenhouse effect and ocean acidification, which had profound consequences for life on land and in the oceans (16–18). An estimated 81% of marine species (19) and 89% of tetrapod genera became extinct as established Permian ecosystems gave way to those of the Triassic. In the ocean, this included the complete extinction of reef-forming tabulate and rugose corals (20, 21) and significant losses in previously diverse ammonoid, brachiopod, and crinoid families (22). On land, many nonmammalian synapsids became extinct (16), and the glossopterid-dominated floras of Gondwana also disappeared (23). Stratigraphic sequences document a global “coral gap” and “coal gap” (24, 25), suggesting reef and forest ecosystems were rare or absent for up to 5 My after the event (26). Continuous fossil-bearing deposits documenting patterns of turnover across the Permian–Triassic transition (PTT) on land (27) and in the oceans (28) are geographically widespread (29, 30), including marine and continental successions that are known from China (31, 32) and India (33). Continental successions are known from Russia (34), Australia (35), Antarctica (36), and South Africa’s Karoo Basin (Fig. 1 and 37–40), the latter providing arguably the most densely sampled and taxonomically scrutinized (41–43) continental record of the PTT. The main extinction has been proposed to occur at the boundary between two biostratigraphic zones with distinctive faunal assemblages, the Daptocephalus and Lystrosaurus declivis assemblage zones (Fig. 1), which marks the traditional placement of the Permian–Triassic geologic boundary [(37) but see ref. 44]. Considerable research has attempted to understand the anatomy of the PTT in South Africa (38, 39, 45–52) and to place it in the context of biodiversity changes across southern Gondwana (53, 54) and globally (29, 31, 32, 44, 47, 55).Open in a separate windowFig. 1.Map of South Africa depicting the distribution of the four tetrapod fossil assemblage zones (Cistecephalus, Daptocephalus, Lystrosaurus declivis, Cynognathus) and our two study sites where fossils were collected in this study (sites A and B). Regional lithostratigraphy and biostratigraphy within the study interval are shown alongside isotope dilution–thermal ionization mass spectrometry dates retrieved by Rubidge et al., Botha et al., and Gastaldo et al. (37, 44, 80). The traditional (dashed red line) and associated PTB hypotheses for the Karoo Basin (37, 44) are also shown. Although traditionally associated with the PTB, the Daptocephalus–Lystrosaurus declivis Assemblage Zone boundary is defined by first appearances of co-occurring tetrapod assemblages, so its position relative to the three PTB hypotheses is unchanged. The Ripplemead member (*) has yet to be formalized by the South African Committee for Stratigraphy.Decades of research have demonstrated the richness of South Africa’s Karoo Basin fossil record, resulting in hundreds of stratigraphically well-documented tetrapod fossils across the PTT (37, 39, 56). This wealth of data has been used qualitatively to identify three extinction phases and an apparent early postextinction recovery phase (39, 45, 51). Furthermore, studies of Karoo community structure and function have elucidated the potential role of the extinction and subsequent recovery in breaking the incumbency of previously dominant clades, including synapsids (11, 57). Nevertheless, understanding patterns of faunal turnover and recovery during the PTT has been limited by the scarcity of quantitative investigations. Previous quantitative studies used coarsely sampled data (i.e., assemblage zone scale, 2 to 3 Ma time intervals) to identify low species richness immediately after the main extinction, potentially associated with multiple “boom and bust” cycles of primary productivity based on δ¹³C variation during the first 5 My of the Triassic (41, 58). However, many details of faunal dynamics in this interval remain unknown. Here, we investigate the dynamics of this major tetrapod extinction at an unprecedented time resolution (on the order of hundreds of thousands of years), using sample-standardized methods to quantify multiple aspects of regional change across the Cistecephalus, Daptocephalus, and Lystrosaurus declivis assemblage zones.     

Genetically edited CD34+ cells derived from human iPS cells in vivo but not in vitro engraft and differentiate into HIV-resistant cells

Genetic editing of induced pluripotent stem (iPS) cells represents a promising avenue for an HIV cure. However, certain challenges remain before bringing this approach to the clinic. Among them, in vivo engraftment of cells genetically edited in vitro needs to be achieved. In this study, CD34⁺ cells derived in vitro from iPS cells genetically modified to carry the CCR5Δ32 mutant alleles did not engraft in humanized immunodeficient mice. However, the CD34⁺ cells isolated from teratomas generated in vivo from these genetically edited iPS cells engrafted in all experiments. These CD34⁺ cells also gave rise to peripheral blood mononuclear cells in the mice that, when inoculated with HIV in cell culture, were resistant to HIV R5-tropic isolates. This study indicates that teratomas can provide an environment that can help evaluate the engraftment potential of CD34⁺ cells derived from the genetically modified iPS cells in vitro. The results further confirm the possibility of using genetically engineered iPS cells to derive engraftable hematopoietic stem cells resistant to HIV as an approach toward an HIV cure.

A major objective of recent HIV research is to develop a “cure” for this virus infection that avoids lifelong adherence to antiretroviral therapy (ART). One of the approaches toward reaching this objective has been to genetically delete or mutate genes encoding for proteins that promote HIV infection and spread. An attractive candidate for this strategy is the Ccr5 gene, for which a genetic mutation causing a 32-bp deletion has been shown to be associated with natural protection from HIV infection and disease (1, 2). The Ccr5 gene encodes CCR5, a human cell-surface chemokine receptor that is a coreceptor for HIV attachment and infection of cells (3, 4). The Ccr5 allele with its 32-bp deletion results in a truncated isoform of the CCR5 receptor, CCR5Δ32, which is not expressed at the cell surface. Thus, entry of the virus into the cell is blocked (5).Induced pluripotent stem (iPS) cells (6), because of their capacity to differentiate into CD34⁺ hematopoietic stem cells (HSCs) (7), can reconstitute a full immune system (8, 9). These iPS cells are therefore a target of choice for genetic engineering. Our group and others have demonstrated that iPS cells generated from the peripheral blood mononuclear cells (PBMC) of both healthy individuals (10) and HIV-infected patients under ART (11) can have their wild-type allele of the Ccr5 gene genetically edited to carry the Ccr5 Δ32 mutation (12, 13). Notably, using CRISPR/Cas9 technology, the Ccr5 gene can be modified to have the naturally occurring Δ32 variant allele that has been associated with resistance to R5-tropic viruses. Moreover, while it is not present at the cell surface, the truncated CCR5Δ32 protein is still expressed and, as such, could have other important physiological roles (14–17).We have confirmed that the genetically modified Ccr5 Δ32 iPS cells can be differentiated into CD34⁺ HSCs in vitro (10, 18). Under appropriate cell culture conditions, they can give rise to various myeloid and lymphoid cell lineages (10, 11, 18). This result can also be observed with the formation of teratomas following the injection of large quantities of iPS cells into mice. Teratomas are multicellular tumors composed of many different cell types including HSCs. Notably, immune cells with the CCR5Δ32 mutation differentiated in vitro from the genetically modified iPS cell-derived HSCs and inoculated with HIV are resistant to R5-tropic virus infection (10, 18).These results have suggested that editing Ccr5 in iPS cells from HIV-infected subjects can be a promising strategy toward an HIV cure. The pluripotent stem cells can be induced from a small number of PBMC from the patients and genetically modified to become resistant to HIV infection (10, 11, 18). In this case, leukapheresis to obtain large amounts of these cells (19) is not required. The edited HSCs could then be transplanted back to the original patient without concern for immune cell rejection. Therefore, because these experiments were performed in cell culture, an important remaining question is whether in vitro-edited iPS cells can differentiate into HSCs that can be transplanted back into a recipient in vivo (20).To address this question, transplantation of the in vitro-derived CD34⁺ cells was attempted under various conditions in animal models of humanized or immunodeficient mice (21). In approaches to obtain sufficient numbers of CD34⁺ cells for transplantation, our ability to grow them in vitro offered an opportunity. However, although we could expand CD34⁺ cells substantially in culture (18), we observed that engraftment of these cell culture-derived CD34⁺ cells in humanized NSG-BLT mice did not occur. Thus, alternatively, to study the genetically edited cells in vivo, we explored the use of differentiated CD34⁺ cells in vivo via the generation of teratomas from iPS cells. We found that not only did these teratomas successfully yield human CD34⁺ cells, but importantly, these CD34⁺ cells could engraft in recipient immunodeficient NSG mice. This observation has been made by Nakauchi and colleagues (22) with different mouse strains. Finally, we confirmed that the PBMC formed in mice from these teratoma-derived genetically edited CD34⁺ cells are resistant to ex vivo R5-tropic HIV infection when they carry the mutant Δ32 Ccr5 allele.     

Identifying hydrophobic protein patches to inform protein interaction interfaces

Interactions between proteins lie at the heart of numerous biological processes and are essential for the proper functioning of the cell. Although the importance of hydrophobic residues in driving protein interactions is universally accepted, a characterization of protein hydrophobicity, which informs its interactions, has remained elusive. The challenge lies in capturing the collective response of the protein hydration waters to the nanoscale chemical and topographical protein patterns, which determine protein hydrophobicity. To address this challenge, here, we employ specialized molecular simulations wherein water molecules are systematically displaced from the protein hydration shell; by identifying protein regions that relinquish their waters more readily than others, we are then able to uncover the most hydrophobic protein patches. Surprisingly, such patches contain a large fraction of polar/charged atoms and have chemical compositions that are similar to the more hydrophilic protein patches. Importantly, we also find a striking correspondence between the most hydrophobic protein patches and regions that mediate protein interactions. Our work thus establishes a computational framework for characterizing the emergent hydrophobicity of amphiphilic solutes, such as proteins, which display nanoscale heterogeneity, and for uncovering their interaction interfaces.

Protein–protein interactions play a crucial role in numerous biological processes, ranging from signal transduction and immune response to protein aggregation and phase behavior (1–3). Consequently, being able to understand, predict, and modulate protein interactions has important implications for understanding cellular processes and mitigating the progression of disease (4, 5). A necessary first step toward this ambitious goal is uncovering the interfaces through which proteins interact (6–8). In principle, identifying hydrophobic protein regions, which interact weakly with water, should be a promising strategy for uncovering protein interaction interfaces (9, 10). Indeed, the release of weakly interacting hydration waters from hydrophobic regions can drive protein interactions, as well as other aqueous assemblies (11–13). However, even when the structure of a protein is available at atomistic resolution, it is challenging to identify its hydrophobic patches because they are not uniformly nonpolar, but display variations in polarity and charge at the nanoscale. Moreover, the emergent hydrophobicity of a protein patch stems from the collective response of protein hydration waters to the nanoscale chemical and topographical patterns displayed by the patch (14–20) and cannot be captured by simply counting the number of nonpolar groups in the patch, or even through more involved additive approaches, such as hydropathy scales or surface-area models (21–28).To address this challenge, we build upon seminal theoretical advances and molecular simulation studies, which have shown that near a hydrophobic surface, it is easier to disrupt surface–water interactions and form interfacial cavities (29–34). To uncover protein regions that have the weakest interactions with water, here, we employ specialized molecular simulations, wherein protein–water interactions are disrupted by systematically displacing water molecules from the protein hydration shell (35–37). By identifying the protein patches that nucleate cavities most readily in our simulations, we are then able to uncover the most hydrophobic protein regions. Interestingly, we find that both hydrophobic and hydrophilic protein patches are highly heterogeneous and contain comparable numbers of nonpolar and polar atoms. Our results thus highlight the nontrivial relationship between the chemical composition of protein patches and their emergent hydrophobicity (24–26), and further emphasize the importance of accounting for the collective solvent response in characterizing protein hydrophobicity (16). We then interrogate whether the most hydrophobic protein patches, which nucleate cavities readily, are also likely to mediate protein interactions. Across five proteins that participate in either homodimer or heterodimer formation, we find that roughly 60 to 70% of interfacial contacts and only about 10 to 20% of noncontacts nucleate cavities. Our work thus provides a versatile computational framework for characterizing hydrophobicity and uncovering interaction interfaces of not just proteins, but also of other complex amphiphilic solutes, such as cavitands, dendrimers, and patchy nanoparticles (38–41).