AMPA receptors (AMPARs) are multimodal transducers of glutamatergic signals throughout the brain. Their diversity is exemplified in the cerebellum: At afferent synapses, AMPARs mediate high-frequency excitation, whereas in Bergmann glia (BG) they support calcium transients that modulate synaptic transmission. This spectrum arises from different combinations of core subunits (GluA1-4), auxiliary proteins, and posttranscriptional modifications. Using mass spectrometry, cryo-electron microscopy, and electrophysiology, we characterize major cerebellar AMPARs in pigs: calcium-impermeable GluA2/A4 heteromers with four transmembrane AMPAR regulatory protein (TARP) subunits, mainly neuronal in origin, and BG-specific, calcium-permeable GluA1/A4 heteromers containing two type II TARPs. We also showed that GluA4 receptors frequently exhibit compact N-terminal domains that promote their synaptic delivery. Our study defines the organizational principles of mammalian cerebellar AMPAR complexes and reveals how different receptor subtypes support cell type-specific functions.

Type-A γ-aminobutyric receptors (GABA A Rs) are fast pentameric ligand gated ion channels (pLGICs) 1-5 . Within a millisecond, saturating agonist concentrations trigger activity bursts consisting of high-frequency fluctuations between conductive and non-conductive states 6,7 . These can last for tens to hundreds of milliseconds until, stochastically, receptors adopt stable, long-lived, desensitised conformations 2,8,9 . This highly dynamic process, known as gating, controls transient passages of chloride ions across plasma membranes to enable neurotransmission and other fundamental processes in animal physiology 10-12 . The conformational transitions inside activity bursts, inferred from electrophysiology, have remained inaccessible to structural biology investigation. Here we describe the gating motions of three GABA A receptor variants imaged within the first 10 milliseconds of agonist application by cryogenic electron microscopy (cryo-EM) 13 . We illustrate how activation and desensitisation proceed through multiple asymmetric states, supported by major secondary, tertiary and quaternary structural rearrangements, and demonstrate that the same gating principles apply to both homomeric and heteromeric GABA A Rs. Furthermore, we show that cholesterol and phospholipids stabilise newly formed inter-subunit interfaces and obstruct channel pores in short-lived desensitised states, while phosphatidylinositol 4,5-bisphosphate (PIP 2 ) precludes the opening of both α1β3 and α1β3γ2 GABA A R channels. Our results provide a novel framework to interpret decades of electrophysiology observations and suggest a broadly applicable approach to investigate mechanistically the vast arrays of physiological and pharmacological modulators of GABA A Rs 5,14 and other fast neurotransmitter receptors 15,16 . Moreover, the subunit interfaces and lipid-binding pockets that form and disappear during GABA A R gating provide new opportunities to discover modulators with improved specificity and therapeutic properties. One sentence summary:Single-particle cryo-EM was used to explore the dynamic conformational landscape of three human GABA A receptor variants within the first 10 milliseconds of interaction with their neurotransmitter agonists.

COVID-19 has stimulated the rapid development of new antibody and small molecule therapeutics to inhibit SARS-CoV-2 infection. Here we describe a third antiviral modality that combines the drug-like advantages of both. Bicycles are entropically constrained peptides stabilized by a central chemical scaffold into a bi-cyclic structure. Rapid screening of diverse bacteriophage libraries against SARS-CoV-2 Spike yielded unique Bicycle binders across the entire protein. Exploiting Bicycles’ inherent chemical combinability, we converted early micromolar hits into nanomolar viral inhibitors through simple multimerization. We also show how combining Bicycles against different epitopes into a single biparatopic agent allows Spike from diverse variants of concern (VoC) to be targeted (Alpha, Beta, Delta and Omicron). Finally, we demonstrate in both male hACE2-transgenic mice and Syrian golden hamsters that both multimerized and biparatopic Bicycles reduce viraemia and prevent host inflammation. These results introduce Bicycles as a potential antiviral modality to tackle new and rapidly evolving viruses.

The COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, has triggered a worldwide health emergency. Here, we show that ferritin-like Dps from hyperthermophilic Sulfolobus islandicus, covalently coupled with SARS-CoV-2 antigens via the SpyCatcher system, forms stable multivalent dodecameric vaccine nanoparticles that remain intact even after lyophilisation. Immunisation experiments in mice demonstrated that the SARS-CoV-2 receptor binding domain (RBD) coupled to Dps (RBD-S-Dps) elicited a higher antibody titre and an enhanced neutralising antibody response compared to monomeric RBD. A single immunisation with RBD-S-Dps completely protected hACE2-expressing mice from serious illness and led to viral clearance from the lungs upon SARS-CoV-2 infection. Our data highlight that multimerised SARS-CoV-2 subunit vaccines are a highly efficacious modality, particularly when combined with an ultra-stable scaffold.

Synthetic excitatory synaptic organizer The human brain contains trillions of synapses within a vast network of neurons. Synapse remodeling is essential to ensure the efficient reception and integration of external stimuli and to store and retrieve information. Building and remodeling of synapses occurs throughout life under the control of synaptic organizer proteins. Errors in this process can lead to neuropsychiatric or neurological disorders. Suzuki et al. combined structural elements of natural synaptic organizers to develop an artificial version called CPTX, which has different binding properties (see the Perspective by Salinas). CPTX could act as a molecular bridge to reconnect neurons and restore excitatory synaptic function in animal models of cerebellar ataxia, familial Alzheimer's disease, and spinal cord injury. The findings illustrate how structure-guided approaches can help to repair neuronal circuits. Science , this issue p. eabb4853 ; see also p. 1052

Thyroglobulin (TG) is the protein precursor of thyroid hormones, which are essential for growth, development and the control of metabolism in vertebrates1,2. Hormone synthesis from TG occurs in the thyroid gland via the iodination and coupling of pairs of tyrosines, and is completed by TG proteolysis3. Tyrosine proximity within TG is thought to enable the coupling reaction but hormonogenic tyrosines have not been clearly identified, and the lack of a three-dimensional structure of TG has prevented mechanistic understanding4. Here we present the structure of full-length human thyroglobulin at a resolution of approximately 3.5 Å, determined by cryo-electron microscopy. We identified all of the hormonogenic tyrosine pairs in the structure, and verified them using site-directed mutagenesis and in vitro hormone-production assays using human TG expressed in HEK293T cells. Our analysis revealed that the proximity, flexibility and solvent exposure of the tyrosines are the key characteristics of hormonogenic sites. We transferred the reaction sites from TG to an engineered tyrosine donor–acceptor pair in the unrelated bacterial maltose-binding protein (MBP), which yielded hormone production with an efficiency comparable to that of TG. Our study provides a framework to further understand the production and regulation of thyroid hormones. The cryo-electron microscopy structure of human thyroglobulin reveals that proximity, flexibility and solvent exposure are key characteristics of its hormonogenic tyrosine pairs, and provides a framework for understanding the formation of thyroid hormones.

Activation of immune cells relies on a dynamic actin cytoskeleton. Despite detailed knowledge of molecular actin assembly, the exact processes governing actin organization during activation remain elusive. Using advanced microscopy, we here show that Rat Basophilic Leukemia (RBL) cells, a model mast cell line, employ an orchestrated series of reorganization events within the cortical actin network during activation. In response to IgE antigen-stimulation of FCε receptors (FCεR) at the RBL cell surface, we observed symmetry breaking of the F-actin network and subsequent rapid disassembly of the actin cortex. This was followed by a reassembly process that may be driven by the coordinated transformation of distinct nanoscale F-actin architectures, reminiscent of self-organizing actin patterns. Actin patterns co-localized with zones of Arp2/3 nucleation, while network reassembly was accompanied by myosin-II activity. Strikingly, cortical actin disassembly coincided with zones of granule secretion, suggesting that cytoskeletal actin patterns contribute to orchestrate RBL cell activation.

Cytoskeletal actin dynamics is essential for T cell activation. Here, we show evidence that the binding kinetics of the antigen engaging the T cell receptor influences the nanoscale actin organization and mechanics of the immune synapse. Using an engineered T cell system expressing a specific T cell receptor and stimulated by a range of antigens, we found that the peak force experienced by the T cell receptor during activation was independent of the unbinding kinetics of the stimulating antigen. Conversely, quantification of the actin retrograde flow velocity at the synapse revealed a striking dependence on the antigen unbinding kinetics. These findings suggest that the dynamics of the actin cytoskeleton actively adjusted to normalize the force experienced by the T cell receptor in an antigen-specific manner. Consequently, tuning actin dynamics in response to antigen kinetics may thus be a mechanism that allows T cells to adjust the lengthscale and timescale of T cell receptor signaling.

The T-cell receptor (TCR) triggers the elimination of pathogens and tumors by T lymphocytes. In order for this to avoid damage to the host, the receptor has to discriminate between thousands of peptide ligands presented by each host cell. Exactly how the TCR does this is unknown. In resting T-cells, the TCR is largely unphosphorylated due to the dominance of phosphatases over kinases expressed at the cell surface. When agonist peptides are presented to the TCR by major histocompatibility complex (MHC) proteins expressed by antigen-presenting cells (APCs), very fast receptor triggering occurs, leading to TCR phosphorylation. Recent work suggests that this depends on the local exclusion of the phosphatases from regions of contact of the T cells with the APCs. Here, we develop and test a quantitative treatment of receptor triggering reliant only upon TCR dwell-time in phosphatase-depleted cell-cell contacts constrained in area by cell topography. Using the model and experimentally-derived parameters, we find that ligand discrimination is possible but that it depends crucially on individual contacts being 400 nm in diameter or smaller, i.e. the size generated by microvilli. The model not only correctly predicts the relative signaling potencies of known agonists and non-agonists, but achieves this in the absence of conventional, multi-step kinetic proof-reading. Our work provides a simple, quantitative and predictive molecular framework for understanding why TCR triggering is so selective and fast, and reveals that for some receptors, cell topography crucially influences signaling outcomes. Significance statement One approach to testing biological theories is to determine if they are predictive. A simple, theoretical treatment of TCR triggering suggests that ligand discrimination by the receptor relies on just two physical principles: (1) the time TCRs spend in cell-cell contacts depleted of large tyrosine phosphatases; and (2) constraints on contact size imposed by T cells using finger-like protrusions to interrogate their targets. The theory not only allows agonistic and non-agonistic TCR ligands to be distinguished but predicts the relative signalling potencies of agonists with remarkable accuracy. This suggests that the theory captures the essential features of receptor triggering.

Structural, biochemical and biophysical studies of eukaryotic soluble and membrane proteins require their production in milligram quantities. Although large-scale protein expression strategies based on transient or stable transfection of mammalian cells are well established, they are associated with high consumable costs, limited transfection efficiency or long and tedious selection of clonal cell lines. Lentiviral transduction is an efficient method for the delivery of transgenes to mammalian cells and unifies the ease of use and speed of transient transfection with the robust expression of stable cell lines. In this protocol, we describe the design and step-by-step application of a lentiviral plasmid suite, termed pHR-CMV-TetO2, for the constitutive or inducible large-scale production of soluble and membrane proteins in HEK293 cell lines. Optional features include bicistronic co-expression of fluorescent marker proteins for enrichment of co-transduced cells using cell sorting and of biotin ligase for in vivo biotinylation. We demonstrate the efficacy of the method for a set of soluble proteins and for the G-protein-coupled receptor (GPCR) Smoothened (SMO). We further compare this method with baculovirus transduction of mammalian cells (BacMam), using the type-A γ-aminobutyric acid receptor (GABAAR) β3 homopentamer as a test case. The protocols described here are optimized for simplicity, speed and affordability; lead to a stable polyclonal cell line and milligram-scale amounts of protein in 3–4 weeks; and routinely achieve an approximately three- to tenfold improvement in protein production yield per cell as compared to transient transduction or transfection. This protocol describes a suite of lentiviral transfer plasmids that can be used for high-yield, time- and cost-efficient, and constitutive or inducible production of soluble and membrane proteins in mammalian cell lines.

The generation of precise synaptic connections between developing neurons is critical to the formation of functional neural circuits. Astrocyte-secreted glypican 4 induces formation of active excitatory synapses by recruiting AMPA glutamate receptors to the postsynaptic cell surface. We now identify the molecular mechanism of how glypican 4 exerts its effect. Glypican 4 induces release of the AMPA receptor clustering factor neuronal pentraxin 1 from presynaptic terminals by signaling through presynaptic protein tyrosine phosphatase receptor δ. Pentraxin then accumulates AMPA receptors on the postsynaptic terminal forming functional synapses. Our findings reveal a signaling pathway that regulates synaptic activity during central nervous system development and demonstrates a role for astrocytes as organizers of active synaptic connections by coordinating both pre and post synaptic neurons. As mutations in glypicans are associated with neurological disorders, such as autism and schizophrenia, this signaling cascade offers new avenues to modulate synaptic function in disease.

Cell-free studies have demonstrated how collective action of actin-associated proteins can organize actin filaments into dynamic patterns, such as vortices, asters and stars. Using complementary microscopic techniques, we here show evidence of such self-organization of the actin cortex in living HeLa cells. During cell adhesion, an active multistage process naturally leads to pattern transitions from actin vortices over stars into asters. This process is primarily driven by Arp2/3 complex nucleation, but not by myosin motors, which is in contrast to what has been theoretically predicted and observed in vitro. Concomitant measurements of mechanics and plasma membrane fluidity demonstrate that changes in actin patterning alter membrane architecture but occur functionally independent of macroscopic cortex elasticity. Consequently, tuning the activity of the Arp2/3 complex to alter filament assembly may thus be a mechanism allowing cells to adjust their membrane architecture without affecting their macroscopic mechanical properties.

T cell activation and especially trafficking of T cell receptor microclusters during immunological synapse formation are widely thought to rely on cytoskeletal remodeling. However, important details on the involvement of actin in the latter transport processes are missing. Using a suite of advanced optical microscopes to analyze resting and activated T cells, we show that, following contact formation with activating surfaces, these cells sequentially rearrange their cortical actin across the entire cell, creating a previously unreported ramifying actin network above the immunological synapse. This network shows all the characteristics of an inward- growing transportation network and its dynamics correlating with T cell receptor rearrangements. This actin reorganization is accompanied by an increase in the nanoscale actin meshwork size and the dynamic adjustment of the turnover times and filament lengths of two differently sized filamentous actin populations, wherein forminmediated long actin filaments support a very flat and stiff contact at the immunological synapse interface. The initiation of immunological synapse formation, as highlighted by calcium release, requires markedly little contact with activating surfaces and no cytoskeletal rearrangements. Our work suggests that incipient signaling in T cells initiates global cytoskeletal rearrangements across the whole cell, including a stiffening process for possibly mechanically supporting contact formation at the immunological synapse interface as well as a central ramified transportation network apparently directed at the consolidation of the contact and the delivery of effector functions.

The discovery of genetic variants influencing sleep patterns can shed light on the physiological processes underlying sleep. As part of a large clinical sequencing project, WGS500, we sequenced a family in which the two male children had severe developmental delay and a dramatically disturbed sleep-wake cycle, with very long wake and sleep durations, reaching up to 106-h awake and 48-h asleep. The most likely causal variant identified was a novel missense variant in the X-linked GRIA3 gene, which has been implicated in intellectual disability. GRIA3 encodes GluA3, a subunit of AMPA-type ionotropic glutamate receptors (AMPARs). The mutation (A653T) falls within the highly conserved transmembrane domain of the ion channel gate, immediately adjacent to the analogous residue in the Grid2 (glutamate receptor) gene, which is mutated in the mouse neurobehavioral mutant, Lurcher. In vitro, the GRIA3(A653T) mutation stabilizes the channel in a closed conformation, in contrast to Lurcher. We introduced the orthologous mutation into a mouse strain by CRISPR-Cas9 mutagenesis and found that hemizygous mutants displayed significant differences in the structure of their activity and sleep compared to wild-type littermates. Typically, mice are polyphasic, exhibiting multiple sleep bouts of sleep several minutes long within a 24-h period. The Gria3A653T mouse showed significantly fewer brief bouts of activity and sleep than the wild-types. Furthermore, Gria3A653T mice showed enhanced period lengthening under constant light compared to wild-type mice, suggesting an increased sensitivity to light. Our results suggest a role for GluA3 channel activity in the regulation of sleep behavior in both mice and humans.

Peter Jönssona,b*, Jennifer Southcombec,d,e*, Ana Mafalda Santosc,d, Jiandong Huoc,d, Ricardo A. Fernandesc,d, James McColla, Melissa Leverf, Edward J. Evansc,d, Alexander Hudsonc,d, Veronica T. Changc,d, Tomáš Hankec, Andrew Godkinh, Paul D. Dunnea, Mathew H. Horrocksa, Matthieu Palayreta, Gavin R. Screatonc, Jan Peterseni,j, Jamie Rossjohnh,i,j, Lars Fuggerc,d,g, Omer Dushekf, Xiaoning Xuc§, Simon J. Davisc,d§ and David Klenermana§

Lymphocyte T cells are responsible for cell-mediated adaptive immune responses, involving transient interactions of the T-cell receptor (TCR) with peptides presented by MHC proteins. A productive interaction triggers the T-cell signaling forming the immunological synapse. Initially, the TCR is phosphorylated by the Lck, a membrane-anchored tyrosine kinase, producing membrane microclusters. Understanding the T-cell pre-synaptic triggering implies comparing resting vs. activated states. Super-resolution imaging techniques (i.e. dSTORM and PALM) suggested protein pre-clustering into "nano-domains" in resting cells, contradicting the classical view of cluster formation upon activation. However, observations with cells touching non-activating surfaces probably don't resemble a true resting state. Instead, avoiding contacts would allow better understanding the resting and early stages of activation. We studied live T-cells in suspension employing a hydrogel in a density gradient and used STED nanoscopy to unravel the plasma membrane distribution and dynamics of Lck in T-cells on glass and in suspension. T-cells suspended in the hydrogel do not triggered calcium, indicating absence of activation; while classically considered resting states triggered calcium in a similar fashion as when cells were deposited onto a surface functionalized by antibody (antiCD3 and antiCD28) coating. In contrast to surface-contacting, suspended T-cells showed mostly a uniform distribution of Lck. Nevertheless, Lck still displayed heterogeneity in mobility in the true resting state. We found several components of mobility rather than a single one. These different diffusion coefficients were remarkably slower when T-cells were in contact with any of the studied surfaces. Our results suggest that pre-clustering of signaling receptors and cell-surface proteins in resting T-cells needs reconsideration and that understanding the T cell activation requires a true resting state, which we can be obtained by means of hydrogels.

The αβ T-cell coreceptor CD4 enhances immune responses more than 1 million-fold in some assays, and yet the affinity of CD4 for its ligand, peptide-major histocompatibility class II (pMHC II) on antigen-presenting cells, is so weak that it was previously unquantifiable. Here, we report that a soluble form of CD4 failed to bind detectably to pMHC II in surface plasmon resonance-based assays, establishing a new upper limit for the solution affinity at 2.5 mM. However, when presented multivalently on magnetic beads, soluble CD4 bound pMHC II-expressing B cells, confirming that it is active and allowing mapping of the native coreceptor binding site on pMHC II. Whereas binding was undetectable in solution, the affinity of the CD4/pMHC II interaction could be measured in 2D using CD4- and adhesion molecule-functionalized, supported lipid bilayers, yielding a 2D Kd of ∼5,000 molecules/μm(2) This value is two to three orders of magnitude higher than previously measured 2D Kd values for interacting leukocyte surface proteins. Calculations indicated, however, that CD4/pMHC II binding would increase rates of T-cell receptor (TCR) complex phosphorylation by threefold via the recruitment of Lck, with only a small, 2-20% increase in the effective affinity of the TCR for pMHC II. The affinity of CD4/pMHC II therefore seems to be set at a value that increases T-cell sensitivity by enhancing phosphorylation, without compromising ligand discrimination.

It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell antigen receptor (TCR) triggering, but how such segregation occurs and whether it can initiate signaling is unclear. Using structural and biophysical analysis, we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying that sites of TCR-ligand engagement would sterically exclude CD45. We also show that the formation of 'close contacts', new structures characterized by spontaneous CD45 and kinase segregation at the submicron-scale, initiates signaling even when TCR ligands are absent. Our work reveals the structural basis for, and the potent signaling effects of, local CD45 and kinase segregation. TCR ligands have the potential to heighten signaling simply by holding receptors in close contacts.

Transmitting signals across the synapse Glutamate receptors located on neuronal cells play a role in mediating electrical signals at excitatory synapses. These glutamatergic synapses are extremely important for nearly all cognitive functions. Elegheert et al. analyzed a complex that bridges the synapse, comprising β-neurexin 1, a cell adhesion molecule on the surface of presynaptic axons; cerebellin 1, a synaptic organizer; and the postsynaptic glutamate receptor GluD2. The structural and functional analysis provides insight into the mechanism of synaptic signaling. Science , this issue p. 295