Correlating super-resolution fluorescence light microscopy with cryo-electron tomography (SRcryoCLEM) is a feasible way of targeting specific proteins of interest for high-resolution cryo-electron tomography (cryoET) imaging within cells. Among different approaches for performing super-resolution fluorescence microscopy on cryogenically preserved samples, cryo-single molecule localization microscopy (cryoSMLM) offers one of the highest imaging resolutions. Thus far, applications of cryoSMLM in SRcryoCLEM have been limited to targeting a single protein structure at a time, as the available palette of cryo-compatible reversibly photoswitchable fluorescent proteins, required for cryoSMLM imaging, is severely limited. Here, we present rsTagRFP and rsEGFP2 as a compatible pair of red and green fluorescent labels that enables dual-colour cryoSMLM, and thus dual-target SRcryoCLEM, in mammalian cells. We demonstrate the simultaneous targeting and identification of two separate structures, MAP2-decorated microtubules and vimentin intermediate filaments, with 30 nm accuracy and within the same cell.

Immunoglobulin G (IgG) is a glycoprotein harboring conserved fragment crystallizable domain glycans, but it can also express variable domain glycans (VDGs). Elevated levels of VDGs are a hallmark of the autoantibodies most specific to rheumatoid arthritis and are also associated with other autoimmune diseases. Nonetheless, the effect of VDGs on IgG function remains poorly understood. This study investigates the impact of VDGs on the ability of antibodies to activate the complement system, a key immune effector mechanism. We demonstrate that VDGs on IgG inhibit the initiation of the classical complement pathway, as evidenced by complement activation assays with IgG displaying varying degrees of VDGs. Structure-function analyses show that VDGs reduce classical complement activation by impeding IgG oligomer formation on antigenic surfaces. As IgG oligomerization is essential for effective C1q binding, VDG-mediated interference with this process lowers IgG's complement activation potential. This impaired ability to recruit complement was further substantiated through direct visualization of IgG oligomer-C1q complexes on antigen-coated vesicles by cryoelectron tomography. Together, these data reveal that VDGs increase the functional diversity of IgG and identify a novel regulatory mechanism that modulates the ability of antibodies to activate one of their main immune effector mechanisms.

Recent discoveries have shown the presence of ribonucleic acid (RNA) on the cell surface, defying the view that RNA only functions intracellularly. However, how RNA is presented on the cell surface and what its biological relevance is are poorly understood. We established Toll-like receptor 7 (TLR7) as a cell-surface RNA (csRNA) probe. Employing it in a genome-wide knockout screening, we identified heparan sulfate (HS) as a crucial factor for csRNA presentation. Cell-surface proximity labeling revealed that HS-associated csRNAs (hepRNAs) are in the vicinity of RNA-binding proteins (RBPs). These observations led us to a model wherein cell-surface HS, RNA, and RBP form ternary complexes, validated by our spatio-selective RNA-protein crosslinking technology in a TLR7-orthogonal manner. We further revealed the identities of hepRNA and found that they can recruit the immune receptor killer cell immunoglobulin-like receptor 2DL5 (KIR2DL5), potentially enhancing receptor-ligand interactions. Employing human cell lines, our findings lay the groundwork for investigating how cell-surface ribonucleoproteins contribute to immune modulation.

Aggregation of RNA-binding proteins (RBPs) is a hallmark of several age-related neuromuscular diseases. However, our understanding of how these aggregates drive dysfunction is often limited by the use of non-disease-relevant models. Oculopharyngeal muscular dystrophy (OPMD) is caused by a short alanine expansion mutation in the PABPN1 gene, which leads to nuclear aggregation of the protein. To investigate how these aggregates impair muscle cell function, we developed a muscle cell model with inducible expression of the pathogenic PABPN1 (A16) variant and confirmed its relevance to OPMD. Using subcellular fractionation combined with mass spectrometry and RNA sequencing, we examined the molecular consequences of nuclear PABPN1 aggregation. In the cytoplasmic fraction, we observed significant impairments in cellular metabolism and biomechanics. In the nuclear fraction, RNA metabolism was broadly disrupted, and additional RBPs were significantly enriched in insoluble aggregates. Importantly, mRNAs trapped within the aggregates were associated with impaired nuclear export and decreased translation efficiency, and the pathogenic PABPN1 variant led to reduced endogenous PABPN1 levels. Our findings support a model in which OPMD pathology arises from reduced levels of soluble PABPN1 due to nuclear aggregation and establish a mechanistic link between RBP aggregation and muscle cell dysfunction, highlighting shared pathological pathways across neuromuscular and neurodegenerative diseases.

Clustering of type-II tumor necrosis factor receptors (TNFRs) is required to induce intracellular signaling. Current methods for receptor clustering lack precise control over ligand valency and spatial organization, potentially limiting optimal TNFR activation, biological insight, and therapeutic efficacy. DNA nanostructures provide nanometer-precise control over molecular arrangement, allowing control of both ligand spacing and valency. Here, we produce a DNA nanostructure decorated with controlled numbers of engineered single-chain TNF-related apoptosis-inducing ligand (sc-TRAIL) trimers, which bind death receptor 5 (DR5) with native affinity and geometry and enable investigation of the geometric parameters influencing apoptotic pathway activation. We show that cell killing is affected by receptor valency and separation and enhanced by superclustering sc-TRAIL trimers, which can induce cell killing in human primary pancreatic and colorectal cancer organoids. Together, our data show that control of receptor superclustering enhances our understanding of receptor activation mechanisms and informs the development of more effective cancer therapies.

Automation and improved hardware have greatly accelerated the rate of data generation in cryoET. As the field moves towards quantitative cryoET, the scale of the resulting datasets presents a significant challenge for analysis and interpretation. To explore ways of handling datasets comprising thousands of tomograms, we investigated a comprehensive segmentation strategy - assigning an ontology-based identity to every voxel in a dataset - that is based on the sequential application of multiple convolutional neural networks. Using an openly available dataset of over 1800 Chlamydomonas reinhardtii tomograms as a test case, we demonstrate the segmentation of 25 different subcellular features across the full dataset, while requiring only a few seconds of processing time per tomogram. We show how the approach enables the representation of large datasets as searchable databases and propose the usage of ontology-based segmentations for improving two common processing tasks in cryoET. First, we explore context-aware particle picking as a method to retain biological context when selecting particles for subtomogram averaging and other downstream analyses. Secondly, we demonstrate area-selective template matching, where we use segmentation-based masks to avoid redundant computations in template matching and enable >500-fold faster processing in specific cases. To illustrate the utility of the approach, all segmentation results have also been made available online via cryopom.streamlit.app. ### Competing Interest Statement The authors have declared no competing interest.

Intracellular protein aggregation is a hallmark of aging and contributes to pathology in some age-associated diseases. In hereditary adult-onset neuromuscular diseases (NMDs), protein aggregates play a key role in disease onset and progression. The wild-type Poly(A) binding protein nuclear 1 (PABPN1) forms benign nuclear aggregates, whereas a short trinucleotide expansion leads to the formation of pathogenic aggregates, a hallmark of Oculopharyngeal Muscular Dystrophy (OPMD). In OPMD, the mutant PABPN1 causes skeletal muscle weakness. So far, the structural differences between benign and pathogenic protein aggregates and their effects on muscle cell biology remain poorly understood. We employed an array of advanced imaging modalities to explore the morphological differences between nuclear aggregates formed by non-pathogenic and pathogenic PABPN1 variants. Through analyses spanning micro- to nanoscale, we identified distinct structural features of aggregates formed by wild-type and expanded PABPN1. We demonstrate that these differences were more pronounced in differentiated muscle cells compared to proliferating cells. We further linked the structural features of PABPN1 aggregates to muscle cell biology, namely alterations in mitochondrial function and proteasomal activity. Our findings provide new insights into the structural distinctions between pathogenic and non-pathogenic aggregates and their implications for cellular dysfunction in NMDs.

Monoclonal antibodies are important modalities in the treatment of cancer. Post-translational modifications of proteins, such as glycosylation, can affect the binding affinity of therapeutic antibodies. Whether other PTMs modulate therapeutic antibody binding to different surface proteins is currently underexplored. Pyroglutamation is the post-translational cyclization of an N-terminal glutamine or glutamic acid residue into a pyroglutamate by glutaminyl cyclase. In this study, we investigated the impact of pyroglutamation on the binding affinity of three therapeutic antibodies targeting CD47 and TRP1. Here, we show that pyroglutamation on CD47 and TRP1 modulates the binding of anti(α)-CD47 magrolimab and αTRP1 TA99 and flanvotumab. Furthermore, the N-terminal glutamine on CD47 is crucial for effective antibody recognition, while pyroglutamation of TRP1 is involved in trafficking to the cell surface. These findings highlight that the pyroglutamation by glutaminyl cyclase can modulate the binding affinity of antibodies with therapeutic potential.

Germline and somatic TP53 variants play a crucial role during tumorigenesis. However, genetic variations that solely affect the alternatively spliced p53 isoforms, p53 beta and p53 gamma, are not fully considered in the molecular diagnosis of Li-Fraumeni syndrome and cancer. In our search for additional cancer predisposing variants, we identify a heterozygous stop-lost variant affecting the p53 beta isoforms (p.*342Serext*17) in four families suspected of an autosomal dominant cancer syndrome with colorectal, breast and papillary thyroid cancers. The stop-lost variant leads to the 17 amino-acid extension of the p53 beta isoforms, which increases oligomerization to canonical p53 alpha and dysregulates the expression of p53's transcriptional targets. Our study reveals the capacity of p53 beta mutants to influence p53 signalling and contribute to the susceptibility of different cancer types. These findings underscore the significance of p53 isoforms and the necessity of comprehensive investigation into the entire TP53 gene in understanding cancer predisposition. Pathogenic germline variants in TP53 predispose to a variety of cancers, but variants solely affecting alternatively spliced isoforms of TP53 are understudied. Here, the authors identify a heterozygous stop-lost variant that specifically affects p53 beta isoforms and predisposes to familial cancer using germline whole-exome sequencing and functional genomics assays.

Structure-inspired peptide design and RaPID selection identifies a macrocyclic peptide with immunotherapeutic potential, and inspires development of complement-modulating drugs.

Complement activation protects against infection but also contributes to pathological mechanisms in a range of clinical conditions such as autoimmune diseases and transplant rejection. Complement-inhibitory drugs, either approved or in development, usually act systemically, thereby increasing the risk for infections. We therefore envisioned a novel class of bispecific antibodies (bsAbs) which are capable of site-directed complement inhibition by bringing endogenous complement regulators in the vicinity of defined cell surface antigens. Here, we analyzed a comprehensive set of obligate bsAbs designed to crosslink a specific target with either complement regulator factor H (FH) or C4b-binding protein (C4BP). The bsAbs were assessed for their capacity to inhibit complement activation and cell lysis in an antigen-targeted manner. We observed that the bsAbs inhibited classical, lectin, and alternative pathway complement activation in which sufficient endogenous serum FH and C4BP could be recruited to achieve local inhibition. Importantly, the bsAbs effectively protected antigen-positive liposomes, erythrocytes, and human leukocytes from complement-mediated lysis. In conclusion, localized complement inhibition by bsAbs capable of recruiting endogenous human complement regulators (such as FH or C4BP) to cell surfaces potentially provides a novel therapeutic approach for the targeted treatment of complement-mediated diseases.

The classical complement pathway is activated by antigen-bound IgG antibodies. Monomeric IgG must oligomerize to activate complement via the hexameric C1q complex, and hexamerizing mutants of IgG appear as promising therapeutic candidates. However, structural data have shown that it is not necessary to bind all six C1q arms to initiate complement, revealing a symmetry mismatch between C1 and the hexameric IgG complex that has not been adequately explained. Here, we use DNA nanotechnology to produce specific nanostructures to template antigens and thereby spatially control IgG valency. These DNA-nanotemplated IgG complexes can activate complement on cell-mimetic lipid membranes, which enabled us to determine the effect of IgG valency on complement activation without the requirement to mutate antibodies. We investigated this using biophysical assays together with 3D cryo-electron tomography. Our data revealed the importance of interantigen distance on antibody-mediated complement activation, and that the cleavage of complement component C4 by the C1 complex is proportional to the number of ideally spaced antigens. Increased IgG valency also translated to better terminal pathway activation and membrane attack complex formation. Together, these data provide insights into how nanopatterning antigen-antibody complexes influence the activation of the C1 complex and suggest routes to modulate complement activation by antibody engineering. Furthermore, to our knowledge, this is the first time DNA nanotechnology has been used to study the activation of the complement system.

Background: Numerous gut microbial metabolites including phenylacetylglutamine (PAGln) are associated with increased cardiovascular disease risk and mortality. However, little is known about the cellular-specific perturbations by which PAGln may drive cardiovascular dysfunction. Aims: To understand if elevated PAGln leads to cardiovascular dysfunction and what direct cardiac- and vascular-specific pathophysiology is occurring. Methods: Herein, we subject C57Bl/6N male mice to pharmacological increases in circulating PAGln (50mg/kg) or vehicle twice daily for 20 days. We performed echocardiography, invasive hemodynamics, and vascular reactivity assays. We also performed ex vivo cardiomyocyte cell physiology experiments in the presence or absence of PAGln. Moreover, to understand vascular effects we subjected endothelial cells to PAGln and looked at expression of proinflammatory and cell adhesion molecules. Results: We first performed LC-MS/MS analysis demonstrating significant elevation in PAGln levels in multiple tissues compared to vehicle. There was no significant alteration in cardiac structure and function through echocardiographic analysis. Invasive hemodynamic assessment of systemic blood pressure showed no change, however left ventricular filling pressures were significantly elevated in PAGln group. Aortic rings subjected to varying concentrations of acetylcholine in the PAGln-treated animals were significantly impaired compared to vehicle controls. Given the elevated filling pressures and aberrant vascular endothelial relaxation we measured nitrite levels. Nitrite was significantly reduced in the plasma, cardiac, and liver tissue in PAGln group. Cardiomyocyte fractional shortening was significantly increased compared to baseline and equivalent to b-adrenergic agonism. PAGln hypercontractility was not inhibited with adrenergic antagonism. Vascular endothelial cells which were exposed to PAGln had an increase in expression of proinflammatory and cell adhesion molecules versus vehicle. Conclusion: Our data demonstrates that PAGln drives cardiovascular dysfunction through cardiomyocyte hypercontractility and inducing vascular coronary endothelial cell activation in vitro .

Background: Heart failure with preserved ejection fraction (HFpEF) is among the leading causes of cardiovascular mortality and morbidity. Recent reports suggest excessive myocardial protein S-nitrosylation (-SNO), a hallmark of nitrosative stress, contributes to HFpEF pathophysiology. However, the role of transnitrosylases, enzymes that induce protein-SNO, or denitrosylases, enzymes that eliminate protein-SNO, have not been investigated in the context of HFpEF. Research Questions and Goals: We sought to investigate the regulation of protein nitrosylation dynamics in a rat model of cardiometabolic HFpEF. Methods: Echocardiographic assessment, invasive hemodynamic measurements, and exercise testing were conducted in WKY or ZSF1 obese (Ob) rats to evaluate HFpEF severity. Nitric oxide (NO) bioavailability and protein-SNO levels were determined in the heart tissue. Endothelial-dependent vascular reactivity was assayed to further evaluate NO signaling. In parallel, single-cell (sc) RNA sequencing was performed to determine the transcriptional changes in trans- and denitrosylases, as well as NO synthases. Results: We observed progressive deterioration in LV diastolic function (significantly elevated E/e’ and LV end-diastolic pressure) and exercise performance in ZSF1 Ob when compared to WKY controls. Notably, ZSF1 Ob rats exhibited an age-dependent elevation in protein-SNO levels, despite significant reduction in NO bioavailability and signaling. Targeted sc transcriptomic analysis revealed significantly elevated expression of transnitrosylases such as hemoglobin subunit α and β in cardiomyocytes, endothelial cells, and cardiac fibroblasts from ZSF1 Ob hearts. In contrast, significantly reduced expression of denitrosylases such as thioredoxin 2 was found in the ZSF1 Ob cardiomyocytes. No change was observed in expression of NO synthases (nNOS, iNOS, eNOS). Conclusion: Herein, we demonstrate a profound disconnect between insufficient NO bioavailability and nitrosative stress in ZSF1 Ob rat model of HFpEF. Our data suggests the pathological accumulation of nitroso - proteins, may be attributed in part to the derangement of transnitrosylases and denitrosylases expression. Restoration of physiological protein nitrosylation dynamics may represent a novel therapeutic approach for HFpEF, and warrants further investigation.

Ageing has a major adverse impact on maintaining cellular proteostasis and age-related dysregulation leads to an increase in protein aggregation. Equivalently, the accumulation of aggregated proteins accelerates proteostasis impairment. Accumulation of protein aggregates and impaired proteostasis are hallmarks of ageing-associated neuromuscular disorders and tissue degeneration is predominantly in post-mitotic muscle and neuronal cells. A short alanine expansion mutation in the Poly(A) binding protein nuclear 1 (PABPN1) causes Oculopharyngeal muscular dystrophy (OPMD), a rare age-associated protein aggregation myopathy. PABPN1 is a vital RNA-binding protein but OPMD pathology is limited to skeletal muscles connected to nuclear aggregates. In contrast to the mutant PABPN1, the wild-type PABPN1 forms age-associated non-pathogenic aggregates. We generated an inducible muscle cell models for mutant and wild-type PABPN1 protein aggregation. By combining four different, but complementary, imaging modalities, covering micro- to nanoscale resolutions, we were able to characterise differences in structure and dynamics between pathogenic and non-pathogenic PABPN1 aggregates in differentiated muscle cells. These data allowed us to correlate the structure of aggregates to cellular function, providing important insights into how aggregates lead to cell dysfunction in post-mitotic cells. ![Figure][1]</img> ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes

Correlated super-resolution fluorescence microscopy and cryo-electron microscopy enables imaging with both high labeling specificity and high resolution. Naturally, combining two sophisticated imaging techniques within one workflow also introduces new requirements on hardware, such as the need for a super-resolution fluorescence capable microscope that can be used to image cryogenic samples. In this chapter, we describe the design and use of the "cryoscope"; a microscope designed for single-molecule localization microscopy (SMLM) of cryoEM samples that fits right into established cryoEM workflows. We demonstrate the results that can be achieved with our microscope by imaging fluorescently labeled vimentin, an intermediate filament, within U2OS cells grown on EM grids, and we provide detailed 3d models that encompass the entire design of the microscope.

Recent discoveries have shown the presence of RNA molecules on the cell surface, defying the traditional view that RNA only functions intracellularly. However, it is not well understood how cell-surface RNA (csRNA) is stably present on the plasma membrane and what functions it performs on the cell surface. By exploiting the RNA-sensing ability of TLR7 as a specific recombinant probe to detect csRNA and coupling it with a genome-wide CRISPR-Cas9-knockout screening to identify genes essential for csRNA presentation on cells, we identified heparan sulfate (HS) as a crucial factor for RNA presentation on cells. Using the TLR7 binding probe, cell surface proximity labelling revealed that csRNA associates mechanistically with a plethora of RNA-binding proteins, and these interactions are crucial for csRNA presentation. Moreover, csRNA modulates receptor-ligand interactions between poliovirus receptor (PVR) and killer cell immunoglobulin-like receptor 2DL5 (KIR2DL5) by acting as a co-binder, recruiting the latter to cell surface. We provide a mechanistic understanding of csRNA presentation and unveil a new layer of complexity in the csRNA-dictated regulation of cell surface receptor-ligand interactions. ### Competing Interest Statement The authors have declared no competing interest.