A subset of spinal dorsal horn interneurons crucial for gating touch-evoked pain-like behavior

A cardinal, intractable symptom of neuropathic pain is mechanical allodynia, pain caused by innocuous stimuli via low-threshold mechanoreceptors such as Aβ fibers. However, the mechanism by which Aβ fiber-derived signals are converted to pain remains incompletely understood. Here we identify a subset of inhibitory interneurons in the spinal dorsal horn (SDH) operated by adeno-associated viral vectors incorporating a neuropeptide Y promoter (AAV-NpyP⁺) and show that specific ablation or silencing of AAV-NpyP⁺ SDH interneurons converted touch-sensing Aβ fiber-derived signals to morphine-resistant pain-like behavioral responses. AAV-NpyP⁺ neurons received excitatory inputs from Aβ fibers and transmitted inhibitory GABA signals to lamina I neurons projecting to the brain. In a model of neuropathic pain developed by peripheral nerve injury, AAV-NpyP⁺ neurons exhibited deeper resting membrane potentials, and their excitation by Aβ fibers was impaired. Conversely, chemogenetic activation of AAV-NpyP⁺ neurons in nerve-injured rats reversed Aβ fiber-derived neuropathic pain-like behavior that was shown to be morphine-resistant and reduced pathological neuronal activation of superficial SDH including lamina I. These findings suggest that identified inhibitory SDH interneurons that act as a critical brake on conversion of touch-sensing Aβ fiber signals into pain-like behavioral responses. Thus, enhancing activity of these neurons may offer a novel strategy for treating neuropathic allodynia.

Damage to the nervous system by cancer, diabetes, chemotherapy, infection, or traumatic injury causes neuropathic pain, a highly debilitating chronic pain condition (1). A cardinal symptom of neuropathic pain is mechanical allodynia, pain that is produced by innocuous mechanical stimulus, such as light touch. The mechanisms underlying mechanical allodynia are poorly understood. Currently available treatments including opioids are largely ineffective.Light mechanical information from the skin is conveyed to the spinal dorsal horn (SDH) via primary afferent low-threshold mechanoreceptors (LTMRs), such as Aβ fibers. These LTMRs are considered to mediate mechanical allodynia (2–7). A major question is where and how touch signals are pathologically converted to pain in the context of nerve damage. One potential region could be the SDH where Aβ fibers and nociceptors interact through interneurons (6, 8–10), as depicted in the gate control theory of pain (11). Over the last 5 y, studies using multiple lines of transgenic mice have identified several subsets of excitatory and inhibitory interneurons in the SDH that are genetically defined (12–18) and shown that these subsets are involved in peripheral nerve injury (PNI)-induced mechanical hypersensitivity (assessed using von Frey filaments). However, the behavioral hypersensitivity by these filaments involves activation not only of LTMRs but also of nociceptors (19–24) and is effectively suppressed by treatment with morphine (18, 25). Thus, the mechanisms underlying LTMR-derived and morphine-resistant neuropathic allodynia are still poorly understood.Using a transgenic rat line W-TChR2V4 in which channelrhodopsin-2 (ChR2) was expressed at nerve endings associated with Merkel cells and lamellar cells to form Meissner’s corpuscle-like structures in the skin (26), we recently reported that following PNI, stimulation of touch-sensing Aβ fibers by illuminating the rats with blue light elicited morphine-resistant mechanical allodynia-like responses (27). Furthermore, Aβ fiber stimulation after PNI causes activation of lamina I neurons, which are normally silent in response to this stimulation. This raises the possibility that alterations in SDH circuits after PNI underscore the conversion of Aβ fiber-derived signals to morphine-resistant pain, but the underlying mechanisms remain to be determined. Considering previous findings (6, 8–11), a possible mechanism for the conversion might involve a loss or reduction of the activity of inhibitory interneurons in the SDH. A single-cell RNA sequencing study has shown that SDH inhibitory interneurons are genetically divided into over 10 subsets, some of which express mRNA encoding neuropeptide Y (NPY) (28). In immunohistochemistry, NPY has been shown to be expressed in inhibitory interneurons (29, 30). Furthermore, previous studies have demonstrated that spinal NPY has inhibitory effects on chronic pain, including PNI-induced mechanical hypersensitivity (31, 32). Thus, to identify a subset of inhibitory SDH interneurons that contributes to the behavioral symptom evoked by optical stimulation of the primary afferent Aβ fibers in the W-TChR2V4 rats after PNI, we focused on the role of inhibitory SDH interneurons operated by an adeno-associated viral (AAV) vector including a Npy promoter (AAV-NpyP). Using the optogenetic approach for neuropathic allodynia (27) combined with chemogenetics, electrophysiology, and transsynaptic tracing, this study reveals that diminished inhibitory tone of these SDH neurons operated by AAV-NpyP contributes to Aβ fiber-derived neuropathic pain-like behavioral responses. Our findings suggest that this subset of neurons could be a therapeutic target for treating neuropathic mechanical allodynia.     

Cell type-specific lipid storage changes in Parkinson’s disease patient brains are recapitulated by experimental glycolipid disturbance

Neurons are dependent on proper trafficking of lipids to neighboring glia for lipid exchange and disposal of potentially lipotoxic metabolites, producing distinct lipid distribution profiles among various cell types of the central nervous system. Little is known of the cellular distribution of neutral lipids in the substantia nigra (SN) of Parkinson’s disease (PD) patients and its relationship to inflammatory signaling. This study aimed to determine human PD SN neutral lipid content and distribution in dopaminergic neurons, astrocytes, and microglia relative to age-matched healthy subject controls. The results show that while total neutral lipid content was unchanged relative to age-matched controls, the levels of whole SN triglycerides were correlated with inflammation-attenuating glycoprotein non-metastatic melanoma protein B (GPNMB) signaling in human PD SN. Histological localization of neutral lipids using a fluorescent probe (BODIPY) revealed that dopaminergic neurons and midbrain microglia significantly accumulated intracellular lipids in PD SN, while adjacent astrocytes had a reduced lipid load overall. This pattern was recapitulated by experimental in vivo inhibition of glucocerebrosidase activity in mice. Agents or therapies that restore lipid homeostasis among neurons, astrocytes, and microglia could potentially correct PD pathogenesis and disease progression.

Both neurons and glia depend on tight regulation and exchange of lipids for proper function. Typically through lipid transport mechanisms (1, 2), the continuous exchange of lipids is essential for maintaining physiological function as brain lipid content, transport, and distribution are complex and critical aspects of neuropathology. Recently, both clinical findings and experimental studies have implicated lipid storage and trafficking in the pathogenesis of Parkinson’s disease (PD) and related disorders (3, 4). Reduced function of lysosomal hydrolases that are associated with lysosomal storage disorders increases the risk for PD and results in brain pathology similar to that seen in most sporadic and genetic forms of the disease (5–11).One of the strongest genetic risk factors for PD is heterozygous loss-of-function mutations in GBA1, encoding the lysosomal hydrolase glucocerebrosidase (GCase) (12–14), a deficiency that causes systemic accumulation of its glycolipid substrate glucosylceramide (GlcCer). GCase activity is reduced with aging of both the human and murine brain (6, 15), and age is the overall greatest risk factor for developing PD (16). In contrast, the more severe loss of GCase activity in homozygous GBA1 mutant carriers causes the lysosomal storage disease Gaucher disease (GD) (17). Conduritol beta epoxide (CBE), an irreversible inhibitor of GCase, causes widespread accumulation of GlcCer and related glycosphingolipids in mice. In this model, there is marked increase of high molecular weight alpha-synuclein (aSYN) and deposition of proteinase K-resistant aSYN resembling that seen in PD (18–20). aSYN is a constituent of the classical Lewy bodies and Lewy neurites (21) found in surviving dopaminergic neurons in postmortem PD materials as a standard pathological criterion for PD (22). aSYN has a lipid-binding domain, and aSYN protein–lipid interactions are potentially perturbed in PD (reviewed in refs. 17, 23, 24). We recently demonstrated that excessive aSYN can deposit into lipid compartments, and that this process is reversible under increased lysosomal β-hexosaminidase expression (25).Several in vitro physiological studies have demonstrated that a reduction in neuronal neutral lipid storage or knockdown of fatty acid desaturases protects cultured neurons from degeneration (26–28). However, as neurons exhibit limited capacity to synthesize, metabolize, and transport lipid species under physiological conditions (29), other resident cells of the substantia nigra (SN), such as glial cells, are required to maintain lipid homeostasis in the brain. Astrocyte health and lipid exchange function—and microglial activation—are potentially central to PD pathogenesis and other age-dependent neurodegenerative diseases (30–33). Brain resident microglia can accumulate and generate lipids, which may propagate inflammatory processes through, for example, TREM2 binding of apolipoproteins (34–38). Proinflammatory cytokines present in chronic conditions are attenuated by the binding of glycoprotein nonmetastatic melanoma protein B (GPNMB) to the CD44 receptor on astrocytes (39). In GD patient serum, GPNMB level is significantly correlated with disease severity (40), and GPNMB is increased in human PD SN and following CBE-induced glycolipid accumulation in mice (41).There are surprisingly little data on lipid distribution patterns in PD-affected cell types given the relevance of lipid homeostasis, aSYN–lipid interactions, and lipidopathy-associated inflammatory signatures as they relate to PD (17, 41). In this study, we measured the differences in total lipid content and cellular distribution between PD and healthy subject (HS) SN and compared them with a lipid-associated neuroinflammatory signal, GPNMB. To quantify cell type-specific intracellular lipid content, colocalization analysis was performed on human postmortem SN sections that were costained for neutral lipids and markers of dopaminergic neurons, astrocytes, and microglia. Compared with HS SN, the lipid content of PD dopaminergic neurons and microglia was significantly higher, and that of astrocytes was significantly lower. To understand a possible mechanistic reason for this lipid distribution pattern, we used an in vivo mouse model of glycolipid dysregulation and attendant aSYN accumulation through GCase inhibition by CBE. This in vivo model recapitulated the lipid distribution pattern that we observed in human PD SN. Based on these data, we propose that PD is characterized by a unique neutral lipid distribution signature in neurons, astrocytes, and microglia that can be recapitulated experimentally by glycolipid dysregulation.     

Wild-type α-synuclein inherits the structure and exacerbated neuropathology of E46K mutant fibril strain by cross-seeding

Heterozygous point mutations of α-synuclein (α-syn) have been linked to the early onset and rapid progression of familial Parkinson’s diseases (fPD). However, the interplay between hereditary mutant and wild-type (WT) α-syn and its role in the exacerbated pathology of α-syn in fPD progression are poorly understood. Here, we find that WT mice inoculated with the human E46K mutant α-syn fibril (hE46K) strain develop early-onset motor deficit and morphologically different α-syn aggregation compared with those inoculated with the human WT fibril (hWT) strain. By using cryo-electron microscopy, we reveal at the near-atomic level that the hE46K strain induces both human and mouse WT α-syn monomers to form the fibril structure of the hE46K strain. Moreover, the induced hWT strain inherits most of the pathological traits of the hE46K strain as well. Our work suggests that the structural and pathological features of mutant strains could be propagated by the WT α-syn in such a way that the mutant pathology would be amplified in fPD.

α-Synuclein (α-Syn) is the main component of Lewy bodies, which serve as the common histological hallmark of Parkinson’s disease (PD) and other synucleinopathies (1, 2). α-Syn fibrillation and cell-to-cell transmission in the brain play essential roles in disease progression (3–5). Interestingly, WT α-syn could form fibrils with distinct polymorphs, which exhibit disparate seeding capability in vitro and induce distinct neuropathologies in mouse models (6–10). Therefore, it is proposed that α-syn fibril polymorphism may underlie clinicopathological variability of synucleinopathies (6, 9). In fPD, several single-point mutations of SNCA have been identified, which are linked to early-onset, severe, and highly heterogeneous clinical symptoms (11–13). These mutations have been reported to influence either the physiological or pathological function of α-syn (14). For instance, A30P weakens while E46K strengthens α-syn membrane binding affinity that may affect its function in synaptic vesicle trafficking (14, 15). E46K, A53T, G51D, and H50Q have been found to alter the aggregation kinetics of α-syn in different manners (15–17). Recently, several cryogenic electron microscopy (cryo-EM) studies revealed that α-syn with these mutations forms diverse fibril structures that are distinct from the WT α-syn fibrils (18–26). Whether and how hereditary mutations induced fibril polymorphism contributes to the early-onset and exacerbated pathology in fPD remains to be elucidated. More importantly, most fPD patients are heterozygous for SNCA mutations (12, 13, 27, 28), which leads to another critical question: could mutant fibrils cross-seed WT α-syn to orchestrate neuropathology in fPD patients?E46K mutation is one of the eight disease-causing mutations on SNCA originally identified from a Spanish family with autosomal-dominant PD (11). E46K-associated fPD features early-onset motor symptoms and rapid progression of dementia with Lewy bodies (11). Studies have shown that E46K mutant has higher neurotoxicity than WT α-syn in neurons and mouse models overexpressing α-syn (29–32). The underlying mechanism is debatable. Some reported that E46K promotes the formation of soluble species of α-syn without affecting the insoluble fraction (29, 30), while others suggested that E46K mutation may destabilize α-syn tetramer and induce aggregation (31, 32). Our previous study showed that E46K mutation disrupts the salt bridge between E46 and K80 in the WT fibril strain and rearranges α-syn into a different polymorph (33). Compared with the WT strain, the E46K fibril strain is prone to be fragmented due to its smaller and less stable fibril core (33). Intriguingly, the E46K strain exhibits higher seeding ability in vitro, suggesting that it might induce neuropathology different from the WT strain in vivo (33).In this study, we found that human E46K and WT fibril strains (referred to as hE46K and hWT strains) induced α-syn aggregates with distinct morphologies in mice. Mice injected with the hE46K strain developed more α-syn aggregation and early-onset motor deficits compared with the mice injected with the hWT strain. Notably, the hE46K strain was capable of cross-seeding both human and mouse WT (mWT) α-syn to form fibrils (named as hWT_cs and mWT_cs). The cross-seeded fibrils replicated the structure and seeding capability of the hE46K template both in vitro and in vivo. Our results suggest that the hE46K strain could propagate its structure as well as the seeding properties to the WT monomer so as to amplify the α-syn pathology in fPD.     

A neural circuit for competing approach and defense underlying prey capture

Predators must frequently balance competing approach and defensive behaviors elicited by a moving and potentially dangerous prey. Several brain circuits supporting predation have recently been localized. However, the mechanisms by which these circuits balance the conflict between approach and defense responses remain unknown. Laboratory mice initially show alternating approach and defense responses toward cockroaches, a natural prey, but with repeated exposure become avid hunters. Here, we used in vivo neural activity recording and cell-type specific manipulations in hunting male mice to identify neurons in the lateral hypothalamus and periaqueductal gray that encode and control predatory approach and defense behaviors. We found a subset of GABAergic neurons in lateral hypothalamus that specifically encoded hunting behaviors and whose stimulation triggered predation but not feeding. This population projects to the periaqueductal gray, and stimulation of these projections promoted predation. Neurons in periaqueductal gray encoded both approach and defensive behaviors but only initially when the mouse showed high levels of fear of the prey. Our findings allow us to propose that GABAergic neurons in lateral hypothalamus facilitate predation in part by suppressing defensive responses to prey encoded in the periaqueductal gray. Our results reveal a neural circuit mechanism for controlling the balance between conflicting approach and defensive behaviors elicited by the same stimulus.

The ability to seek and capture prey adeptly is a conserved behavior essential to the survival of numerous animal species. However, attacking prey brings risks for the predator, and efficient hunting requires a skillful balance between responding to threatening and appetitive prey cues. The neural circuits involved in this balance are unknown. Rodents naturally hunt and consume a variety of insects, and hunting behavior has been studied in the laboratory using rats given cockroaches or mice (1) and, more recently, in laboratory mice given crickets (2–5). In early studies, immediate early gene mapping during hunting identified the recruitment of lateral hypothalamus (LHA) (1, 6, 7) and periaqueductal gray (PAG) (7, 8), and electrical stimulation of either LHA (9–12) or PAG (13, 14) could initiate avid predatory attacks in rats and cats, suggesting the presence of neuronal cell populations that promote hunting in both these brain structures.LHA has been historically regarded as a central node in the neural system controlling seeking behaviors, including feeding and predatory hunting (14–16). Recent circuit neuroscience work has confirmed this role by finding that stimulation of Vgat+ GABAergic neurons in LHA can trigger predation as well as feeding (5, 17–20). However, the role of these neurons in predation may be indirect, as any manipulation that increases food seeking is expected to promote motivation to hunt as well. Moreover, some studies in which LHA GABAergic neurons were optogenetically stimulated did not observe the full repertoire of feeding behaviors but instead found increased chewing activity, digging, or general motor activity (21–23), suggesting that the link between LHA-driven feeding and hunting may be more complex. One possible confound in these studies is their use of two different Cre driver lines (Vgat::Cre versus Gad2::Cre) that have been shown to target distinct LHA GABAergic neurons (23). Thus, it remains unclear to what extent different populations of LHA GABAergic neurons specifically encode and control predatory behaviors.A link between LHA and PAG in predation was made by a recent study showing that activation of LHA to PAG projections can promote predatory hunting in mice (5), and the central importance of the PAG in hunting is further strengthened by several studies identifying a series of PAG afferents arising from different brain structures—including the central nucleus of the amygdala, medial preoptic area, and zona incerta—that promote predation (3, 4, 24, 25). However, the precise functional role of the PAG in hunting remains unclear. For example, it was found that inhibition of glutamatergic neurons in the lateral and ventrolateral PAG (l/vlPAG) by GABAergic inputs from the central nucleus of the amygdala promotes predatory behavior (3), suggesting that PAG neurons might function to suppress rather than promote predation. At the same time, other studies have shown that activation of glutamatergic neurons in l/vlPAG induces defensive behaviors (26), a finding that is consistent with an extensive literature linking PAG to both innate and learned defensive behavior (27–29). One explanation for these observations is that PAG has an antagonistic role in hunting by promoting defensive behaviors toward prey early in the encounter when familiarity with the prey is low. Suppression of these defensive responses by GABAergic inputs would therefore facilitate unimpeded predation.Here, we identify Gad2+ neurons in LHA as those specifically recruited during predatory chasing and attack, and both are sufficient and necessary to drive hunting, but not feeding or social behavior, in male mice. Activation of LHA Gad2+ neuron projections to PAG decreased defensive responses to prey and promoted hunting during the early phases of predation when male mice learned to hunt cockroaches. Finally, in vivo calcium endoscopy identified a neural population in PAG that encoded risk assessment and flight behaviors elicited by prey. Notably, we failed to detect PAG neurons responsive to predatory pursuit or attack. These results point to a circuit in which activity in LHA Gad2+ neurons promotes hunting in part by suppressing defensive responses encoded in PAG.     

CYK-1/Formin activation in cortical RhoA signaling centers promotes organismal left–right symmetry breaking

Proper left–right symmetry breaking is essential for animal development, and in many cases, this process is actomyosin-dependent. In Caenorhabditis elegans embryos active torque generation in the actomyosin layer promotes left–right symmetry breaking by driving chiral counterrotating cortical flows. While both Formins and Myosins have been implicated in left–right symmetry breaking and both can rotate actin filaments in vitro, it remains unclear whether active torques in the actomyosin cortex are generated by Formins, Myosins, or both. We combined the strength of C. elegans genetics with quantitative imaging and thin film, chiral active fluid theory to show that, while Non-Muscle Myosin II activity drives cortical actomyosin flows, it is permissive for chiral counterrotation and dispensable for chiral symmetry breaking of cortical flows. Instead, we find that CYK-1/Formin activation in RhoA foci is instructive for chiral counterrotation and promotes in-plane, active torque generation in the actomyosin cortex. Notably, we observe that artificially generated large active RhoA patches undergo rotations with consistent handedness in a CYK-1/Formin–dependent manner. Altogether, we conclude that CYK-1/Formin–dependent active torque generation facilitates chiral symmetry breaking of actomyosin flows and drives organismal left–right symmetry breaking in the nematode worm.

The emergence of left–right asymmetry is essential for normal animal development and, in the majority of animal species, one type of handedness is dominant (1). The actin cytoskeleton plays an instrumental role in establishing the left–right asymmetric body plan of invertebrates like fruit flies (2–6), nematodes (7–11), and pond snails (12–15). Moreover, an increasing number of studies showed that vertebrate left–right patterning also depends on a functional actomyosin cytoskeleton (13, 16–22). Actomyosin-dependent chiral behavior has even been reported in isolated cells (23–28) and such cell-intrinsic chirality has been shown to promote left–right asymmetric morphogenesis of tissues (29, 30), organs (21, 31), and entire embryonic body plans (12, 13, 32, 33). Active force generation in the actin cytoskeleton is responsible for shaping cells and tissues during embryo morphogenesis. Torques are rotational forces with a given handedness and it has been proposed that in plane, active torque generation in the actin cytoskeleton drives chiral morphogenesis (7, 8, 34, 35).What could be the molecular origin of these active torques? The actomyosin cytoskeleton consists of actin filaments, actin-binding proteins, and Myosin motors. Actin filaments are polar polymers with a right-handed helical pitch and are therefore chiral themselves (36, 37). Due to the right-handed pitch of filamentous actin, Myosin motors can rotate actin filaments along their long axis while pulling on them (33, 38–42). Similarly, when physically constrained, members of the Formin family rotate actin filaments along their long axis while elongating them (43). In both cases the handedness of this rotation is determined by the helical nature of the actin polymer. From this it follows that both Formins and Myosins are a potential source of molecular torque generation that could drive cellular and organismal chirality. Indeed, chiral processes across different length scales, and across species, are dependent on Myosins (19), Formins (13–15, 26), or both (7, 8, 21, 44). It is, however, unclear how Formins and Myosins contribute to active torque generation and the emergence chiral processes in developing embryos.In our previous work we showed that the actomyosin cortex of some Caenorhabditis elegans embryonic blastomeres undergoes chiral counterrotations with consistent handedness (7, 35). These chiral actomyosin flows can be recapitulated using active chiral fluid theory that describes the actomyosin layer as a thin-film, active gel that generates active torques (7, 45, 46). Chiral counterrotating cortical flows reorient the cell division axis, which is essential for normal left–right symmetry breaking (7, 47). Moreover, cortical counterrotations with the same handedness have been observed in Xenopus one-cell embryos (32), suggesting that chiral counterrotations are conserved among distant species. Chiral counterrotating actomyosin flow in C. elegans blastomeres is driven by RhoA signaling and is dependent on Non-Muscle Myosin II motor proteins (7). Moreover, the Formin CYK-1 has been implicated in actomyosin flow chirality during early polarization of the zygote as well as during the first cytokinesis (48, 49). Despite having identified a role for Myosins and Formins, the underlying mechanism by which active torques are generated remains elusive.Here we show that the Diaphanous-like Formin, CYK-1/Formin, is a critical determinant for the emergence of actomyosin flow chirality, while Non-Muscle Myosin II (NMY-2) plays a permissive role. Our results show that cortical CYK-1/Formin is recruited by active RhoA signaling foci and promotes active torque generation, which in turn tends to locally rotate the actomyosin cortex clockwise. In the highly connected actomyosin meshwork, a gradient of these active torques drives the emergence of chiral counterrotating cortical flows with uniform handedness, which is essential for proper left–right symmetry breaking. Together, these results provide mechanistic insight into how Formin-dependent torque generation drives cellular and organismal left–right symmetry breaking.     

The β-encapsulation cage of rearrangement hotspot (Rhs) effectors is required for type VI secretion

Bacteria deploy rearrangement hotspot (Rhs) proteins as toxic effectors against both prokaryotic and eukaryotic target cells. Rhs proteins are characterized by YD-peptide repeats, which fold into a large β-cage structure that encapsulates the C-terminal toxin domain. Here, we show that Rhs effectors are essential for type VI secretion system (T6SS) activity in Enterobacter cloacae (ECL). ECL rhs⁻ mutants do not kill Escherichia coli target bacteria and are defective for T6SS-dependent export of hemolysin-coregulated protein (Hcp). The RhsA and RhsB effectors of ECL both contain Pro−Ala−Ala−Arg (PAAR) repeat domains, which bind the β-spike of trimeric valine−glycine repeat protein G (VgrG) and are important for T6SS activity in other bacteria. Truncated RhsA that retains the PAAR domain is capable of forming higher-order, thermostable complexes with VgrG, yet these assemblies fail to restore secretion activity to ∆rhsA ∆rhsB mutants. Full T6SS-1 activity requires Rhs that contains N-terminal transmembrane helices, the PAAR domain, and an intact β-cage. Although ∆rhsA ∆rhsB mutants do not kill target bacteria, time-lapse microscopy reveals that they assemble and fire T6SS contractile sheaths at ∼6% of the frequency of rhs⁺ cells. Therefore, Rhs proteins are not strictly required for T6SS assembly, although they greatly increase secretion efficiency. We propose that PAAR and the β-cage provide distinct structures that promote secretion. PAAR is clearly sufficient to stabilize trimeric VgrG, but efficient assembly of T6SS-1 also depends on an intact β-cage. Together, these domains enforce a quality control checkpoint to ensure that VgrG is loaded with toxic cargo before assembling the secretion apparatus.

Bacteria use many strategies to compete against other microorganisms in the environment. Research over the past 15 y has uncovered several distinct mechanisms by which bacteria deliver inhibitory toxins directly into neighboring competitors (1–8). Cell contact-dependent competition systems have been characterized most extensively in Gram-negative bacteria, and the most widespread mechanism is mediated by the type VI secretion system (T6SS) (9). T6SSs are multiprotein complexes related in structure and function to the contractile tails of Myoviridae bacteriophages. T6SS loci vary considerably between bacterial species, but all encode 13 core type VI secretion (Tss) proteins that are required to build a functional apparatus. TssJ, TssL, and TssM form a multimeric complex that spans the cell envelope and serves as the secretion conduit. The phage-like baseplate is composed of TssE, TssF, TssG, and TssK proteins, which form a sixfold symmetrical array surrounding a central “hub” of trimeric valine−glycine repeat protein G (VgrG/TssI). VgrG is structurally homologous to the gp27−gp5 tail spike of phage T4 (10, 11). The T4 tail spike is further acuminated with gp5.4, a small protein that forms a sharpened apex at the tip of the gp5 spike (12). Proline−alanine−alanine−arginine (PAAR) repeat proteins form an orthologous structure on VgrG; and PAAR is thought to facilitate penetration of the target cell outer membrane (13). The T6SS duty cycle begins when the baseplate docks onto TssJLM at the cytoplasmic face of the inner membrane (14). The baseplate then serves as the assembly origin for the contractile sheath and inner tube. The sheath is built from TssB−TssC subunits, and the tube is formed by stacked hexameric rings of hemolysin-coregulated protein (Hcp/TssD). TssA coordinates this assembly process to ensure that the sheath and tube are polymerized at equivalent rates (15). After elongating across the width of the cell, the sheath undergoes rapid contraction to expel the PAAR•VgrG-capped Hcp tube through the transenvelope complex. The ejected tube impales neighboring cells and delivers a variety of toxic effector proteins into the target. After firing, the contracted sheath is disassembled by the ClpV (TssH) ATPase (16), and the recycled TssBC subunits are used to support additional rounds of sheath assembly and contraction.T6SSs were originally identified through their ability to intoxicate eukaryotic host cells (17), and VgrG proteins were the first effectors to be recognized. VgrG-1 from Vibrio cholerae V52 carries a C-terminal domain that cross-links actin and blocks macrophage phagocytosis (10). Similarly, the VgrG1 protein from Aeromonas hydrophila American Type Culture Collection (ATCC) 7966 carries a C-terminal actin adenosine 5′-diphosphate (ADP) ribosyltransferase domain that disrupts the host cytoskeleton (18). Although the T6SS clearly plays a role in pathogenesis, most of the systems characterized to date deliver toxic effectors into competing bacteria. Because antibacterial effectors are potentially autoinhibitory, these latter toxins are invariably encoded with specific immunity proteins. Antibacterial effectors commonly disrupt the integrity of the bacterial cell envelope. VgrG-3 from V. cholerae carries a lysozyme-like domain that degrades the peptidoglycan cell wall (19, 20). Other peptidoglycan-cleaving amidase toxins are packaged within the lumen of Hcp hexamers for T6SS-mediated delivery (21–24). Phospholipase toxins collaborate with peptidoglycan degrading enzymes to lyse target bacteria (25–27). Other T6SS effectors act in the cytosol to degrade nucleic acids and nicotinamide adenine dinucleotide cofactors (3, 28, 29). Most recently, Whitney and coworkers described a novel T6SS effector that produces the inhibitory nucleotide ppApp (30). These latter toxins are commonly delivered through noncovalent interactions with VgrG. Many effectors contain PAAR domains, which enable direct binding to the C-terminal β-spike of VgrG (13), whereas others are indirectly tethered to VgrG through adaptor proteins (31–33). This combinatorial strategy allows multiple different toxins to be delivered with each firing event.Rearrangement hotspot (Rhs) proteins are potent effectors deployed by many T6SS⁺ bacteria (3, 34–37). T6SS-associated Rhs effectors range from ∼150 kDa to 180 kDa in mass and carry highly variable C-terminal toxin domains. The N-terminal region of Rhs proteins often contains two predicted transmembrane (TM) helix regions followed by a PAAR domain. The central region is composed of many Rhs/YD-peptide repeats, which form a β-cage structure that fully encapsulates the toxin domain (38). Genes coding for Rhs were first identified in Escherichia coli K-12 as elements that promote chromosomal duplication (39, 40). This genomic rearrangement was the result of unequal recombination between the rhsA and rhsB loci, which share 99.4% sequence identity over some 3,700 nucleotides. Subsequently, Hill and coworkers recognized that rhs genes are genetic composites (41), and that the variable C-terminal extension domains inhibit cell growth (42). Although E. coli K-12 encodes four full-length Rhs proteins, it lacks a T6SS, and there is no evidence that it deploys Rhs in competition. However, other Rhs/YD-peptide repeat proteins are known to deliver toxins in a T6SS-independent manner. Gram-positive bacteria export antibacterial YD-repeat proteins through the Sec pathway (3), and the tripartite insecticidal toxin complexes released by Photorhabdus and Yersinia species contain subunits with Rhs/YD repeats (38, 43). Thus, the Rhs encapsulation structure has been incorporated into at least three different toxin delivery platforms.Here, we report that Rhs effectors are critical for the activity of the T6SS-1 locus of Enterobacter cloacae ATCC 13047 (ECL). ECL encodes two Rhs effectors—RhsA and RhsB—which are each exported in a constitutive manner by T6SS-1 (35). Deletion of either rhs gene has little effect on T6SS-1 activity, but mutants lacking both rhsA and rhsB are defective for Hcp1 secretion and no longer inhibit target bacteria. Although ∆rhsA ∆rhsB mutants lose T6SS-1−mediated inhibition activity, they still assemble and fire contractile sheaths at a significantly reduced frequency. We further show that truncated RhsA that retains the PAAR domain still interacts with cognate VgrG2, but the resulting complex does not support Hcp1 secretion or target-cell killing. Full T6SS-1 function requires wild-type Rhs effectors that retain the N-terminal TM helices and PAAR domain together with an intact β-cage. These findings suggest that the Rhs β-cage mediates a quality control checkpoint on T6SS-1 assembly to ensure that VgrG is loaded with a toxic effector prior to export.     

Molecular switch architecture determines response properties of signaling pathways

Many intracellular signaling pathways are composed of molecular switches, proteins that transition between two states—on and off. Typically, signaling is initiated when an external stimulus activates its cognate receptor that, in turn, causes downstream switches to transition from off to on using one of the following mechanisms: activation, in which the transition rate from the off state to the on state increases; derepression, in which the transition rate from the on state to the off state decreases; and concerted, in which activation and derepression operate simultaneously. We use mathematical modeling to compare these signaling mechanisms in terms of their dose–response curves, response times, and abilities to process upstream fluctuations. Our analysis elucidates several operating principles for molecular switches. First, activation increases the sensitivity of the pathway, whereas derepression decreases sensitivity. Second, activation generates response times that decrease with signal strength, whereas derepression causes response times to increase with signal strength. These opposing features allow the concerted mechanism to not only show dose–response alignment, but also to decouple the response time from stimulus strength. However, these potentially beneficial properties come at the expense of increased susceptibility to upstream fluctuations. We demonstrate that these operating principles also hold when the models are extended to include additional features, such as receptor removal, kinetic proofreading, and cascades of switches. In total, we show how the architecture of molecular switches govern their response properties. We also discuss the biological implications of our findings.

Several molecules involved in intracellular signaling pathways act as molecular switches. These are proteins that can be temporarily modified to transition between two conformations, one corresponding to an on (active) state and another to an off (inactive) state. Two prominent examples of such switches are proteins that are modified by phosphorylation and dephosphorylation and GTPases that bind nucleotides. For phosphorylation–dephosphorylation cycles, it is common for the covalent addition of a phosphate by a kinase to cause activation of the modified protein. A phosphatase removes the phosphate to turn the protein off. In the GTPase cycle, the protein is on when bound to guanosine triphosphate (GTP) and off when bound to guanosine diphosphate (GDP). The transition from the GDP-bound state to the GTP-bound state requires nucleotide exchange, whereas the transition from the GTP-bound to the GDP-bound state is achieved via hydrolysis of the γ phosphate on GTP. The basal rates of nucleotide exchange and hydrolysis are often small. These reaction rates are increased severalfold by Guanine Exchange Factors (GEFs) and GTPase Accelerating Proteins (GAPs), respectively (1, 2).A signaling pathway is often initiated upon recognition of a stimulus by its cognate receptor, which then activates a downstream switch. In principle, a switch may be turned on by three mechanisms: (a) activation, by increasing the transition rate from the off state to the on state; (b) derepression, by decreasing the transition rate from the on state to the off state; and (c) concerted activation and derepression. Examples of these three mechanisms are found in the GTPase cycles in different organisms. In animals, signaling through many pathways is initiated by G-protein-coupled receptors (GPCRs) that respond to a diverse set of external stimuli. These receptors act as GEFs to activate heterotrimeric G proteins (3–6). Thus, pathway activation relies upon increasing the transition rate from the off state to the on state. There are no GPCRs in plants and other bikonts; the nucleotide exchange occurs spontaneously, without requiring GEF activity (7–9). G proteins are kept in the off state by a repressor such as a GAP or some other protein that holds the self-activating G protein in its inactive state. In this scenario, the presence of a stimulus results in derepression, i.e., removal of the repressing activity (10–12). Concerted activation and dererpression occur in the GTPase cycle of the yeast mating-response pathway (13, 14), in which the inactive GPCRs recruit a GAP protein and act to repress, whereas active receptors have GEF activity and act to activate. Thus, perception of a stimulus leads to concerted activation and derepression by increasing GEF activity while decreasing GAP activity.These three mechanisms of signaling through molecular switches also occur in many other systems. For example, the activation mechanism described here is a simpler abstraction of a linear signaling cascade, a classical framework used to study general properties of signaling pathways (15–19), as well as to model specific signaling pathways (20–22). While derepression may seem like an unusual mechanism, it occurs in numerous important signaling pathways in plants (e.g., auxin, ethylene, gibberellin, and phytochrome), as well as gene regulation (23–27). In many of these cases, derepression occurs through a decrease in the degradation rate of a component instead of its deactivation rate. Concerted mechanisms are found in bacterial two-component systems, wherein the same component acts as kinase and phosphatase (28–35).Many previous studies have focused on the properties of a single switch mechanism without drawing comparisons between the three potential ways for initiating signaling. For example, the classical Goldbeter–Koshland model studied zero-order ultrasensitivity of an activation mechanism (15). Further analyses examined the effect of receptor numbers (36–38), feedback mechanisms (39, 40), and removal of active receptors via endocytosis and degradation (41, 42). Similarly, important properties of the concerted mechanism have been elucidated, such as its ability to perform ratiometric signaling (13, 14), to align dose responses at different stages of the signaling pathway (43), as well as its robustness (29, 44). The derepression mechanism is relatively less studied. Although there are models of G-signaling in Arabidopsis thaliana (45–47), these models have a large number of states and parameters and do not specifically examine distinct behaviors conferred by derepression.What are the evolutionary constraints that may favor activation over derepression and vice versa? Seminal studies have investigated this question for gene-regulatory networks (48–50). However, an analysis of differences in the functional characteristics of activation, derepression, and concerted mechanisms in the context of cell signaling is still lacking. To address this deficiency, we perform a systematic comparison of the three mechanisms using the following metrics: 1) dose–response, 2) response time, and 3) ability to suppress or filter stochastic fluctuations in upstream components. The rationale behind comparing dose–response curves is that they provide information about the input sensitivity range and the output dynamic range, both of which are of pharmacological importance. We supplement this comparison with response times, which provide information about the dynamics of the signaling activity. The third metric of comparison is motivated from the fact that signaling pathways are subject to intrinsic fluctuations that occur due to the stochastic nature of biochemical reactions (51–56).We construct and analyze both deterministic ordinary differential equation (ODE) models and stochastic models based on continuous-time Markov chains. We show that activation has the following two effects: It makes the switch response more sensitive than that of the receptor, and it speeds up the response with the stimulus strength. In contrast, derepression makes the switch response less sensitive than the receptor occupancy and slows down the response speed as stimulus strength increases. These counteracting behaviors of activation and derepression lead to intermediate sensitivity and intermediate response time for the concerted mechanism. In the special case of a perfect concerted mechanism (equal activation and repression), the dose–response curve of the pathway aligns with the receptor occupancy, and the response time does not depend upon the stimulus level. The noise comparison reveals that the concerted mechanism is more susceptible to fluctuations than the activation and derepression mechanisms, which perform similarly. We further show that these results qualitatively hold for more complex models, such as those incorporating receptor removal and proofreading. We finally discuss our findings to suggest reasons that might have led biological systems to evolve one of these mechanisms over the others, a question that has received considerable attention in the context of gene regulation (48–50).     

From the Cover: Mechanistic transmission modeling of COVID-19 on the Diamond Princess cruise ship demonstrates the importance of aerosol transmission

Several lines of existing evidence support the possibility of airborne transmission of coronavirus disease 2019 (COVID-19). However, quantitative information on the relative importance of transmission pathways of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains limited. To evaluate the relative importance of multiple transmission routes for SARS-CoV-2, we developed a modeling framework and leveraged detailed information available from the Diamond Princess cruise ship outbreak that occurred in early 2020. We modeled 21,600 scenarios to generate a matrix of solutions across a full range of assumptions for eight unknown or uncertain epidemic and mechanistic transmission factors. A total of 132 model iterations met acceptability criteria (R² > 0.95 for modeled vs. reported cumulative daily cases and R² > 0 for daily cases). Analyzing only these successful model iterations quantifies the likely contributions of each defined mode of transmission. Mean estimates of the contributions of short-range, long-range, and fomite transmission modes to infected cases across the entire simulation period were 35%, 35%, and 30%, respectively. Mean estimates of the contributions of larger respiratory droplets and smaller respiratory aerosols were 41% and 59%, respectively. Our results demonstrate that aerosol inhalation was likely the dominant contributor to COVID-19 transmission among the passengers, even considering a conservative assumption of high ventilation rates and no air recirculation conditions for the cruise ship. Moreover, close-range and long-range transmission likely contributed similarly to disease progression aboard the ship, with fomite transmission playing a smaller role. The passenger quarantine also affected the importance of each mode, demonstrating the impacts of the interventions.

Understanding the importance of each transmission pathway for COVID-19 is critical to informing public health guidelines for effectively managing the spread of the disease. Although information and guidance on the likely routes of transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to evolve, quantitative information on the relative importance of specific transmission pathways remains limited (1). The current position of the World Health Organization (WHO) is that the COVID-19 virus is transmitted primarily through respiratory droplets (assumed >5 to 10 µm in diameter) and direct and indirect contact routes, while airborne transmission of the COVID-19 virus via smaller aerosols (assumed <5 µm) is likely not a major route of transmission other than in settings in which aerosol-generating procedures are occurring (2). Similarly, the US Centers for Disease Control and Prevention (CDC) has updated their position multiple times and currently maintains that “COVID-19 is thought to spread mainly through close contact from person-to-person” (which CDC defines as within about 1.8 m) and that fomite transmission and inhalation of respiratory droplets are likely not the main ways that the virus spreads (3). CDC has also acknowledged that airborne transmission by smaller droplets traveling more than 1.8 m away from infected individual(s) can sometimes occur (4).Since the beginning of the pandemic, numerous researchers (5–15) and professional societies [e.g., American Society of Heating, Refrigerating and Air-Conditioning Engineers (16)] have raised concerns that transmission of SARS-CoV-2 can occur from both symptomatic and asymptomatic (or presymptomatic) individuals to others beyond close-range contact through a combination of larger respiratory droplets that are carried further than 1 to 2 m via airflow patterns and smaller inhalable aerosols that can remain suspended and easily transport over longer distances. These concerns arise from a growing understanding of human respiratory emissions (17, 18), known transmission pathways of other respiratory viruses (19), recent empirical evidence detecting SARS-CoV-2 in aerosol and surface samples in health care settings (20–25), and recent case studies demonstrating the likely importance of longer-range aerosol transmission in some settings (26–28).In the absence of empirical studies using controlled exposures to elucidate transmission pathways (29), mathematical modeling approaches can offer insights into the likely importance of the different modes of disease transmission among human populations (30–34), provided that sufficiently accurate inputs are available. To help fill these knowledge gaps, this work uses a mechanistic modeling approach to investigate the relative importance of multiple transmission routes of SARS-CoV-2 among individuals aboard the Diamond Princess cruise ship, which experienced a major outbreak of COVID-19 in early 2020.     

Orthosteric–allosteric dual inhibitors of PfHT1 as selective antimalarial agents

Artemisinin-resistant malaria parasites have emerged and have been spreading, posing a significant public health challenge. Antimalarial drugs with novel mechanisms of action are therefore urgently needed. In this report, we exploit a “selective starvation” strategy by inhibiting Plasmodium falciparum hexose transporter 1 (PfHT1), the sole hexose transporter in P. falciparum, over human glucose transporter 1 (hGLUT1), providing an alternative approach to fight against multidrug-resistant malaria parasites. The crystal structure of hGLUT3, which shares 80% sequence similarity with hGLUT1, was resolved in complex with C3361, a moderate PfHT1-specific inhibitor, at 2.3-Å resolution. Structural comparison between the present hGLUT3-C3361 and our previously reported PfHT1-C3361 confirmed the unique inhibitor binding-induced pocket in PfHT1. We then designed small molecules to simultaneously block the orthosteric and allosteric pockets of PfHT1. Through extensive structure–activity relationship studies, the TH-PF series was identified to selectively inhibit PfHT1 over hGLUT1 and potent against multiple strains of the blood-stage P. falciparum. Our findings shed light on the next-generation chemotherapeutics with a paradigm-shifting structure-based design strategy to simultaneously target the orthosteric and allosteric sites of a transporter.

Plasmodium falciparum is the deadliest species of Plasmodium, responsible for around 50% of human malaria cases and nearly all malarial death (1). Despite intensive malaria-eradication efforts to control the spread of this disease, malaria prevalence remains alarmingly high, with 228 million cases and a fatality tally of 405,000 in 2018 alone (2). The situation has become even more daunting as resistance to the first-line antimalarial agents has emerged and is rapidly spreading. For instance, artemisinin resistance, primarily mediated by P. falciparum Kelch13 (PF3D7_1343700) propeller domain mutations (3, 4), severely compromises the campaign of antimalarial chemotherapy (5–9). Novel antimalarial agents overcoming the drug resistance are therefore urgently needed (10).The blood-stage malaria parasites depend on a constant glucose supply as their primary source of energy (11). P. falciparum hexose transporter 1 (PfHT1; PF3D7_0204700) (12) is transcribed from a single-copy gene with no close paralogue (13) and has been genetically validated as essential for the survival of the blood-stage parasite (14). A possible approach to kill the parasite is to “starve it out” by the chemical intervention of the parasite hexose transporter (13, 15). The feasibility of this approach would depend on the successful development of selective PfHT1 inhibitors that do not affect the activities of human hexose transporter orthologs (e.g., human glucose transporter 1 [hGLUT1]).Previously, Compound 3361 (C3361) (15), a glucose analog, has been reported to moderately inhibit PfHT1 and suppress the growth of blood-stage parasites in vitro (16). Nonetheless, the modest potency and selectivity of C3361 had limited its further development. Structural determination of PfHT1 and human glucose transporters provides an unprecedented opportunity for rational design of PfHT1-specific inhibitors (17–20). While hGLUT1 is the primary glucose transporter in erythrocyte, its structure was determined only in the inward-open state (17). Fortunately, the neuronal glucose transporter hGLUT3, which shares over 80% sequence similarity with hGLUT1, was captured in both outward-open and outward-occluded conformations (18). A reliable homology model of outward-facing hGLUT1 could thus be generated based on the structure of hGLUT3.Comparing the structures of PfHT1 (19, 20) and hGLUT1, we identified an additional pocket adjacent to the substrate-binding site. Coadministration of allosteric and orthosteric drugs is generally applied to tackle drug resistance when these two pockets were spatially separated (21). However, this discovery led to a hypothesis that simultaneously targeting the orthosteric and allosteric sites by tethering a pharmacophore to the carbohydrate core might render selective inhibitors for PfHT1. Based on this hypothesis, we designed a class of small molecules containing a sugar moiety and an allosteric pocket-occupying motif connected by a flexible linker. Among them, TH-PF01, TH-PF02, and TH-PF03 have exhibited selective biophysical and antiplasmodial activities with moderate cytotoxicity. Furthermore, in silico computational simulations also confirmed their binding mode, lending further support to the dual-inhibitor design. Taken together, our studies validated an antimalaria development strategy that simultaneously targets the orthosteric and allosteric sites of PfHT1.     

Mesoscale networks and corresponding transitions from self-assembly of block copolymers

A series of cubic network phases was obtained from the self-assembly of a single-composition lamellae (L)-forming block copolymer (BCP) polystyrene-block-polydimethylsiloxane (PS-b-PDMS) through solution casting using a PS-selective solvent. An unusual network phase in diblock copolymers, double-primitive phase (DP) with space group of Im3¯m, can be observed. With the reduction of solvent evaporation rate for solution casting, a double-diamond phase (DD) with space group of Pn3¯m can be formed. By taking advantage of thermal annealing, order–order transitions from the DP and DD phases to a double-gyroid phase (DG) with space group of Ia3¯d can be identified. The order–order transitions from DP (hexapod network) to DD (tetrapod network), and finally to DG (trigonal planar network) are attributed to the reduction of the degree of packing frustration within the junction (node), different from the predicted Bonnet transformation from DD to DG, and finally to DP based on enthalpic consideration only. This discovery suggests a new methodology to acquire various network phases from a simple diblock system by kinetically controlling self-assembling process.

From constituted molecules to polymers, finally ordered hierarchical superstructures, self-assembled solids cover a vast area of nanostructures where the characters of building blocks direct the progress of self-assembly (1, 2). In nature, fascinating periodic network structures and morphologies from different species are appealing in nanoscience and nanotechnology due to their superior properties, especially for photonic crystal structures (3–7). For gyroid, trigonal planar network with chirality demonstrates its potential as chiropitc metamaterial (8–10). Beyond the splendid colors, networks either in macroscale or mesoscale mechanically strengthen their skeletons and protect those fragile but vital organs from impact (11, 12). Inspired by nature, biomimicking materials with mesoscale network may exceed the limitation of the intrinsic properties (13). The topology of networks could further improve their adaptability, allowing extreme deformation for energy dissipation (14). Moreover, network materials from hybridization of self-assembled block copolymers (BCPs) have been exploited to the design of mesoscale quantum metamaterials (15, 16). With the desire to acquire network textures for biomimicking nanomaterials, BCPs with immiscible constituted segments covalently joined together give the accessibility to the formation of nanonetwork morphologies via balancing enthalpic penalty from the repulsive interaction of constituted blocks and entropic penalty from the stretching of polymer chains (17–21). By taking advantage of precise synthesis procedures, it is feasible to obtain the aimed network phases from the self-assembly of BCPs such as Fddd (O⁷⁰) (22–24), gyroid (Q²¹⁴, Q²³⁰) (20, 21, 25–27), and diamond (Q²²⁴, Q²²⁷) (28–31) experimentally and theoretically. On the basis of theoretical prediction, the junction points (nodes) in the network phases could be coordinated with three, four, or six neighbors in three-dimensional space, resulting in the enhancement of packing frustration (31). Topologically, all these phases match the coordination number to neighbors (n = 3, 4, 6), showing no special case of quasicrystal. Accordingly, an order–order transition from double-diamond phase (DD, tetrapod) to double-gyroid phase (DG, trigonal planar network) has been observed (29). Yet, there is no DP phase being found in simple diblock systems except for liquid crystals (32, 33) or organic–inorganic nanocomposites from the mixtures of BCP with inorganic precursors (34, 35). Searching the rare occurrence of network phases and the corresponding phase transitions among phases will be essential to the demands for application by considering the deliberate structuring effects on aimed properties but the approaches remain challenging (8, 36–40). For instance, viewing the narrow window for network morphologies in diblock copolymer phase diagram, it demands harsh requirements for syntheses (2, 41). Recently, by taking advantage of using selective solvent for solution casting, it is feasible to acquire DG phase and even inverted DG phase from the self-assembly of lamellae (L)-forming polystyrene-block-polydimethylsiloxane (PS-b-PDMS) (42). Apart from that, a triclinic DG phase was recently discovered from the PS-b-PDMS which is commonly believed nonexisting in the conventional phase diagram (43). As a result, the phase diagram of BCPs with high interaction parameter is worthy of study for searching the metastable phases with unique network textures (44). Herein, we aim to acquire network phases from a simple diblock system by kinetically controlling the transformation mechanisms of self-assembly. As exemplified by using the PS-b-PDMS for solution casting, with the use of a PS-selective solvent (chloroform), a DP phase and a DD phase could be formed through controlled self-assembly, giving unique network phases simply from solution casting. Moreover, a DG phase can be also acquired from phase transformation. Consequently, a series of network phases with hexapod, tetrapod, and trigonal planar building units could be successfully obtained by using a single-composition L-forming PS-b-PDMS for self-assembly. The corresponding order–order transitions among these network phases examined by temperature-resolved in situ small-angle X-ray scattering (SAXS) combining with electron tomography results provide insights of network phase formation and the corresponding phase transformation mechanisms in the self-assembly of BCPs.     

The retromer is co-opted to deliver lipid enzymes for the biogenesis of lipid-enriched tombusviral replication organelles

Biogenesis of viral replication organelles (VROs) is critical for replication of positive-strand RNA viruses. In this work, we demonstrate that tomato bushy stunt virus (TBSV) and the closely related carnation Italian ringspot virus (CIRV) hijack the retromer to facilitate building VROs in the surrogate host yeast and in plants. Depletion of retromer proteins, which are needed for biogenesis of endosomal tubular transport carriers, strongly inhibits the peroxisome-associated TBSV and the mitochondria-associated CIRV replication in yeast and in planta. In vitro reconstitution revealed the need for the retromer for the full activity of the viral replicase. The viral p33 replication protein interacts with the retromer complex, including Vps26, Vps29, and Vps35. We demonstrate that TBSV p33-driven retargeting of the retromer into VROs results in delivery of critical retromer cargoes, such as 1) Psd2 phosphatidylserine decarboxylase, 2) Vps34 phosphatidylinositol 3-kinase (PI3K), and 3) phosphatidylinositol 4-kinase (PI4Kα-like). The recruitment of these cellular enzymes by the co-opted retromer is critical for de novo production and enrichment of phosphatidylethanolamine phospholipid, phosphatidylinositol-3-phosphate [PI(3)P], and phosphatidylinositol-4-phosphate [PI(4)P] phosphoinositides within the VROs. Co-opting cellular enzymes required for lipid biosynthesis and lipid modifications suggest that tombusviruses could create an optimized lipid/membrane microenvironment for efficient VRO assembly and protection of the viral RNAs during virus replication. We propose that compartmentalization of these lipid enzymes within VROs helps tombusviruses replicate in an efficient milieu. In summary, tombusviruses target a major crossroad in the secretory and recycling pathways via coopting the retromer complex and the tubular endosomal network to build VROs in infected cells.

Viruses are intracellular parasites which co-opt cellular resources to produce abundant viral progeny. Positive-strand (+)RNA viruses replicate on subcellular membranes by forming viral replication organelles (VROs) (1–5). VROs sequester the viral proteins and viral RNAs together with co-opted host factors to provide an optimal subcellular environment for the assembly of numerous viral replicase complexes (VRCs), which are then responsible for robust viral RNA replication. VROs also spatially and temporally organize viral replication. Importantly, the VROs hide the viral RNAs from cellular defense mechanisms as well (5, 6). VROs consist of extensively remodeled membranes with unique lipid composition. How viruses achieve these membrane remodeling and lipid modifications and lipid enrichment is incompletely understood. Therefore, currently, there is a major ongoing effort to dissect the VRC assembly process and to understand the roles of viral and host factors in driving the biogenesis of VROs (1, 3, 7).Tomato bushy stunt virus (TBSV), a plant-infecting tombusvirus, has been shown to induce complex rearrangements of cellular membranes and alterations in lipid and other metabolic processes during infections (8–10). The VROs formed during TBSV infections include extensive membrane contact sites (vMCSs) and harbor numerous spherules (containing VRCs), which are vesicle-like invaginations in the peroxisomal membranes (8, 11–13). A major gap in our understanding of the biogenesis of VROs, including vMCSs and VRCs, is how the cellular lipid-modifying enzymes are recruited to the sites of viral replication.Tombusviruses belong to the large Flavivirus-like supergroup that includes important human, animal, and plant pathogens. Tombusviruses have a small single-component (+)RNA genome of ∼4.8 kb that codes for five proteins. Among those, there are two essential replication proteins, namely p33 and p92^pol, the latter of which is the RdRp protein and it is translated from the genomic RNA via readthrough of the translational stop codon in p33 open reading frame (14). The smaller p33 replication protein is an RNA chaperone, which mediates the selection of the viral (+)RNA for replication (14–16). Altogether, p33 is the master regulator of VRO biogenesis (3). We utilize a TBSV replicon (rep)RNA, which contains four noncontiguous segments from the genomic RNA, and it can efficiently replicate in yeast and plant cells expressing p33 and p92^pol (14, 17).Tombusviruses hijack various cellular compartments and pathways for VRO biogenesis (18). These include peroxisomes by TBSV or mitochondria (in the case of the closely related carnation Italian ringspot virus [CIRV]), the endoplasmic reticulum (ER) network, Rab1-positive COPII vesicles, and the Rab5-positive endosomes (8, 19–23). Tombusviruses also induce membrane proliferation, new lipid synthesis, and enrichment of lipids, most importantly phosphatidylethanolamine (PE), sterols, phosphatidylinositol-4-phosphate [PI(4)P], and phosphatidylinositol-3-phosphate [PI(3)P] phosphoinositides in peroxisomal or mitochondrial membranes for different tombusviruses (13, 24–27). This raised the question that how TBSV could hijack lipid synthesis enzymes from other subcellular locations that leads to enrichment of critical lipids in the large VROs in model yeast and plant hosts.The endosomal network (i.e., early, late, and recycling endosomes) is a collection of pleomorphic organelles which sort membrane-bound proteins and lipids either for vacuolar/lysosomal degradation or recycling to other organelles. With the help of the so-called retromer complex, tubular transport carriers formed from the endosomes recycle cargoes to the Golgi and ER or to the plasma membrane (28–31). The core retromer complex consists of three conserved proteins, Vps26, Vps29, and Vps35, which are involved in cargo sorting and selection. The retromer complex affects several cellular processes, including autophagy through the maturation of lysosomes (32), neurodegenerative diseases (33), plant root hair growth (34), and plant immunity (35).The cellular retromer is important for several pathogen–host interactions. For example, the retromer is targeted by Brucella, Salmonella, and Legionella bacteria (36–39) and the rice blast fungus (40). The retromer is also involved in the intracellular transport of the Shigella and Cholera toxins and the plant ricin toxin. The NS5A replication protein of hepatitis C virus (HCV) interacts with Vps35 and this interaction is important for HCV replication in human cells (41). The cytoplasmic tail of the Env protein of HIV-1 binds to the retromer components Vps35 and Vps26, which is required for Env trafficking and infectious HIV-1 morphogenesis (42). Moreover, the retromer complex affects the morphogenesis of vaccinia virus (43) and HPV16 human papillomavirus entry and delivery to the trans-Golgi network (44). Despite the importance of the retromer in pathogen–host interactions, the mechanistic insights are far from complete.In the case of tombusviruses, enrichment of PE and PI(3)P within VROs is facilitated by co-opting the endosomal Rab5 small GTPase and Vps34 PI3K (20, 24), suggesting that the endosome-mediated trafficking pathway might be involved in viral replication in host cells. However, the actual mechanism of how tombusviruses exploit the endosomal/endocytic pathway and induce lipid enrichment within VROs is not yet dissected. Therefore, in this work, we targeted the retromer complex, based on previous genome-wide screens using yeast gene-deletion libraries, which led to the identification of VPS29 and VPS35 as host genes affecting TBSV replication and recombination, respectively (45, 46). These proteins are components of the retromer complex (28–31). We found TBSV and the closely related CIRV co-opt the retromer complex for the biogenesis of VROs in yeast and plants. We observed that depletion of retromer proteins strongly inhibited TBSV and CIRV replication. The recruitment of the retromer is driven by the viral p33 replication protein, which interacts with Vps26, Vps29, and Vps35 retromer proteins. We show that the retromer helps delivering critical cargo proteins, such as Psd2 phosphatidylserine decarboxylase, Vps34 phosphatidylinositol 3-kinase (PI3K), and Stt4 phosphatidylinositol 4-kinase (PI4Kα-like). These co-opted cellular enzymes are then involved in de novo production and enrichment of PE phospholipid, PI(3)P, and PI(4)P phosphoinositides within the VROs. Altogether, these virus-driven activities create an optimized membrane microenvironment within VROs to support efficient tombusvirus replication.     

Microbiome reduction and endosymbiont gain from a switch in sea urchin life history

Animal gastrointestinal tracts harbor a microbiome that is integral to host function, yet species from diverse phyla have evolved a reduced digestive system or lost it completely. Whether such changes are associated with alterations in the diversity and/or abundance of the microbiome remains an untested hypothesis in evolutionary symbiosis. Here, using the life history transition from planktotrophy (feeding) to lecithotrophy (nonfeeding) in the sea urchin Heliocidaris, we demonstrate that the lack of a functional gut corresponds with a reduction in microbial community diversity and abundance as well as the association with a diet-specific microbiome. We also determine that the lecithotroph vertically transmits a Rickettsiales that may complement host nutrition through amino acid biosynthesis and influence host reproduction. Our results indicate that the evolutionary loss of a functional gut correlates with a reduction in the microbiome and the association with an endosymbiont. Symbiotic transitions can therefore accompany life history transitions in the evolution of developmental strategies.

Animal gastrointestinal tracts contain microbial communities that are integral to host metabolism, immunity, and development (1, 2). Symbioses between animals and their gut microbiome have deep evolutionary origins (1, 2), often exhibit phylosymbiosis (3), and can serve as a physiological buffer to heterogeneous environments (2). Despite the necessity of the gastrointestinal tract and benefits of the gut microbiome (3), species in various phyla have lost a functional digestive system (4, 5). Loss of a functional gut should, in theory, cascade into a reduction in microbial diversity and the loss of diet-induced shifts in microbiome composition. These nutritional shifts may then provide a niche for functionally important endosymbionts, such as the chemoautotrophic bacteria commonly associated with gutless invertebrates (6, 7).Major life history transitions are driven by tradeoffs in reproduction and development that, in turn, impact fitness (8). These tradeoffs are particularly evident in benthic marine invertebrates whose developmental stages broadly group into two alternative nutritional strategies (4, 9). The first—planktotrophy—typically includes the production of a high number of small, energy-poor eggs that develop into larvae with feeding structures used to collect and process exogenous resources required to reach metamorphic competency (4, 9). The second—lecithotrophy—involves the production of fewer large, energy-rich eggs and nonfeeding larvae that undergo metamorphosis without the requirement of external nutrients through feeding (4, 9). Life history transitions between these developmental modes have occurred in several major animal lineages, with rapid evolutionary shifts from planktotrophy to lecithotrophy being well documented in echinoderms (4, 5, 10–13). It is thought that an increase in the eggs energetic content relaxes the selective pressure maintaining the feeding structures (e.g., the larval arms and a functional gastrointestinal tract) and that development to metamorphosis is accelerated once these are lost (5).One of the most comprehensively studied systems for life history transitions among marine invertebrates involves species in the sea urchin genus Heliocidaris. A speciation event ∼5 Mya resulted in two sister species with alternative life history strategies: Heliocidaris tuberculata is planktotrophic while Heliocidaris erythrogramma is lecithotrophic (14). Typical of planktotrophs, H. tuberculata develops from small eggs into feeding larvae that exhibit morphological plasticity in response to food limitation (15), which is correlated with compositional shifts in the microbiome (16, 17). H. erythrogramma, on the other hand, develops from eggs ∼53× to 86× the volume of H. tuberculata (18), lacks the morphological structures required for feeding, and has a reduced, nonfunctional digestive tract (11). This life history switch and heterochronic shift in development (11) corresponds with a rewiring of the gene regulatory network (19), reorganization of cell fates (20), and modification to gametogenesis (21).Here, we compare the bacterial communities of these Heliocidaris species and test two hypotheses. First, we test whether the loss of gut function coincides with a reduction in microbial symbiont diversity, and second, by simulating the natural range in food availability, we also test that the loss in gut function coincides with a loss in diet-related shifts in the microbiome. We report major reductions in microbiome diversity and abundance as well as the absence of bacterial communities correlated with food availability for the lecithotrophic H. erythrogramma. Moreover, we find that this species vertically transmits a Rickettsiales that encodes pathways for the biosynthesis of essential amino acids, proteins with pivotal roles in host reproduction, and enzymes to metabolize diacylglycerol ethers, the major lipid group responsible for the increase in egg size in H. erythrogramma and that is used to fuel growth and development (18, 22).