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The cryo-EM structure and physical basis for anesthetic inhibition of the THIK1 K2P channel
THIK1 tandem pore domain (K2P) potassium channels regulate microglial surveillance of the central nervous system and responsiveness to inflammatory insults. With microglia recognized as critical to the pathogenesis of neurodegenerative diseases, THIK1 channels are putative therapeutic targets to control microglia dysfunction. While THIK channels can principally be distinguished from other K2Ps by their distinctive inhibitory response to volatile anesthetics (VAs), molecular details governing THIK channel gating remain largely unexplored. Here, we report a 3.2 Å cryo-electron microscopy structure of the THIK1 channel in a closed conformation. A central pore gate located directly below the THIK1 selectivity filter is formed by inward-facing TM4 helix tyrosine residues that occlude the ion conduction pathway. VA inhibition of THIK requires closure of this central pore gate. Using a combination of anesthetic photolabeling, electrophysiology, and molecular dynamics simulation, we identify a functionally critical THIK1 VA binding site positioned between the central gate and a structured section of the THIK1 TM2/TM3 loop. Our results demonstrate the molecular architecture of the THIK1 channel and elucidate critical structural features involved in regulation of THIK1 channel gating and anesthetic inhibition.
An unusual potassium conductance protects <i>Caenorhabditis elegans</i> pharyngeal muscle rhythms against environmental noise
The nematode Caenorhabditis elegans feeds by rhythmic contraction and relaxation of a neuromuscular organ called the pharynx, which draws in and filters water and bacterial food. This behavior is driven by myogenic plateau potentials, long-lasting depolarizations of the pharyngeal muscle, which are timed by neuronal input from a dedicated pharyngeal nervous system. While the timing of these plateaus’ initiation has received significant attention, their mechanisms of termination remain incompletely understood. In particular, it is unclear how plateaus resist early termination by hyperpolarizing current noise. Here, we present a computational model of pharyngeal plateaus against a noisy background. We propose that an unusual, rapidly inactivating potassium conductance confers exceptional noise robustness on the system. We further investigate the possibility that a similar mechanism in other systems permits switching between plateau and spiking behavior under noisy conditions.
Dual genetic tracing demonstrates the heterogeneous differentiation and function of neuromesodermal progenitors in vivo
In recent decades, the traditional paradigm of three distinct germ layers formed during gastrulation has been revised with the identification of neuromesodermal progenitors (NMPs). These progenitors emerge during gastrulation and contribute to both the neural ectoderm, particularly the spinal cord, and the adjacent paraxial mesoderm [D. Henrique et al. , Development 142 , 2864–2875 (2015); R. J. Garriock et al. , Development 142 , 1628–1638 (2015); E. Tzouanacou et al. , Dev. Cell 17 , 365–376 (2009)]. However, effective genetic tools for lineage tracing and functional assessments of NMPs in vivo are currently lacking. Here, we developed a dual recombinase–mediated genetic system to specifically trace and ablate Brachyury + Sox2 + NMPs. Our genetic tracing and single-cell RNA sequencing analyses revealed that NMPs consist of three distinct unipotent and bipotent progenitor populations that progressively differentiate into neural and mesodermal fates. Genetic depletion of NMPs demonstrated their critical role in trunk and tail formation. This study provides in vivo genetic evidence supporting the heterogeneity of NMPs in terms of cell fate determination and their functional roles in the developing embryo.
The Hippo pathway and p27 <sup>Kip1</sup> cooperate to suppress mitotic regeneration in the organ of Corti and the retina
The mature mammalian auditory sensory organ, the organ of Corti (OC), lacks the capacity for regenerating hair cells, leading to permanent hearing impairment. In contrast, the vestibular system has a limited capacity for hair cell regeneration, which we have shown to be further enhanced by inhibiting the Hippo pathway. Here, we demonstrate that, despite similar transcriptional responses, only vestibular and not auditory supporting cells proliferate as a result of Yap activation following Hippo inhibition. Mechanistically, we identify p27 Kip1 , a cell cycle kinase inhibitor encoded by Cdkn1b , as an additional barrier preventing cell cycle reentry specifically in the OC. We show that while in both systems Yap stimulates p27 Kip1 degradation through activation of its direct target gene Skp2 , this protein-level control is antagonized by an unusually high level of Cdkn1b transcription in the cochlea. Consequently, p27 Kip1 activity is maintained in the OC even in the presence of constitutively active Yap5SA, counteracting its mitogenic effects. Supporting this model, inactivation of the Hippo pathway in the Cdkn1b -deficient background is sufficient to induce adult auditory supporting cell proliferation in vivo. Furthermore, we show that the synergistic interaction between Hippo and p27 Kip1 is conserved in the retina where inhibition of both pathways potently induces Müller glia proliferation and initiates neuronal regeneration. Our work uncovers the molecular mechanism preventing quiescent adult sensory progenitor cells, supporting cells in the ear and Müller glia in the eye, from reentering the cell cycle after damage—the key step toward sensory receptor regeneration blocked in mammals.
Searching permutations for constructing uniformly distributed point sets
Uniformly distributed point sets of low discrepancy are heavily used in experimental design and across a very wide range of applications such as numerical integration, computer graphics, and finance. Recent methods based on Graph Neural Networks [T. K. Rusch, N. Kirk, M. M. Bronstein, C. Lemieux, D. Rus, Proc. Natl. Acad. Sci. U.S.A. 121, e2409913121 (2024).] and solver-based optimization identified point sets having much lower discrepancy than previously known constructions. We show in this note that further substantial improvements are possible by separating the construction of low-discrepancy point sets into i) the relative position of the points, and ii) the optimal placement respecting these relationships. Using tailored permutations, we construct point sets that are of 20% smaller discrepancy on average than those proposed by Rusch et al. In terms of inverse discrepancy, our sets reduce the number of points in dimension 2 needed to obtain a discrepancy of 0.005 from more than 500 points to less than 350. For applications where the sets are used to query time-consuming models, this is a significant reduction.
AcrIE7 inhibits the CRISPR-Cas system by directly binding to the R-loop single-stranded DNA
The CRISPR-Cas system is a well-known adaptive immune system in bacteria, and a prominent mechanism for evading this immunity involves anti-CRISPR (Acr) proteins, which employ various methods to neutralize the CRISPR-Cas system. In this study, using structural and biochemical analyses, we revealed that AcrIE7 binds to the single-stranded DNA in the R-loop formed when Cascade encounters the target DNA, thereby preventing Cas3 from cleaving the DNA. This represents a different inhibition strategy distinct from previously reported Acr mechanisms and offers insights into CRISPR-Cas inhibition.
microRNA-218-5p coordinates scaling of excitatory and inhibitory synapses during homeostatic synaptic plasticity
Homeostatic synaptic plasticity (HSP) is a neuronal mechanism that allows networks to compensate for prolonged changes in activity by adjusting synaptic strength. This process is crucial for maintaining stable brain function and has been implicated in memory consolidation during sleep. While scaling of both excitatory and inhibitory synapses plays an important role during homeostatic synaptic plasticity, molecules coordinating these processes are unknown. In this study, we investigate the role of miR-218-5p as a regulator of inhibitory and excitatory synapses in the context of picrotoxin (PTX)-induced homeostatic synaptic downscaling (HSD) in rat hippocampal neurons. Using enrichment analysis of microRNA-binding sites in genes changing upon PTX-induced HSD, we bioinformatically predict and experimentally validate increased miR-218-5p activity upon PTX treatment. By electrophysiological recordings and confocal microscopy, we demonstrate that inhibiting miR-218-5p activity exerts a dual effect during HSD: It occludes the downscaling of excitatory synapses and dendritic spines, while at the same time attenuating inhibitory synapse upscaling. Furthermore, we identify the Neuroligin2 interacting molecule Mdga1 as a direct miR-218-5p target which potentially mediates the effect of miR-218-5p on homeostatic upscaling of inhibitory synapses. By performing long-term electroencephalographic recordings, we further reveal that local inhibition of miR-218-5p in the somatosensory cortex reduces local slow-wave activity during non-rapid-eye-movement sleep. In summary, this study uncovers miR-218-5p as a key player in coordinating inhibitory and excitatory synapses during homeostatic plasticity and sleep. Our findings contribute to a deeper understanding of how neural circuits maintain stability in the face of activity-induced perturbations, with implications for pathophysiology.
QnAs with Inara S. Tareque, Jorge Guzman, and Dan J. Wang
Allosterically switchable network orients <i>β</i> -flap in <i>Clostridioides difficile</i> toxins
Allosteric proteins exhibit a functional response upon ligand binding far from the active site. Clostridioides difficile toxins use allosteric binding by the endogenous cofactor myo -inositol hexakisphosphate to orchestrate self-cleavage from within the target cell. This binding event induces a conformational shift, primarily effecting a lever-like β -flap region, with two known orientations. We uncovered a mechanism for this allosteric transition using extensive atomistic molecular dynamics simulations and computational and experimental mutagenesis. The mechanism relies on a switchable interaction network. The most prominent interaction pair is K600-E743, with K600 interactions explaining ∼70% of the allosteric effect. Rather than gradually morphing between two end states, the interaction network adopts two mutually exclusive configurations in the active and inactive state. Similar switchable networks may explain allostery more broadly. This mechanism in particular could aid in drug development targeting the C. difficile toxins autoproteolysis.
Structural switching dynamically controls the doubly pseudoknotted Rous sarcoma virus–programmed ribosomal frameshifting element
A hallmark of retrovirus replication is the translation of two different polyproteins from one RNA through programmed –1 frameshifting. This is a mechanism in which the actively translating ribosome is induced to slip in the 5′ direction at a defined codon and then continues translating in the new reading frame. Programmed frameshifting controls the stoichiometry of viral proteins and is therefore under stringent evolutionary selection. Forty years ago, the first frameshifting stimulatory element was discovered in the Rous sarcoma virus. The ~120 nt RNA segment was predicted to contain a pseudoknot, but its 3D structure has remained elusive. Now, we have determined cryoEM and X-ray crystallographic structures of this classic retroviral element, finding that it adopts a butterfly-like double-pseudoknot fold. One “wing” contains a dynamic pyrimidine-rich helix, observed crystallographically in two conformations and in a third conformation via cryoEM. The other wing encompasses the predicted pseudoknot, which interacts with a second unexpected pseudoknot through a toggle residue, A2546. This key purine switches conformations between structural states and tunes the stability of interacting residues in the two wings. We find that its mutation can modulate frameshifting by as much as 50-fold, likely by altering the relative abundance of different structural states in the conformational ensemble of the RNA. Taken together, our structure–function analyses reveal how a dynamic double pseudoknot junction stimulates frameshifting by taking advantage of conformational heterogeneity, supporting a multistate model in which high Shannon entropy enhances frameshifting efficiency.
Unveiling Cas8 dynamics and regulation within a transposon-encoded Cascade–TniQ complex
The Vibrio cholerae Cascade–TniQ complex unveiled a new paradigm in biology, demonstrating that CRISPR-associated proteins can direct DNA transposition. Despite the tremendous potential of “knocking-in” genes at desired sites, the mechanisms underlying DNA binding and transposition remain elusive. In this system, a conformational change of the Cas8 protein is essential for DNA binding, yet how it occurs is unclear. Here, structural modeling and free energy simulations reconstruct the Cas8 helical bundle and reveal an open–closed conformational change that is key for the complex’s function. We show that when Cascade–TniQ binds RNA, the Cas8 bundle changes conformation mediated by the interaction with the Cas7.1 protein. This interaction promotes the bundle’s transition toward the open state, priming the complex for DNA binding. As the target DNA binds the guide RNA, the opening of the Cas8 bundle becomes more favorable, exposing positively charged residues and facilitating their interaction with DNA, which ultimately leads the DNA-binding process to completion. These outcomes provide a dynamic representation of a critical conformational change in one of the largest CRISPR systems and illustrate its role at critical steps of the Cascade–TniQ biophysical function, advancing our understanding of nucleic acid binding and transposition mechanisms.
Structural basis for neutralizing antibody binding to pertussis toxin
Pertussis toxin (PT) is a key protective antigen in vaccine- and natural immunity-mediated protection from Bordetella pertussis infection. Despite its importance, no PT-neutralizing epitopes have been characterized structurally. To define neutralizing epitopes and identify key structural elements to preserve during PT antigen design, we determined a 3.6 Å cryoelectron microscopy structure of genetically detoxified PT (PTg) bound to hu11E6 and hu1B7, two potently neutralizing anti-PT antibodies with complementary mechanisms: disruption of toxin adhesion to cells and intracellular activities, respectively. Hu11E6 binds the paralogous S2 and S3 subunits of PTg via a conserved epitope but surprisingly did not span the previously identified sialic acid–binding site implicated in toxin adhesion. Hu11E6 specifically prevented PTg binding to sialylated N-glycans and a sialylated model receptor, as demonstrated by high-throughput glycan array analysis and ELISA, while a T cell activation assay showed that it blocks PTg mitogenic activities to define its neutralizing mechanism. Hu1B7 bound a quaternary epitope spanning the S1 and S5 subunits, although functional studies of hu1B7 variants suggested that S5 binding is not involved in its PT neutralization mechanism. These results structurally define neutralizing epitopes on PT, improving our molecular understanding of immune protection from B. pertussis and providing key information for the future development of PT immunogens.
Digital phenotyping using smartphones could help steer mental health treatment
DDX54 downregulation enhances anti-PD1 therapy in immune-desert lung tumors with high tumor mutational burden
High tumor mutational burden (TMB-H) is a predictive biomarker for the responsiveness of cancer to immune checkpoint inhibitor (ICI) therapy that indicates whether immune cells can sufficiently recognize cancer cells as nonself. However, about 30% of all cancers from The Cancer Genome Atlas (TCGA) are classified as immune-desert tumors lacking T cell infiltration despite TMB-H. Since the underlying mechanism of these immune-desert tumors has yet to be unraveled, there is a pressing need to transform such immune-desert tumors into immune-inflamed tumors and thereby enhance their responsiveness to anti-PD1 therapy. Here, we present a systems framework for identifying immuno-oncotargets, based on analysis of gene regulatory networks, and validating the effect of these targets in transforming immune-desert into immune-inflamed tumors. In particular, we identify DEAD-box helicases 54 (DDX54) as a master regulator of immune escape in immune-desert lung cancer with TMB-H and show that knockdown of DDX54 can increase immune cell infiltration and lead to improved sensitivity to anti-PD1 therapy.
Phosphatidylinositol 4,5-bisphosphate activation mechanism of human KCNQ5
The human voltage-gated potassium channels KCNQ2, KCNQ3, and KCNQ5 can form homo- and heterotetrameric channels that are responsible for generating the neuronal M current and maintaining the membrane potential stable. Activation of KCNQ channels requires both the depolarization of membrane potential and phosphatidylinositol 4,5-bisphosphate (PIP 2 ). Here, we report cryoelectron microscopy structures of the human KCNQ5–calmodulin (CaM) complex in the apo, PIP 2 -bound, and both PIP 2 - and the activator HN37-bound states in either a closed or an open conformation. In the closed conformation, a PIP 2 molecule binds in the middle of the groove between two adjacent voltage-sensing domains (VSDs), whereas in the open conformation, one additional PIP 2 binds to the interface of VSD and the pore domain, accompanying structural rearrangement of the cytosolic domain of KCNQ and CaM. The structures, along with electrophysiology analyses, reveal the two different binding modes of PIP 2 and elucidate the PIP 2 activation mechanism of KCNQ5.
Nuclear Galectin-1 promotes <i>KRAS</i> -dependent activation of pancreatic cancer stellate cells
Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, primarily due to its complex tumor microenvironment (TME), which drives both disease progression and therapy resistance. Understanding the molecular mechanisms governing TME dynamics is essential for developing new treatment strategies for this devastating disease. In this study, we uncover an oncogenic role for Galectin-1 (Gal1), a glycan-binding protein abundantly expressed by activated pancreatic stellate cells (PSCs), a key component of the PDAC TME that orchestrates tumor progression. Our findings reveal that Gal1 expression is elevated in the nucleus of human PSCs in both tissue samples and cultured cell lines. Using chromatin immunoprecipitation followed by sequencing analysis (ChIP-seq), we identify Gal1 occupancy at the promoters of several cancer-associated genes, including KRAS , a pivotal oncogene involved in PDAC pathogenesis. We demonstrate that Gal1 binds to the KRAS promoter, sustaining KRAS expression in PSCs, which, in turn, maintains PSC activation and promotes the secretion of protumorigenic cytokines. Mechanistically, Gal1 is required to preserve histone H3 lysine 4 monomethylation levels and to recruit the histone methyltransferase MLL1 to target promoters. Collectively, our findings define a nuclear function of Gal1 in modulating the transcriptional landscape of cancer-associated genes in PSCs within the PDAC TME, mediated through an epigenetic mechanism. These insights enhance our understanding of PDAC pathology and open potential avenues for therapeutic interventions targeting intracellular Gal1.
A conserved ARF–DNA interface underlies auxin-triggered transcriptional response
Auxin Response Factor (ARF) plant transcription factors are the key effectors in auxin signaling. Their DNA-Binding Domain (DBD) contains a B3 domain that allows base-specific interactions with Auxin Response Elements (AuxREs) in DNA target sites. Land plants encode three phylogenetically distinct ARF classes: the closely related A- and B-classes have overlapping DNA binding properties, contrasting with the different DNA-binding properties of the divergent C-class ARFs. ARF DNA-binding divergence likely occurred early in the evolution of the gene family, but the molecular determinants underlying it remain unclear. Here, we show that the B3 DNA-binding residues are deeply conserved in ARFs, and variability within these is only present in tracheophytes, correlating with greatly expanded ARF families. Using the liverwort Marchantia polymorpha , we confirm the essential role of conserved DNA-contacting residues for ARF function. We further show that ARF B3–AuxRE interfaces are not mutation-tolerant, suggesting low evolvability that has led to the conservation of the B3–DNA interface between ARF classes. Our data support the almost complete interchangeability between A/B-class ARF B3 by performing interspecies domain swaps in M. polymorpha , even between ARF lineages that diverged over half a billion years ago. Our analysis further suggests that C-class ARF DNA-binding specificity diverged early during ARF evolution in a common streptophyte ancestor, followed by strong selection in A and B-class ARFs as part of a competition-based auxin response system.
Phenotypic changes of auditory nerve fibers after excitotoxicity
There is a substantial body of evidence elucidating the pathophysiological aspects of excitotoxicity in the mammalian cochlea. However, the question of whether the resultant damage is reversible remains unresolved. To replicate an excitotoxic event, we investigated the long-term effects of kainate application in gerbil cochleae. Surprisingly, despite persistent synapse loss, the compound action potential of the auditory nerve fully recovered. This functional retrieval was associated with a phenotypic change in auditory nerve fibers. Thresholds were improved along the tonotopic axis. High-spontaneous rate (SR) fibers largely populated the apical region, while low-SR fibers from the basal region exhibited sound-driven activity indistinguishable from control high-SR fibers. This functional phenotype change may support the full recovery of neural response thresholds and amplitudes after excitotoxicity. Furthermore, hyperresponsiveness of the auditory nerve fibers could be a crucial factor in the development of hyperactivity in the central auditory pathways, a common occurrence following acoustic overstimulation.