The ZAR1 resistosome is a calcium-permeable channel triggering plant immune signaling.

Nucleotide-binding, leucine-rich repeat receptors (NLRs) are major immune receptors in plants and animals. Upon activation, the Arabidopsis NLR protein ZAR1 forms a pentameric resistosome in vitro and triggers immune responses and cell death in plants. In this study, we employed single-molecule imaging to show that the activated ZAR1 protein can form pentameric complexes in the plasma membrane. The ZAR1 resistosome displayed ion channel activity in Xenopus oocytes in a manner dependent on a conserved acidic residue Glu11 situated in the channel pore. Pre-assembled ZAR1 resistosome was readily incorporated into planar lipid-bilayers and displayed calcium-permeable cation-selective channel activity. Furthermore, we show that activation of ZAR1 in the plant cell led to Glu11-dependent Ca2+ influx, perturbation of subcellular structures, production of reactive oxygen species, and cell death. The results thus support that the ZAR1 resistosome acts as a calcium-permeable cation channel to trigger immunity and cell death.

Full-Length P2X7 Structures Reveal How Palmitoylation Prevents Channel Desensitization.

P2X receptors are trimeric, non-selective cation channels activated by extracellular ATP. The P2X7 receptor subtype is a pharmacological target because of involvement in apoptotic, inflammatory, and tumor progression pathways. It is the most structurally and functionally distinct P2X subtype, containing a unique cytoplasmic domain critical for the receptor to initiate apoptosis and not undergo desensitization. However, lack of structural information about the cytoplasmic domain has hindered understanding of the molecular mechanisms underlying these processes. We report cryoelectron microscopy structures of full-length rat P2X7 receptor in apo and ATP-bound states. These structures reveal how one cytoplasmic element, the C-cys anchor, prevents desensitization by anchoring the pore-lining helix to the membrane with palmitoyl groups. They show a second cytoplasmic element with a unique fold, the cytoplasmic ballast, which unexpectedly contains a zinc ion complex and a guanosine nucleotide binding site. Our structures provide first insights into the architecture and function of a P2X receptor cytoplasmic domain.

Phase transition of spindle-associated protein regulate spindle apparatus assembly.

Spindle assembly required during mitosis depends on microtubule polymerization. We demonstrate that the evolutionarily conserved low-complexity protein, BuGZ, undergoes phase transition or coacervation to promote assembly of both spindles and their associated components. BuGZ forms temperature-dependent liquid droplets alone or on microtubules in physiological buffers. Coacervation in vitro or in spindle and spindle matrix depends on hydrophobic residues in BuGZ. BuGZ coacervation and its binding to microtubules and tubulin are required to promote assembly of spindle and spindle matrix in Xenopus egg extract and in mammalian cells. Since several previously identified spindle-associated components also contain low-complexity regions, we propose that coacervating proteins may be a hallmark of proteins that comprise a spindle matrix that functions to promote assembly of spindles by concentrating its building blocks.

GM130 Regulates Golgi-Derived Spindle Assembly by Activating TPX2 and Capturing Microtubules.

Spindle assembly requires the coordinated action of multiple cellular structures to nucleate and organize microtubules in a precise spatiotemporal manner. Among them, the contributions of centrosomes, chromosomes, and microtubules have been well studied, yet the involvement of membrane-bound organelles remains largely elusive. Here, we provide mechanistic evidence for a membrane-based, Golgi-derived microtubule assembly pathway in mitosis. Upon mitotic entry, the Golgi matrix protein GM130 interacts with importin α via a classical nuclear localization signal that recruits importin α to the Golgi membranes. Sequestration of importin α by GM130 liberates the spindle assembly factor TPX2, which activates Aurora-A kinase and stimulates local microtubule nucleation. Upon filament assembly, nascent microtubules are further captured by GM130, thus linking Golgi membranes to the spindle. Our results reveal an active role for the Golgi in regulating spindle formation to ensure faithful organelle inheritance.

Repair of a DNA-protein crosslink by replication-coupled proteolysis.

DNA-protein crosslinks (DPCs) are caused by environmental, endogenous, and chemotherapeutic agents and pose a severe threat to genome stability. We use Xenopus egg extracts to recapitulate DPC repair in vitro and show that this process is coupled to DNA replication. A DPC on the leading strand template arrests the replisome by stalling the CMG helicase. The DPC is then degraded on DNA, yielding a peptide-DNA adduct that is bypassed by CMG. The leading strand subsequently resumes synthesis, stalls again at the adduct, and then progresses past the adduct using DNA polymerase ζ. A DPC on the lagging strand template only transiently stalls the replisome, but it too is degraded, allowing Okazaki fragment bypass. Our experiments describe a versatile, proteolysis-based mechanism of S phase DPC repair that avoids replication fork collapse.

Structural features of nucleosomes in interphase and metaphase chromosomes.

Structural heterogeneity of nucleosomes in functional chromosomes is unknown. Here, we devise the template-, reference- and selection-free (TRSF) cryo-EM pipeline to simultaneously reconstruct cryo-EM structures of protein complexes from interphase or metaphase chromosomes. The reconstructed interphase and metaphase nucleosome structures are on average indistinguishable from canonical nucleosome structures, despite DNA sequence heterogeneity, cell-cycle-specific posttranslational modifications, and interacting proteins. Nucleosome structures determined by a decoy-classifying method and structure variability analyses reveal the nucleosome structural variations in linker DNA, histone tails, and nucleosome core particle configurations, suggesting that the opening of linker DNA, which is correlated with H2A C-terminal tail positioning, is suppressed in chromosomes. High-resolution (3.4-3.5 Å) nucleosome structures indicate DNA-sequence-independent stabilization of superhelical locations ±0-1 and ±3.5-4.5. The linker histone H1.8 preferentially binds to metaphase chromatin, from which chromatosome cryo-EM structures with H1.8 at the on-dyad position are reconstituted. This study presents the structural characteristics of nucleosomes in chromosomes.

A checkpoint function for Nup98 in nuclear pore formation suggested by novel inhibitory nanobodies.

Nuclear pore complex (NPC) biogenesis is a still enigmatic example of protein self-assembly. We now introduce several cross-reacting anti-Nup nanobodies for imaging intact nuclear pore complexes from frog to human. We also report a simplified assay that directly tracks postmitotic NPC assembly with added fluorophore-labeled anti-Nup nanobodies. During interphase, NPCs are inserted into a pre-existing nuclear envelope. Monitoring this process is challenging because newly assembled NPCs are indistinguishable from pre-existing ones. We overcame this problem by inserting Xenopus-derived NPCs into human nuclear envelopes and using frog-specific anti-Nup nanobodies for detection. We further asked whether anti-Nup nanobodies could serve as NPC assembly inhibitors. Using a selection strategy against conserved epitopes, we obtained anti-Nup93, Nup98, and Nup155 nanobodies that block Nup-Nup interfaces and arrest NPC assembly. We solved structures of nanobody-target complexes and identified roles for the Nup93 α-solenoid domain in recruiting Nup358 and the Nup214·88·62 complex, as well as for Nup155 and the Nup98 autoproteolytic domain in NPC scaffold assembly. The latter suggests a checkpoint linking pore formation to the assembly of the Nup98-dominated permeability barrier.

Tiki1 is required for head formation via Wnt cleavage-oxidation and inactivation.

Secreted Wnt morphogens are signaling molecules essential for embryogenesis, pathogenesis, and regeneration and require distinct modifications for secretion, gradient formation, and activity. Whether Wnt proteins can be posttranslationally inactivated during development and homeostasis is unknown. Here we identify, through functional cDNA screening, a transmembrane protein Tiki1 that is expressed specifically in the dorsal Spemann-Mangold Organizer and is required for anterior development during Xenopus embryogenesis. Tiki1 antagonizes Wnt function in embryos and human cells via a TIKI homology domain that is conserved from bacteria to mammals and acts likely as a protease to cleave eight amino-terminal residues of a Wnt protein, resulting in oxidized Wnt oligomers that exhibit normal secretion but minimized receptor-binding capability. Our findings identify a Wnt-specific protease that controls head formation, reveal a mechanism for morphogen inactivation through proteolysis-induced oxidation-oligomerization, and suggest a role of the Wnt amino terminus in evasion of oxidizing inactivation. TIKI proteins may represent potential therapeutic targets.

TMEM16F forms a Ca2+-activated cation channel required for lipid scrambling in platelets during blood coagulation.

Collapse of membrane lipid asymmetry is a hallmark of blood coagulation. TMEM16F of the TMEM16 family that includes TMEM16A/B Ca(2+)-activated Cl(-) channels (CaCCs) is linked to Scott syndrome with deficient Ca(2+)-dependent lipid scrambling. We generated TMEM16F knockout mice that exhibit bleeding defects and protection in an arterial thrombosis model associated with platelet deficiency in Ca(2+)-dependent phosphatidylserine exposure and procoagulant activity and lack a Ca(2+)-activated cation current in the platelet precursor megakaryocytes. Heterologous expression of TMEM16F generates a small-conductance Ca(2+)-activated nonselective cation (SCAN) current with subpicosiemens single-channel conductance rather than a CaCC. TMEM16F-SCAN channels permeate both monovalent and divalent cations, including Ca(2+), and exhibit synergistic gating by Ca(2+) and voltage. We further pinpointed a residue in the putative pore region important for the cation versus anion selectivity of TMEM16F-SCAN and TMEM16A-CaCC channels. This study thus identifies a Ca(2+)-activated channel permeable to Ca(2+) and critical for Ca(2+)-dependent scramblase activity during blood coagulation. PAPERFLICK:

The permeability of reconstituted nuclear pores provides direct evidence for the selective phase model.

Nuclear pore complexes (NPCs) maintain a permeability barrier between the nucleus and the cytoplasm through FG-repeat-containing nucleoporins (Nups). We previously proposed a "selective phase model" in which the FG repeats interact with one another to form a sieve-like barrier that can be locally disrupted by the binding of nuclear transport receptors (NTRs), but not by inert macromolecules, allowing selective passage of NTRs and associated cargo. Here, we provide direct evidence for this model in a physiological context. By using NPCs reconstituted from Xenopus laevis egg extracts, we show that Nup98 is essential for maintaining the permeability barrier. Specifically, the multivalent cohesion between FG repeats is required, including cohesive FG repeats close to the anchorage point to the NPC scaffold. Our data exclude alternative models that are based solely on an interaction between the FG repeats and NTRs and indicate that the barrier is formed by a sieve-like FG hydrogel.

Mosaicism-independent mechanisms contribute to Pcdh19-related epilepsy and repetitive behaviors in Xenopus.

Protocadherin19 (PCDH19)-related epilepsy syndrome is a rare disorder characterized by early-onset epilepsy, intellectual disability, and autistic behaviors. PCDH19 is located on the X chromosome and encodes a calcium-dependent single-pass transmembrane protein, which regulates cell-to-cell adhesion through homophilic binding. In human, 90% of heterozygous females, containing PCDH19 wild-type and mutant cells due to random X inactivation, are affected, whereas mutant males, containing only mutant cells, are typically not. The current view, the cellular interference, is that the altered interactions between wild-type and mutant cells during development, rather than loss of function itself, are responsible. However, studies using Pcdh19 knockout mice showed that the complete loss of function also causes autism-like behaviors both in males and females, suggesting that other functions of PCDH19 may also contribute to pathogenesis. To address whether mosaicism is required for PCDH19-related epilepsy, we generated Xenopus tropicalis tadpoles with complete or mosaic loss of function by injecting antisense morpholino oligonucleotides into the blastomeres of neural lineage at different stages of development. We found that either mosaic or complete knockdown results in seizure-like behaviors, which could be rescued by antiseizure medication, and repetitive behaviors. Our results suggest that the loss of PCDH19 function itself, in addition to cellular interference, may also contribute to PCDH19-related epilepsy.

Selective bypass of a lagging strand roadblock by the eukaryotic replicative DNA helicase.

The eukaryotic replicative DNA helicase, CMG, unwinds DNA by an unknown mechanism. In some models, CMG encircles and translocates along one strand of DNA while excluding the other strand. In others, CMG encircles and translocates along duplex DNA. To distinguish between these models, replisomes were confronted with strand-specific DNA roadblocks in Xenopus egg extracts. An ssDNA translocase should stall at an obstruction on the translocation strand but not the excluded strand, whereas a dsDNA translocase should stall at obstructions on either strand. We found that replisomes bypass large roadblocks on the lagging strand template much more readily than on the leading strand template. Our results indicate that CMG is a 3' to 5' ssDNA translocase, consistent with unwinding via "steric exclusion." Given that MCM2-7 encircles dsDNA in G1, the data imply that formation of CMG in S phase involves remodeling of MCM2-7 from a dsDNA to a ssDNA binding mode.

A regulatory archipelago controls Hox genes transcription in digits.

The evolution of digits was an essential step in the success of tetrapods. Among the key players, Hoxd genes are coordinately regulated in developing digits, where they help organize growth and patterns. We identified the distal regulatory sites associated with these genes by probing the three-dimensional architecture of this regulatory unit in developing limbs. This approach, combined with in vivo deletions of distinct regulatory regions, revealed that the active part of the gene cluster contacts several enhancer-like sequences. These elements are dispersed throughout the nearby gene desert, and each contributes either quantitatively or qualitatively to Hox gene transcription in presumptive digits. We propose that this genetic system, which we call a "regulatory archipelago," provides an inherent flexibility that may partly underlie the diversity in number and morphology of digits across tetrapods, as well as their resilience to drastic variations.

Katanin contributes to interspecies spindle length scaling in Xenopus.

Bipolar spindles must separate chromosomes by the appropriate distance during cell division, but mechanisms determining spindle length are poorly understood. Based on a 2D model of meiotic spindle assembly, we predicted that higher localized microtubule (MT) depolymerization rates could generate the shorter spindles observed in egg extracts of X. tropicalis compared to X. laevis. We found that katanin-dependent MT severing was increased in X. tropicalis, which, unlike X. laevis, lacks an inhibitory phosphorylation site in the katanin p60 catalytic subunit. Katanin inhibition lengthened spindles in both species. In X. tropicalis, k-fiber MT bundles that connect to chromosomes at their kinetochores extended through spindle poles, disrupting them. In both X. tropicalis extracts and the spindle simulation, a balance between k-fiber number and MT depolymerization is required to maintain spindle morphology. Thus, mechanisms have evolved in different species to scale spindle size and coordinate regulation of multiple MT populations in order to generate a robust steady-state structure.

Recognition of a mononucleosomal histone modification pattern by BPTF via multivalent interactions.

Little is known about how combinations of histone marks are interpreted at the level of nucleosomes. The second PHD finger of human BPTF is known to specifically recognize histone H3 when methylated on lysine 4 (H3K4me2/3). Here, we examine how additional heterotypic modifications influence BPTF binding. Using peptide surrogates, three acetyllysine ligands are indentified for a PHD-adjacent bromodomain in BPTF via systematic screening and biophysical characterization. Although the bromodomain displays limited discrimination among the three possible acetyllysines at the peptide level, marked selectivity is observed for only one of these sites, H4K16ac, in combination with H3K4me3 at the mononucleosome level. In support, these two histone marks constitute a unique trans-histone modification pattern that unambiguously resides within a single nucleosomal unit in human cells, and this module colocalizes with these marks in the genome. Together, our data call attention to nucleosomal patterning of covalent marks in dictating critical chromatin associations.

Decoding the signaling of a GPCR heteromeric complex reveals a unifying mechanism of action of antipsychotic drugs.

Atypical antipsychotic drugs, such as clozapine and risperidone, have a high affinity for the serotonin 5-HT(2A) G protein-coupled receptor (GPCR), the 2AR, which signals via a G(q) heterotrimeric G protein. The closely related non-antipsychotic drugs, such as ritanserin and methysergide, also block 2AR function, but they lack comparable neuropsychological effects. Why some but not all 2AR inhibitors exhibit antipsychotic properties remains unresolved. We now show that a heteromeric complex between the 2AR and the G(i)-linked GPCR, metabotropic glutamate 2 receptor (mGluR2), integrates ligand input, modulating signaling output and behavioral changes. Serotonergic and glutamatergic drugs bind the mGluR2/2AR heterocomplex, which then balances Gi- and Gq-dependent signaling. We find that the mGluR2/2AR-mediated changes in Gi and Gq activity predict the psychoactive behavioral effects of a variety of pharmocological compounds. These observations provide mechanistic insight into antipsychotic action that may advance therapeutic strategies for disorders including schizophrenia and dementia.

Evolution of the endothelin pathway drove neural crest cell diversification.

Neural crest cells (NCCs) are migratory, multipotent embryonic cells that are unique to vertebrates and form an array of clade-defining adult features. The evolution of NCCs has been linked to various genomic events, including the evolution of new gene-regulatory networks1,2, the de novo evolution of genes3 and the proliferation of paralogous genes during genome-wide duplication events4. However, conclusive functional evidence linking new and/or duplicated genes to NCC evolution is lacking. Endothelin ligands (Edns) and endothelin receptors (Ednrs) are unique to vertebrates3,5,6, and regulate multiple aspects of NCC development in jawed vertebrates7-10. Here, to test whether the evolution of Edn signalling was a driver of NCC evolution, we used CRISPR-Cas9 mutagenesis11 to disrupt edn, ednr and dlx genes in the sea lamprey, Petromyzon marinus. Lampreys are jawless fishes that last shared a common ancestor with modern jawed vertebrates around 500 million years ago12. Thus, comparisons between lampreys and gnathostomes can identify deeply conserved and evolutionarily flexible features of vertebrate development. Using the frog Xenopus laevis to expand gnathostome phylogenetic representation and facilitate side-by-side analyses, we identify ancient and lineage-specific roles for Edn signalling. These findings suggest that Edn signalling was activated in NCCs before duplication of the vertebrate genome. Then, after one or more genome-wide duplications in the vertebrate stem, paralogous Edn pathways functionally diverged, resulting in NCC subpopulations with different Edn signalling requirements. We posit that this new developmental modularity facilitated the independent evolution of NCC derivatives in stem vertebrates. Consistent with this, differences in Edn pathway targets are associated with differences in the oropharyngeal skeleton and autonomic nervous system of lampreys and modern gnathostomes. In summary, our work provides functional genetic evidence linking the origin and duplication of new vertebrate genes with the stepwise evolution of a defining vertebrate novelty.

Structure of nucleosome-bound DNA methyltransferases DNMT3A and DNMT3B.

CpG methylation by de novo DNA methyltransferases (DNMTs) 3A and 3B is essential for mammalian development and differentiation and is frequently dysregulated in cancer1. These two DNMTs preferentially bind to nucleosomes, yet cannot methylate the DNA wrapped around the nucleosome core2, and they favour the methylation of linker DNA at positioned nucleosomes3,4. Here we present the cryo-electron microscopy structure of a ternary complex of catalytically competent DNMT3A2, the catalytically inactive accessory subunit DNMT3B3 and a nucleosome core particle flanked by linker DNA. The catalytic-like domain of the accessory DNMT3B3 binds to the acidic patch of the nucleosome core, which orients the binding of DNMT3A2 to the linker DNA. The steric constraints of this arrangement suggest that nucleosomal DNA must be moved relative to the nucleosome core for de novo methylation to occur.

Cryo-EM structure of SWI/SNF complex bound to a nucleosome.

The chromatin-remodelling complex SWI/SNF is highly conserved and has critical roles in various cellular processes, including transcription and DNA-damage repair1,2. It hydrolyses ATP to remodel chromatin structure by sliding and evicting histone octamers3-8, creating DNA regions that become accessible to other essential factors. However, our mechanistic understanding of the remodelling activity is hindered by the lack of a high-resolution structure of complexes from this family. Here we report the cryo-electron microscopy structure of Saccharomyces cerevisiae SWI/SNF bound to a nucleosome, at near-atomic resolution. In the structure, the actin-related protein (Arp) module is sandwiched between the ATPase and the rest of the complex, with the Snf2 helicase-SANT associated (HSA) domain connecting all modules. The body contains an assembly scaffold composed of conserved subunits Snf12 (also known as SMARCD or BAF60), Snf5 (also known as SMARCB1, BAF47 or INI1) and an asymmetric dimer of Swi3 (also known as SMARCC, BAF155 or BAF170). Another conserved subunit, Swi1 (also known as ARID1 or BAF250), resides in the core of SWI/SNF, acting as a molecular hub. We also observed interactions between Snf5 and the histones at the acidic patch, which could serve as an anchor during active DNA translocation. Our structure enables us to map and rationalize a subset of cancer-related mutations in the human SWI/SNF complex and to propose a model for how SWI/SNF recognizes and remodels the +1 nucleosome to generate nucleosome-depleted regions during gene activation9.

Insights into the assembly and activation of the microtubule nucleator γ-TuRC.

Microtubules are dynamic polymers of α- and ß-tubulin and have crucial roles in cell signalling, cell migration, intracellular transport and chromosome segregation1. They assemble de novo from αß-tubulin dimers in an essential process termed microtubule nucleation. Complexes that contain the protein γ-tubulin serve as structural templates for the microtubule nucleation reaction2. In vertebrates, microtubules are nucleated by the 2.2-megadalton γ-tubulin ring complex (γ-TuRC), which comprises γ-tubulin, five related γ-tubulin complex proteins (GCP2-GCP6) and additional factors3. GCP6 is unique among the GCP proteins because it carries an extended insertion domain of unknown function. Our understanding of microtubule formation in cells and tissues is limited by a lack of high-resolution structural information on the γ-TuRC. Here we present the cryo-electron microscopy structure of γ-TuRC from Xenopus laevis at 4.8 Å global resolution, and identify a 14-spoked arrangement of GCP proteins and γ-tubulins in a partially flexible open left-handed spiral with a uniform sequence of GCP variants. By forming specific interactions with other GCP proteins, the GCP6-specific insertion domain acts as a scaffold for the assembly of the γ-TuRC. Unexpectedly, we identify actin as a bona fide structural component of the γ-TuRC with functional relevance in microtubule nucleation. The spiral geometry of γ-TuRC is suboptimal for microtubule nucleation and a controlled conformational rearrangement of the γ-TuRC is required for its activation. Collectively, our cryo-electron microscopy reconstructions provide detailed insights into the molecular organization, assembly and activation mechanism of vertebrate γ-TuRC, and will serve as a framework for the mechanistic understanding of fundamental biological processes associated with microtubule nucleation, such as meiotic and mitotic spindle formation and centriole biogenesis4.