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Stress granules (SGs) are large ribonucleoprotein assemblies that form in response to acute stress in eukaryotes. SG formation is thought to be initiated by liquid-liquid phase separation (LLPS) of key proteins and RNA. These molecules serve as a scaffold for recruitment of client molecules. LLPS of scaffold proteins in vitro is highly concentration-dependent, yet biomolecular condensates in vivo contain hundreds of unique proteins, most of which are thought to be clients rather than scaffolds. Many proteins that localize to SGs contain low-complexity, prion-like domains (PrLDs) that have been implicated in LLPS and SG recruitment. The degree of enrichment of proteins in biomolecular condensates such as SGs can vary widely, but the underlying basis for these differences is not fully understood. Here, we develop a toolkit of model PrLDs to examine the factors that govern efficiency of PrLD recruitment to stress granules. Recruitment was highly sensitive to amino acid composition: enrichment in SGs could be tuned through subtle changes in hydrophobicity. By contrast, SG recruitment was largely insensitive to PrLD concentration at both a population level and single-cell level. These observations point to a model wherein PrLDs are enriched in SGs through either simple solvation effects or interactions that are effectively non-saturable even at high expression levels., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Coiled-coil domain-containing 124 protein is a multifunctional RNA-binding factor, and it was previously reported to interact with various biomolecular complexes localized at diverse subcellular locations, such as the ribosome, centrosome, midbody, and nucleoli. We aimed to better characterize the subcellular CCDC124 translocation by labelling this protein with a fluorescent tag, followed by laser scanning confocal microscopy methods. As traditional GFP-tagging of small proteins such as CCDC124 often faces limitations like potential structural perturbations of labeled proteins, and interference of the fluorescent-tag with their endogenous cellular functions, we aimed to label CCDC124 with the smallest possible split-GFP associated protein-tagging system (GFP11/GFP1-10) for better characterization of its subcellular localizations and its translocation dynamics. By recombinant DNA techniques we generated CCDC124-constructs labelled with either single of four tandem copies of GFP11 (GFP11 ×  1 ::CCDC124, GFP11 ×  4 ::CCDC124, or CCDC124::GFP11 ×  4 ). We then cotransfected U2OS cells with these split-GFP constructs (GFP11 ×  1(or X4) ::CCDC124/GFP1-10) and analyzed subcellular localization of CCDC124 protein by laser scanning confocal microscopy. Tagging CCDC124 with four tandem copies of a 16-amino acid short GFP-derived peptide-tag (GFP11  × 4 ::CCDC124) allowed better characterization of the subcellular localization of CCDC124 protein in our model human bone osteosarcoma (U2OS) cells. Thus, by this novel methodology we successfully identified GFP11 ×  4 ::CCDC124 molecules in G3BP1-overexpression induced stress-granules by live cell protein imaging for the first time. Our findings propose CCDC124 as a novel component of the stress granule which is a membraneless organelle involved in translational shut-down in response to cellular stress., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Stress granules (SGs) are conserved cytoplasmic biomolecular condensates mainly formed by proteins and RNA molecules assembled by liquid-liquid phase separation. Isolation of SGs components has been a major challenge in the field due to the dynamic and transient nature of stress granule shells. Here, we describe the methodology for the isolation and visualization of SGs proteins from Arabidopsis thaliana plants using a scaffold component as the target. The protocol consists of the first immunoprecipitation of GFP-tagged scaffold protein, followed by an on-beads enzymatic digestion and previous mass spectrometry identification. Finally, the localization of selected SGs candidates is visualized in Nicotiana benthamiana mesophyll protoplasts., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cells respond to stress by blocking translation, rewiring metabolism and forming transient messenger ribonucleoprotein assemblies called stress granules (SGs). After stress release, re-establishing homeostasis and disassembling SGs requires ATP-consuming processes. However, the molecular mechanisms whereby cells restore ATP production and disassemble SGs after stress remain poorly understood. Here we show that upon stress, the ATP-producing enzyme Cdc19 forms inactive amyloids, and that their rapid re-solubilization is essential to restore ATP production and disassemble SGs in glucose-containing media. Cdc19 re-solubilization is initiated by the glycolytic metabolite fructose-1,6-bisphosphate, which directly binds Cdc19 amyloids, allowing Hsp104 and Ssa2 chaperone recruitment and aggregate re-solubilization. Fructose-1,6-bisphosphate then promotes Cdc19 tetramerization, which boosts its activity to further enhance ATP production and SG disassembly. Together, these results describe a molecular mechanism that is critical for stress recovery and directly couples cellular metabolism with SG dynamics via the regulation of reversible Cdc19 amyloids., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Mutations in the RNA binding protein, Fused in Sarcoma (FUS), lead to amyotrophic lateral sclerosis (ALS), the most frequent form of motor neuron disease. Cytoplasmic aggregation and defective DNA repair machinery are etiologically linked to mutant FUS-associated ALS. Although FUS is involved in numerous aspects of RNA processing, little is understood about the pathophysiological mechanisms of mutant FUS. Here, we employed RNA-sequencing technology in Drosophila brains expressing FUS to identify significantly altered genes and pathways involved in FUS-mediated neurodegeneration. We observed the expression levels of DEAD-Box Helicase 17 (DDX17) to be significantly downregulated in response to mutant FUS in Drosophila and human cell lines. Mutant FUS recruits nuclear DDX17 into cytoplasmic stress granules and physically interacts with DDX17 through the RGG1 domain of FUS. Ectopic expression of DDX17 reduces cytoplasmic mislocalization and sequestration of mutant FUS into cytoplasmic stress granules. We identified DDX17 as a novel regulator of the DNA damage response pathway whose upregulation repairs defective DNA damage repair machinery caused by mutant neuronal FUS ALS. In addition, we show DDX17 is a novel modifier of FUS-mediated neurodegeneration in vivo. Our findings indicate DDX17 is downregulated in response to mutant FUS, and restoration of DDX17 levels suppresses FUS-mediated neuropathogenesis and toxicity in vivo., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

RNA helicase A/DHX9 is required for diverse RNA-related essential cellular functions and antiviral responses and is hijacked by RNA viruses to support their replication. Here, we show that during the late replication stage in human cancer cells of myxoma virus (MYXV), a member of the double-stranded DNA (dsDNA) poxvirus family that is being developed as an oncolytic virus, DHX9, forms unique granular cytoplasmic structures, which we named "DHX9 antiviral granules." These DHX9 antiviral granules are not formed if MYXV DNA replication and/or late protein synthesis is blocked. When formed, DHX9 antiviral granules significantly reduced nascent protein synthesis in the MYXV-infected cancer cells. MYXV late gene transcription and translation were also significantly compromised, particularly in nonpermissive or semipermissive human cancer cells where MYXV replication is partly or completely restricted. Directed knockdown of DHX9 significantly enhanced viral late protein synthesis and progeny virus formation in normally restrictive cancer cells. We further demonstrate that DHX9 is not a component of the canonical cellular stress granules. DHX9 antiviral granules are induced by MYXV, and other poxviruses, in human cells and are associated with other known cellular components of stress granules, dsRNA and virus encoded dsRNA-binding protein M029, a known interactor with DHX9. Thus, DHX9 antiviral granules function by hijacking poxviral elements needed for the cytoplasmic viral replication factories. These results demonstrate a novel antiviral function for DHX9 that is recruited from the nucleus into the cytoplasm, and this step can be exploited to enhance oncolytic virotherapy against the subset of human cancer cells that normally restrict MYXV. IMPORTANCE The cellular DHX9 has both proviral and antiviral roles against diverse RNA and DNA viruses. In this article, we demonstrate that DHX9 can form unique antiviral granules in the cytoplasm during myxoma virus (MYXV) replication in human cancer cells. These antiviral granules sequester viral proteins and reduce viral late protein synthesis and thus regulate MYXV, and other poxviruses, that replicate in the cytoplasm. In addition, we show that in the absence of DHX9, the formation of DHX9 antiviral granules can be inhibited, which significantly enhanced oncolytic MYXV replication in human cancer cell lines where the virus is normally restricted. Our results also show that DHX9 antiviral granules are formed after viral infection but not by common nonviral cellular stress inducers. Thus, our study suggests that DHX9 has antiviral activity in human cancer cells, and this pathway can be targeted for enhanced activity of oncolytic poxviruses against even restrictive cancer cells.

Germ granules are protein-RNA condensates that segregate with the embryonic germline. In Caenorhabditis elegans embryos, germ (P) granule assembly requires MEG-3, an intrinsically disordered protein that forms RNA-rich condensates on the surface of PGL condensates at the core of P granules. MEG-3 is related to the GCNA family and contains an N-terminal disordered region (IDR) and a predicted ordered C-terminus featuring an HMG-like motif (HMGL). We find that MEG-3 is a modular protein that uses its IDR to bind RNA and its C-terminus to drive condensation. The HMGL motif mediates binding to PGL-3 and is required for co-assembly of MEG-3 and PGL-3 condensates in vivo. Mutations in HMGL cause MEG-3 and PGL-3 to form separate condensates that no longer co-segregate to the germline or recruit RNA. Our findings highlight the importance of protein-based condensation mechanisms and condensate-condensate interactions in the assembly of RNA-rich germ granules., Competing Interests: HS, AP, DR No competing interests declared, GS serves on the Scientific Advisory Board of Dewpoint Therapeutics, Inc, (© 2021, Schmidt et al.)

Knee osteoarthritis (KOA) represents a clinical challenge due to poor potential for spontaneous healing of cartilage lesions. Several treatment options are available for KOA, including oral nonsteroidal anti-inflammatory drugs, physical therapy, braces, activity modification, and finally operative treatment. Intra-articular (IA) injections are usually used when the non-operative treatment is not effective, and when the surgery is not yet indicated. More and more studies suggesting that IA injections are as or even more efficient and safe than NSAIDs. Recently, research to improve intra-articular homeostasis has focused on biologic adjuncts, such as platelet-rich plasma (PRP). The catabolic and inflammatory intra-articular processes that exists in knee osteoarthritis (KOA) may be influenced by the administration of PRP and its derivatives. PRP can induce a regenerative response and lead to the improvement of metabolic functions of damaged structures. However, the positive effect on chondrogenesis and proliferation of mesenchymal stem cells (MSC) is still highly controversial. Recommendations from in vitro and animal research often lead to different clinical outcomes because it is difficult to translate non-clinical study outcomes and methodology recommendations to human clinical treatment protocols. In recent years, significant progress has been made in understanding the mechanism of PRP action. In this review, we will discuss mechanisms related to inflammation and chondrogenesis in cartilage repair and regenerative processes after PRP administration in in vitro and animal studies. Furthermore, we review clinical trials of PRP efficiency in changing the OA biomarkers in knee joint.

Cells organize biochemical processes into biological condensates. P-bodies are cytoplasmic condensates that are enriched in enzymes important for mRNA degradation and have been identified as sites of both storage and decay. How these opposing outcomes can be achieved in condensates remains unresolved. mRNA decapping immediately precedes degradation, and the Dcp1/Dcp2 decapping complex is enriched in P-bodies. Here, we show that Dcp1/Dcp2 activity is modulated in condensates and depends on the interactions promoting phase separation. We find that Dcp1/Dcp2 phase separation stabilizes an inactive conformation in Dcp2 to inhibit decapping. The activator Edc3 causes a conformational change in Dcp2 and rewires the protein-protein interactions to stimulate decapping in condensates. Disruption of the inactive conformation dysregulates decapping in condensates. Our results indicate that the regulation of enzymatic activity in condensates relies on a coupling across length scales ranging from microns to ångstroms. We propose that this regulatory mechanism may control the functional state of P-bodies and related phase-separated compartments.

Lysosome-related organelles (LROs) are endosomal compartments carrying tissue-specific proteins, which become enlarged in Chediak-Higashi syndrome (CHS) due to mutations in LYST. Here, we show that Drosophila Mauve, a counterpart of LYST, suppresses vesicle fusion events with lipid droplets (LDs) during the formation of yolk granules (YGs), the LROs of the syncytial embryo, and opposes Rab5, which promotes fusion. Mauve localizes on YGs and at spindle poles, and it co-immunoprecipitates with the LDs' component and microtubule-associated protein Minispindles/Ch-TOG. Minispindles levels are increased at the enlarged YGs and diminished around centrosomes in mauve-derived mutant embryos. This leads to decreased microtubule nucleation from centrosomes, a defect that can be rescued by dominant-negative Rab5. Together, this reveals an unanticipated link between endosomal vesicles and centrosomes. These findings establish Mauve/LYST's role in regulating LRO formation and centrosome behavior, a role that could account for the enlarged LROs and centrosome positioning defects at the immune synapse of CHS patients., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The organelle-like structures of Xanthomonas citri, a bacterial pathogen that causes citrus canker, were investigated using an analytical transmission electron microscope. After high-pressure freezing, the bacteria were then freeze-substituted for imaging and element analysis. Miniscule electron-dense structures of varying shapes without a membrane enclosure were frequently observed near the cell poles in a 3-day culture. The bacteria formed cytoplasmic electron-dense spherical structures measuring approximately 50 nm in diameter. Furthermore, X. citri produced electron-dense or translucent ellipsoidal intracellular or extracellular granules. Single- or double-membrane-bound vesicles, including outer-inner membrane vesicles, were observed both inside and outside the cells. Most cells had been lysed in the 3-week X. citri culture, but they harbored one or two electron-dense spherical structures. Contrast-inverted scanning transmission electron microscopy images revealed distinct white spherical structures within the cytoplasm of X. citri. Likewise, energy-dispersive X-ray spectrometry showed the spatial heterogeneity and co-localization of phosphorus, oxygen, calcium, and iron only in the cytoplasmic electron-dense spherical structures, thus corroborating the nature of polyphosphate granules., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Many of the genes whose mutation causes Amyotrophic Lateral Sclerosis (ALS) are RNA-binding proteins which localize to stress granules, while others impact the assembly, stability, and elimination of stress granules. This has led to the hypothesis that alterations in the dynamics of stress granules and RNA biology cause ALS. Genetic mutations in Superoxide Dismutase 1 (SOD1) also cause ALS. Evidence demonstrates that SOD1 harboring ALS-linked mutations is recruited to stress granules, induces changes in alternative splicing, and could be an RNA-binding protein. Whether SOD1 inclusions contain RNA in disease models and whether SOD1 directly binds RNA remains uncertain. We applied methods including cross-linking immunoprecipitation and in vitro gel shift assays to detect binding of SOD1 to RNA in vitro, in cells with and without stress granules, and in mice expressing human SOD1 G93A. We find that SOD1 localizes to RNA-rich structures including stress granules, and SOD1 inclusions in mice contain mRNA. However, we find no evidence that SOD1 directly binds RNA. This suggests that SOD1 may impact stress granules, alternative splicing and RNA biology without binding directly to RNA., (© 2020 International Society for Neurochemistry.)

Background: Allogeneic platelet (PLT) infusion is a strategy to raise Factor V (FV) levels in patients with congenital FV deficiency. However, since FV is labile in vitro, we hypothesized that FV activity could be low in PLT units., Study Design and Methods: FV activity was tested using a prothrombin time-based platform in the supernatant and platelet lysate (PL) of apheresis PLT units (16 units stored in PLT additive solution with acetate and phosphate [PAS-C] and 10 units stored in plasma only), on post-collection days 3-6. Statistical analysis was performed using Student's t test (P < .05)., Results: FV activity was severely diminished in PAS-C PLTs (N = 16) supernatant (3.70% ± 1.02%) and PL (3.26% ± 1.02%). FV activity in plasma-only PLTs (N = 10) was lower in both supernatant (44.55% ± 6.46%) and lysate (39.67% ± 6.33%) relative to normal plasma levels, but both were significantly higher (P < .0001) compared to PAS-C PLTs. In a separate set of experiments, FV activity in PAS-C PLTs examined serially over storage time (N = 3 for these experiments) showed that FV levels were reduced by day 3 and not significantly different by day 5 of storage (Day 3 supernatant 5.03% ± 1.41%; Day 5 supernatant: 3.10% ± 0.57%; P = .2; Day 3 lysate: 3.89% ± 1.03%; Day 5 lysate: 2.61% ± 0.41%; P = .4)., Conclusion: Plasma should be considered over PLTs as first-line therapy for non-complex FV deficiency-associated hemorrhage. If PLTs are considered for transfusion, plasma-only PLT units should be preferentially utilized, as PAS-C PLT have near-absent FV activity., (© 2020 AABB.)

Eosinophils are important for tissue homeostasis and host responses to pathogens and allergens. The impact of eosinophils within tissues depends in part on whether cytotoxic proteins in crystalloid granules are released. Determinants of eosinophil motility and loss of granule contents are incompletely understood. The goal of this chapter is to present methods to study the effects of potential mediators on purified human blood eosinophils interacting with adhesive proteins found in extracellular matrix. We show that differential interference contrast video-enhanced microscopy and a bead-clearing assay provide complementary information about how different mediator-adhesive protein combinations direct eosinophil motility and granule fate. The former method is rich in information about cell shape, pattern of movement, and state of granules whereas the latter method lends itself to quantification and interrogation of multiple conditions in replicate.

Protein quality control is maintained by a number of integrated cellular pathways that monitor the folding and functionality of the cellular proteome. Defects in these pathways lead to the accumulation of misfolded or faulty proteins that may become insoluble and aggregate over time. Protein aggregates significantly contribute to the development of a number of human diseases such as amyotrophic lateral sclerosis, Huntington's disease, and Alzheimer's disease. In vitro, imaging-based, cellular studies have defined key biomolecular components that recognize and clear aggregates; however, no unifying method is available to quantify cellular aggregates, limiting our ability to reproducibly and accurately quantify these structures. Here we describe an ImageJ macro called AggreCount to identify and measure protein aggregates in cells. AggreCount is designed to be intuitive, easy to use, and customizable for different types of aggregates observed in cells. Minimal experience in coding is required to utilize the script. Based on a user-defined image, AggreCount will report a number of metrics: (i) total number of cellular aggregates, (ii) percentage of cells with aggregates, (iii) aggregates per cell, (iv) area of aggregates, and (v) localization of aggregates (cytosol, perinuclear, or nuclear). A data table of aggregate information on a per cell basis, as well as a summary table, is provided for further data analysis. We demonstrate the versatility of AggreCount by analyzing a number of different cellular aggregates including aggresomes, stress granules, and inclusion bodies caused by huntingtin polyglutamine expansion., (Copyright © 2020 © 2020 Klickstein et al. Published by Elsevier Inc. All rights reserved.)

Biomolecular condensates play a key role in organizing cellular fluids such as the cytoplasm and nucleoplasm. Most of these non-membranous organelles show liquid-like properties both in cells and when studied in vitro through liquid-liquid phase separation (LLPS) of purified proteins. In general, LLPS of proteins is known to be sensitive to variations in pH, temperature and ionic strength, but the role of crowding remains underappreciated. Several decades of research have shown that macromolecular crowding can have profound effects on protein interactions, folding and aggregation, and it must, by extension, also impact LLPS. However, the precise role of crowding in LLPS is far from trivial, as most condensate components have a disordered nature and exhibit multiple weak attractive interactions. Here, we discuss which factors determine the scope of LLPS in crowded environments, and we review the evidence for the impact of macromolecular crowding on phase boundaries, partitioning behavior and condensate properties. Based on a comparison of both in vivo and in vitro LLPS studies, we propose that phase separation in cells does not solely rely on attractive interactions, but shows important similarities to segregative phase separation.

To date, the recently discovered SARS-CoV-2 virus has afflicted >6.9 million people worldwide and disrupted the global economy. Development of effective vaccines or treatments for SARS-CoV-2 infection will be aided by a molecular-level understanding of SARS-CoV-2 proteins and their interactions with host cell proteins. The SARS-CoV-2 nucleocapsid (N) protein is highly homologous to the N protein of SARS-CoV, which is essential for viral RNA replication and packaging into new virions. Emerging models indicate that nucleocapsid proteins of other viruses can form biomolecular condensates to spatiotemporally regulate N protein localization and function. Our bioinformatic analyses, in combination with pre-existing experimental evidence, suggest that the SARS-CoV-2 N protein is capable of forming or regulating biomolecular condensates in vivo by interaction with RNA and key host cell proteins. We discuss multiple models, whereby the N protein of SARS-CoV-2 may harness this activity to regulate viral life cycle and host cell response to viral infection., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Recent advances in microscopy of living cells have established membraneless organelles as critical elements of diverse biological processes. The body of experimental work suggests that formation of such organelles is driven by liquid-liquid phase separation, a physical process that has been studied extensively for both simple liquids and mixtures of polymers. Here, we combine molecular dynamics simulations with polymer theory to show that the thermodynamic behavior of one particular biomolecular condensate-fused in sarcoma (FUS)-can be quantitatively accounted for at the level of the chain collapse theory. First, we show that a particle-based molecular dynamics model can reproduce known phase separation properties of a FUS condensate, including its critical concentration and susceptibility to mutations. Next, we obtain a polymer physics representation of a FUS condensate by examining the behavior of a single FUS protein as a function of temperature. We use the chain collapse theory to determine the thermodynamic properties of the condensate and to characterize changes in the single-chain conformation at the onset of phase separation. Altogether, our findings suggest that the phase behavior of FUS condensates can be explained by the properties of individual FUS proteins and that the change in the FUS conformation is the main force driving for the phase separation.

Intracellular bodies such as nucleoli, Cajal bodies and various signalling assemblies represent membraneless organelles, or condensates, that form via liquid-liquid phase separation (LLPS) 1,2 . Biomolecular interactions-particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions-are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems 3-6 have led to the concept that a single fixed saturation concentration is a defining feature of endogenous LLPS 7-9 , and has been suggested as a mechanism for intracellular concentration buffering 2,7,8,10 . However, the assumption of a fixed saturation concentration remains largely untested within living cells, in which the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed saturation concentration. As the concentration of individual components is varied, their partition coefficients change in a manner that can be used to determine the thermodynamic free energies that underlie LLPS. We find that heterotypic interactions among protein and RNA components stabilize various archetypal intracellular condensates-including the nucleolus, Cajal bodies, stress granules and P-bodies-implying that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA-processing condensates such as the nucleolus, this manifests in the selective exclusion of fully assembled ribonucleoprotein complexes, providing a thermodynamic basis for vectorial ribosomal RNA flux out of the nucleolus. This methodology is conceptually straightforward and readily implemented, and can be broadly used to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deeper understanding of the thermodynamics of multicomponent intracellular phase behaviour and its interplay with the nonequilibrium activity that is characteristic of endogenous condensates.

Transfection is a powerful tool that enables introducing foreign nucleic acids into living cells in order to study the function of a gene product. Ever since the discovery of transfection many side effects or artifacts caused by transfection reagents have been reported. Here, we show that the transfection reagent, JetPRIME alters the localization of the splicing protein SC35 widely used as a nuclear speckle marker. We demonstrate that transfection of plasmids with JetPRIME leads to enlarged SC35 speckles and SC35 cytoplasmic granules. By contrast, transfection of the same plasmid with Lipofectamine 3000 does not have any effect on SC35 localization. The formation of SC35 cytoplasmic granules by JetPRIME-mediated transfection is independent of exogenous expression by plasmid and although similar in morphology they are distinct from P-bodies and stress granules. This method of transfection affected only SC35 and phosphorylated SR proteins but not the nuclear speckles. The JetPRIME-mediated transfection also showed compromised transcription in cells with enlarged SC35 speckles. Our work indicates that the use of JetPRIME alters SC35 localization and can affect gene expression and alternative splicing. Therefore, caution should be exercised when interpreting results after the use of a transient transfection system, particularly when the subject of the study is the function of a protein in the control of gene expression or mRNA splicing., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Previous studies demonstrated spontaneous type 2 airway inflammation with eosinophilia in juvenile Scnn1b (sodium channel, non-voltage-gated 1, β-subunit)-transgenic ( Scnn1b -Tg) mice with muco-obstructive lung disease. IL-1 receptor (IL-1R) signaling has been implicated in allergen-driven airway disease; however, its role in eosinophilic inflammation in muco-obstructive lung disease remains unknown. In this study, we examined the role of IL-1R signaling in the development of airway eosinophilia and type 2 inflammation in juvenile Scnn1b -Tg mice. We determined effects of genetic deletion of Il1r1 (IL-1 receptor type I) on eosinophil counts, transcript levels of key type 2 cytokines, markers of eosinophil activation and apoptosis, and tissue morphology in lungs of Scnn1b -Tg mice at different time points during neonatal development. Furthermore, we measured endothelial surface expression of intercellular adhesion molecule 1 (ICAM-1), an integrin involved in eosinophil transendothelial migration, and determined effects of eosinophil depletion using an anti-IL-5 antibody on lung morphology. Lack of IL-1R reduced airway eosinophilia and structural lung damage, but it did not reduce concentrations of type 2 cytokines and associated eosinophil activation in Scnn1b -Tg mice. Structural lung damage in Scnn1b -Tg mice was also reduced by eosinophil depletion. Lack of IL-1R was associated with reduced expression of ICAM-1 on lung endothelial cells and reduced eosinophil counts in lungs from Scnn1b -Tg mice. We conclude that IL-1R signaling is implicated in airway eosinophilia independent of type 2 cytokines in juvenile Scnn1b -Tg mice. Our data suggest that IL-1R signaling may be relevant in the pathogenesis of eosinophilic airway inflammation in muco-obstructive lung diseases, which may be mediated in part by ICAM-1-dependent transmigration of eosinophils into the lungs.

Red spherule cells (RSCs) are considered one of the prime immune cells of sea urchins, but their detailed biological role during immune responses is not well elucidated. Lack of pure populations accounts for one of the major challenges of studying these cells. In this study, we have demonstrated that live RSCs exhibit strong, multi-colour autofluorescence distinct from other coelomocytes, and with the help of fluorescence-activated cell sorting (FACS), a pure population of live RSCs was successfully separated from other coelomocytes in the green sea urchin, Strongylocentrotus droebachiensis. This newly developed RSCs isolation method has allowed profiling of the naphthoquinone content in these cells. With the use of ultra high-performance liquid chromatography, UV absorption spectra, and high-resolution tandem mass spectrometry, it was possible to identify sulphated derivatives of spinochrome C, D, E and spinochrome dimers, which suggests that the RSCs may play an important biological role in the biogenesis of naphthoquinone compounds and regulating their bioactivity.

Staining cells or tissues with basic dyes was the mainstay of mast cell and basophil detection methods for more than a century following the first identification of these cell types using such methods. These techniques have now been largely supplanted by immunohistochemical procedures with monoclonal antibodies directed against unique constituents of these cell types. Immunohistochemistry with antibodies specific for the granule protease tryptase provides a more sensitive and discriminating means for detecting mast cells than using the classical histochemical procedures, and using antibodies specific for products of basophils (2D7 antigen and basogranulin) has allowed detection of basophils that infiltrate into tissues. The application of immunohistochemistry to detect more than one marker in the same cell has underpinned concepts of mast cell heterogeneity based on differential expression of chymase and other proteases. The double labeling procedures employed have also provided a means for investigating the expression of cytokines and a range of other products. Protocols are here set out that have been used for immunohistochemical detection of mast cells and basophils and their subpopulations in human tissues. Consideration is given to pitfalls to avoid and to a range of alternative approaches.

The intracellular environment is partitioned into functionally distinct compartments containing specific sets of molecules and reactions. Biomolecular condensates, also referred to as membrane-less organelles, are diverse and abundant cellular compartments that lack membranous enclosures. Molecules assemble into condensates by phase separation; multivalent weak interactions drive molecules to separate from their surroundings and concentrate in discrete locations. Biomolecular condensates exist in all eukaryotes and in some prokaryotes, and participate in various essential house-keeping, stress-response and cell type-specific processes. An increasing number of recent studies link abnormal condensate formation, composition and material properties to a number of disease states. In this review, we discuss current knowledge and models describing the regulation of condensates and how they become dysregulated in neurodegeneration and cancer. Further research on the regulation of biomolecular phase separation will help us to better understand their role in cell physiology and disease., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Electrical activity in neurons and other excitable cells is a result of complex interactions between the system of ion channels, involving both global coupling (e.g., via voltage or bulk cytosolic Ca 2+ concentration) of the channels, and local coupling in ion channel complexes (e.g., via local Ca 2+ concentration surrounding Ca 2+ channels (CaVs), the so-called Ca 2+ nanodomains). We recently devised a model of large-conductance BK Ca potassium currents, and hence BK Ca -CaV complexes controlled locally by CaVs via Ca 2+ nanodomains. We showed how different CaV types and BK Ca -CaV stoichiometries affect whole-cell electrical behavior. Ca 2+ nanodomains are also important for triggering exocytosis of hormone-containing granules, and in this regard, we implemented a strategy to characterize the local interactions between granules and CaVs. In this study, we coupled electrical and exocytosis models respecting the local effects via Ca 2+ nanodomains. By simulating scenarios with BK Ca -CaV complexes with different stoichiometries in pituitary cells, we achieved two main electrophysiological responses (continuous spiking or bursting) and investigated their effects on the downstream exocytosis process. By varying the number and distance of CaVs coupled with the granules, we found that bursting promotes exocytosis with faster rates than spiking. However, by normalizing to Ca 2+ influx, we found that bursting is only slightly more efficient than spiking when CaVs are far away from granules, whereas no difference in efficiency between bursting and spiking is observed with close granule-CaV coupling.

Small cytoplasmic mRNP granules are implicated in mRNA transport, translational control, and decay. Using super-resolution microscopy and fluorescence correlation spectroscopy, we analyzed the molecular composition and dynamics of single cytoplasmic YBX1&#95;IMP1 mRNP granules in live cells. Granules appeared elongated and branched, with patches of IMP1 and YBX1 distributed along mRNA, reflecting the attachment of the two RNA-binding proteins in cis. Particles form at the nuclear pore and do not associate with translating ribosomes, so the mRNP is a repository for mRNAs awaiting translation. In agreement with the average number of mRNA-binding sites derived from crosslinked immunoprecipitation (CLIP) analyses, individual mRNPs contain 5-15 molecules of YBX1 and IMP1 and a single poly(A) tail identified by PABPC1. Taken together, we conclude that small cytoplasmic mRNP granules are mRNA singletons, thus depicting the cellular transcriptome. Consequently, expression of functionally related mRNAs in RNA regulons is unlikely to result from coordinated assembly., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

During degranulation, mast cells secrete a specific set of mediators defined as "secretome" including the preformed mediators that have already been synthesized by a cell and contained in the cytoplasmic granules. This group includes serine proteases, in particular, chymase and tryptase. Biological significance of chymase depends on the mechanisms of degranulation and is characterized by selective effects on the cellular and non-cellular components of the specific tissue microenvironment. Chymase is known to be closely involved in the mechanisms of inflammation and allergy, angiogenesis, and oncogenesis, remodeling of the extracellular matrix of the connective tissue and changes in organ histoarchitectonics. Number of chymase-positive mast cells in the intra-organ population, and the mechanisms of biogenesis and secretome degranulation appear to be the informative criteria for interpreting the state of the internal organs, characterizing not only the diagnostic efficacy but also the properties of targets of pharmacotherapy. In this review, we discussed the current state of knowledge about mast cell chymase as one of the mast cell secretome proteases. Main issues of the reviewed publications are highlighted with our microscopic images of mast cell chymase visualized using immunohistochemical staining.

Intrinsically disordered proteins (IDPs) are ubiquitous in proteomes and serve in a range of cellular functions including signaling, regulation, transport and enzyme function. IDP misfunction and aggregation are also associated with several diseases including neurodegenerative diseases and cancer. During the past decade, single-molecule methods have become popular for detailed biophysical and structural studies of these complex proteins. This work has included recent applications to cellular liquid-liquid phase separation (LLPS), relevant for functional dynamics of membraneless organelles such as the nucleolus and stress granules. In this concise review, we cover the conceptual motivations for development and application of single-molecule fluorescence methods for such IDP studies. We follow with a few key examples of systems and biophysical problems that have been addressed, and conclude with thoughts for emerging and future directions., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Platelet α-granules play important roles in platelet function. They contain hundreds of proteins that are synthesized by the megakaryocyte or taken up by endocytosis. The trafficking pathways that mediate platelet α-granule biogenesis are incompletely understood, especially with regard to cargo synthesized by the megakaryocyte. Vacuolar-protein sorting 33B (VPS33B) and VPS16B are essential proteins for α-granule biogenesis, but they are largely uncharacterized. Here, we adapted a powerful method to directly map the pathway followed by newly synthesized cargo proteins to reach α-granules. Using this method, we revealed the recycling endosome as a key intermediate compartment in α-granule biogenesis. We then used CRISPR/Cas9 gene editing to knock out VPS33B in pluripotent stem cell-derived immortalized megakaryocyte cells (imMKCLs). Consistent with the observations in platelets from patients with VPS33B mutation, VPS33B-knockout (KO) imMKCLs have drastically reduced levels of α-granule proteins platelet factor 4, von Willebrand factor, and P-selectin. VPS33B and VPS16B form a distinct and small complex in imMKCLs with the same hydrodynamic radius as the recombinant VPS33B-VPS16B heterodimer purified from bacteria. Mechanistically, the VPS33B-VPS16B complex ensures the correct trafficking of α-granule proteins. VPS33B deficiency results in α-granule cargo degradation in lysosomes. VPS16B steady-state levels are significantly lower in VPS33B-KO imMKCLs, suggesting that VPS16B is destabilized in the absence of its partner. Exogenous expression of green fluorescent protein-VPS33B in VPS33B-KO imMKCLs reconstitutes the complex, which localizes to the recycling endosome, further defining this compartment as a key intermediate in α-granule biogenesis. These results advance our understanding of platelet α-granule biogenesis and open new avenues for the study of these organelles., (© 2019 by The American Society of Hematology.)

The accumulation of lipofuscin-like granules in liver, kidneys, and spleen cells in mice and rats of different lines receiving 30% sugar solutions (fructose, glucose, their mixture, and sucrose) in addition to balanced semisynthetic diet for 62 or 122 days was studied by the method of laser scanning confocal microscopy. The granules were detected by their autofluorescence at maximum λ ex =570-600 nm and λ ex =488 nm. In the kidneys of rats receiving glucose and, especially, the mixture of glucose and fructose, significant accumulation of lipofuscin-like granules was found that was absent in the control group animals receiving water. Intensive accumulation of the granules was observed in the kidneys of all groups of mice receiving sugars (except for glucose). Lipofuscin-like granules were located in the cytoplasm of epithelial cells of the distal and proximal convoluted tubules. In the liver of rats and mice, the signs of accumulation of lipofuscin-like granules were absent or minimal. In rat spleen, lipofuscinlike granules were found in the red pulp in all groups, but their accumulation significantly increased in animals receiving the diet enriched with glucose and sucrose.

Liquid-liquid phase separation is thought to be a key organizing principle in eukaryotic cells to generate highly concentrated dynamic assemblies, such as the RNP granules. Numerous in vitro approaches have validated this model, yet a missing aspect is to take into consideration the complex molecular mixture and promiscuous interactions found in vivo. Here we report the versatile scaffold ArtiG to generate concentration-dependent RNA-protein condensates within living cells, as a bottom-up approach to study the impact of co-segregated endogenous components on phase separation. We demonstrate that intracellular RNA seeds the nucleation of the condensates, as it provides molecular cues to locally coordinate the formation of endogenous high-order RNP assemblies. Interestingly, the co-segregation of intracellular components ultimately impacts the size of the phase-separated condensates. Thus, RNA arises as an architectural element that can influence the composition and the morphological outcome of the condensate phases in an intracellular context.

Liquid granules rich in intrinsically disordered proteins and RNA play key roles in critical cellular functions such as RNA processing and translation. Many details of the mechanism via which this occurs remain to be elucidated. Motivated by the lacuna in the field and by the prospects of developing de novo artificial granules that provide extrinsic control of translation, we report a bottom-up approach to engineer ribonucleoprotein granules composed of a recombinant RNA-binding IDP that exhibits phase behavior in water. We developed a kinetic model to illustrate that these granules inhibit translation through reversible or irreversible sequestration of mRNA. Within monodisperse droplets capable of transcription and translation, we experimentally demonstrate temporal inhibition of translation by using designer IDPs that exhibit tunable phase behavior. This work lays the foundation for developing artificial granules that promise to further our mechanistic understanding of their naturally occurring counterparts., (Copyright © 2019 Elsevier Inc. All rights reserved.)

N 6 -methyladenosine (m 6 A) is the most prevalent modified nucleotide in mRNA 1,2 , with around 25% of mRNAs containing at least one m 6 A. Methylation of mRNA to form m 6 A is required for diverse cellular and physiological processes 3 . Although the presence of m 6 A in an mRNA can affect its fate in different ways, it is unclear how m 6 A directs this process and why the effects of m 6 A can vary in different cellular contexts. Here we show that the cytosolic m 6 A-binding proteins-YTHDF1, YTHDF2 and YTHDF3-undergo liquid-liquid phase separation in vitro and in cells. This phase separation is markedly enhanced by mRNAs that contain multiple, but not single, m 6 A residues. Polymethylated mRNAs act as a multivalent scaffold for the binding of YTHDF proteins, juxtaposing their low-complexity domains and thereby leading to phase separation. The resulting mRNA-YTHDF complexes then partition into different endogenous phase-separated compartments, such as P-bodies, stress granules or neuronal RNA granules. m 6 A-mRNA is subject to compartment-specific regulation, including a reduction in the stability and translation of mRNA. These studies reveal that the number and distribution of m 6 A sites in cellular mRNAs can regulate and influence the composition of the phase-separated transcriptome, and suggest that the cellular properties of m 6 A-modified mRNAs are governed by liquid-liquid phase separation principles.

Membraneless organelles are distinct compartments within a cell that are not enclosed by a traditional lipid membrane and instead form through a process called liquid-liquid phase separation. Examples of these non-membrane-bound organelles include nucleoli, stress granules, P bodies, pericentriolar material and germ granules. Many recent studies have used Caenorhabditis elegans germ granules, known as P granules, to expand our understanding of the formation of these unique cellular compartments. From this work, we know that proteins with intrinsically disordered regions (IDRs) play a critical role in the process of phase separation. IDR phase separation is further tuned through their interactions with RNA and through protein modifications such as phosphorylation and methylation. These findings from C elegans, combined with work done in other model organisms, continue to provide insight into the formation of membraneless organelles and the important role they play in compartmentalizing cellular processes., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Leishmania parasites lack pathways for de novo purine biosynthesis. The depletion of purines induces differentiation into virulent metacyclic forms. In vitro, the parasites can survive prolonged periods of purine withdrawal changing their morphology to long and slender cells with an extended flagellum, and decreasing their translation rates. Reduced translation leads to the appearance of discrete granules that contain LeishIF4E-3, one of the six eIF4E paralogs encoded by the Leishmania genome. We hypothesize that each is responsible for a different function during the life cycle. LeishIF4E-3 is a weak cap-binding protein paralog, but its involvement in translation under normal conditions cannot be excluded. However, in response to nutritional stress, LeishIF4E-3 concentrates in specific cytoplasmic granules. LeishIF4E-3 granulation can be induced by the independent elimination of purines, amino acids and glucose. As these granules contain mature mRNAs, we propose that these bodies store inactive transcripts until recovery from stress occurs. In attempt to examine the content of the nutritional stress-induced granules, they were concentrated over sucrose gradients and further pulled-down by targeting in vivo tagged LeishIF4E-3. Proteomic analysis highlighted granule enrichment with multiple ribosomal proteins, suggesting that ribosome particles are abundant in these foci, as expected in case of translation inhibition. RNA-binding proteins, RNA helicases and metabolic enzymes were also enriched in the granules, whereas no degradation enzymes or P-body markers were detected. The starvation-induced LeishIF4E-3-containing granules, therefore, appear to store stalled ribosomes and ribosomal subunits, along with their associated mRNAs. Following nutritional stress, LeishIF4E-3 becomes phosphorylated at position S75, located in its less-conserved N-terminal extension. The ability of the S75A mutant to form granules was reduced, indicating that cellular signaling regulates LeishIF4E-3 function., Competing Interests: The authors have declared that no competing interests exist.

Chlamydomonas reinhardtii represents an ideal model microbial system to decipher starch metabolism. In this green algae, in cells growing in photosynthetic conditions, starch mainly accumulates as a sheath surrounding the pyrenoid while in cells subjected to a nutrient starvation, numerous starch granules are filling up the plastid stroma. The mechanisms underlying and regulating this switch from photosynthetic to storage starch metabolisms are not known. In this work, we have isolated a Chlamydomonas mutant strain containing a deletion in chromosome 2 which displays abnormal starch granule distribution. Under nitrogen starvation, this strain contains an additional starch granules population. These granules are twice as big as the wild-type granules and display characteristics of photosynthetic starch. Genetic and functional complementation analyses allowed us to identify the gene responsible for this original phenotype which was called BSG1 for "Bimodal Starch Granule". Possible roles of BSG1 in starch metabolism modifications during the transition from photosynthetic to starved growth conditions are discussed.

Polyhydroxyalkanoates (PHA) are storage polymers accumulated by numerous prokaryotes in form of intracellular granules. Native PHA granules are formed by amorphous polymer which reveals considerably higher elasticity and flexibility as compared to crystalline pure PHA polymers. The fact that bacteria store PHA in amorphous state has great biological consequences. It is not clear which mechanisms protect amorphous polymer in native granules from transition into thermodynamically favorable crystalline state. Here, we demonstrate that exposition of bacterial cells to particular stressors induces granules aggregation, which is the first but not sufficient condition for PHA crystallization. Crystallization of the polymer occurs only when the stressed bacterial cells are subsequently dried. The fact that both granules aggregation and cell drying must occur to induce crystallization of PHA indicates that both previously suggested hypotheses about mechanisms of stabilization of amorphous state of native PHA are valid and, in fact, both effects participate synergistically. It seems that the amorphous state of the polymer is stabilized kinetically by the low rate of crystallization in limited volume in small PHA granules and, moreover, water present in PHA granules seems to function as plasticizer protecting the polymer from crystallization, as confirmed experimentally for the first time by the present work.

Toxoplasma gondii is an opportunistic pathogen infecting humans and a variety of vertebrate animals. Secretory dense-granule proteins (GRAs) play diverse roles in the mediation of host-parasite interactions and facilitate parasitism, but many of them still remain to be identified. Here, we used two proximity-based protein labeling techniques to identify novel GRA proteins. Taking GRA1 as bait, transgenic strains expressing GRA1-BirA* or GRA1-APEX were constructed to biotinylate GRAs. Using these methods, a total of 46 proteins were identified, 20 of which were known GRA proteins. Among these 46, 17 were identified by both strategies, and 14 out of the 17 were known GRAs. The other three were all confirmed to localize to dense granules. Nonetheless a significant portion of the proteins were only identified by either APEX or BirA*, indicating that there are differences between these methods. Of the 26 novel GRAs, 5 were validated as bona fide GRAs by localization studies. The majority of these novel GRAs are only present in coccidian parasites and are likely dispensable for parasite growth in vitro; they may play roles during animal infections. The identification of novel GRAs laid the foundation for further studies investigating the mechanisms underlying parasite-host interactions.

The secretory granules of pancreatic beta cells are specialized organelles responsible for the packaging, storage and secretion of the vital hormone insulin. The insulin secretory granules also contain more than 100 other proteins including the proteases involved in proinsulin-to insulin conversion, other precursor proteins, minor co-secreted peptides, membrane proteins involved in cell trafficking and ion translocation proteins essential for regulation of the intragranular environment. The synthesis, transport and packaging of these proteins into nascent granules must be carried out in a co-ordinated manner to ensure correct functioning of the granule. The process is regulated by many circulating nutrients such as glucose and can change under different physiological states. This chapter discusses the various processes involved in insulin granule biogenesis with a focus on the granule composition in health and disease.

Ribonucleoprotein (RNP) condensations through liquid-liquid phase separation play vital roles in the dynamic formation-dissolution of stress granules (SGs). These condensations are, however, usually assumed to be linked to pathologic fibrillation. Here, we show that physiologic condensation and pathologic fibrillation of RNPs are independent processes that can be unlinked with the chemical chaperone trimethylamine N-oxide (TMAO). Using the low-complexity disordered domain of the archetypical SG-protein TDP-43 as a model system, we show that TMAO enhances RNP liquid condensation yet inhibits protein fibrillation. Our results demonstrate effective decoupling of physiologic condensation from pathologic aggregation and suggest that selective targeting of protein fibrillation (without altering condensation) can be employed as a therapeutic strategy for RNP aggregation-associated degenerative disorders.

We studied proliferative activity of multipotent mesenchymal stromal cells on collagen matrices containing platelets stabilized with silver nanoparticles. Dose-dependent stimulation of the growth of stromal cells without their structural damage was observed in the presence of stabilized platelets in proportion of 20-120 mln per 10 5 multipotent mesenchymal stromal cells. The same doses of non-stabilized platelets produced less pronounced stimulation effect. In higher doses (≥180 mln) stabilized platelets inhibited cell proliferation and induced their damage. Stabilized platelets enhanced migration of multipotent mesenchymal stromal cells; after formation of the monolayer, they actively colonized deep layer of the collagen matrix. The formed monolayer of multipotent mesenchymal stromal cells survived over 14 days without appreciable cell damage.

RNA localization mechanisms have been intensively studied and include localized protection of mRNA from degradation, diffusion-coupled local entrapment of mRNA, and directed transport of mRNAs along the cytoskeleton. While it is well understood how cells utilize these three mechanisms to organize mRNAs within the cytoplasm, a newly appreciated mechanism of RNA localization has emerged in recent years in which mRNAs phase-separate and form liquid-like droplets. mRNAs both contribute to condensation of proteins into liquid-like structures and are themselves regulated by being incorporated into membraneless organelles. This ability to condense into droplets is in many instances contributing to previously appreciated mRNA localization phenomena. Here we review how phase separation enables mRNAs to selectively and efficiently colocalize and be coregulated, allowing control of gene expression in time and space.

The pineal gland is a neuroendocrine organ associated with photoperiodic regulation in mammals. The aim of this study was to evaluate the pineal gland at the pre-pubertal, pubertal and post-pubertal periods by means of morphology and stereology. The study examined at total of 24 ovine pineal glands collected from healthy female Akkaraman breed. Thick sections (40 μm) were cut and treated with synaptophysin. Following each thick section, six consecutive sections at a thickness of 5 μm were cut. Each thin section was stained with one of the following dyes: Crossman's modified triple dye, glial fibrillary acidic protein (GFAP), melatonin marker, periodic acid-Schiff, Von Kossa and AgNOR. The pineal gland volume was measured using Cavalieri's method. The optical fractionator was used to estimate the total number of pinealocytes. The percentage of parenchyma and connective tissue and degree of vascularization were estimated by the area fraction fractionator method. The pineal gland volumes in the pre-pubertal, pubertal and post-pubertal groups were 7.53 ± 1.715 mm 3 , 11.20 ± 1.336 mm 3 and 17.75 ± 1.188 mm 3 , respectively (p < .5). The number of pinealocytes in the pre-pubertal, pubertal and post-pubertal groups was 3,244,000 ± 228,076, 4,438,000 ± 243,610, 7,381,766 ± 406,223, respectively (p < .05). The glands of the post-pubertal group contained the highest amount of connective tissue (11.49 ± 2.103%; p < .5) and the largest GFAP staining area (p < .05). The melatonin staining density was the highest in the pubertal group. The density of lipofuscin staining was higher in the pubertal and post-pubertal groups., (© 2018 Blackwell Verlag GmbH.)

Tissue-specific autoimmunity occurs when selected antigens presented by susceptible alleles of the major histocompatibility complex are recognized by T cells. However, the reason why certain specific self-antigens dominate the response and are indispensable for triggering autoreactivity is unclear. Spontaneous presentation of insulin is essential for initiating autoimmune type 1 diabetes in non-obese diabetic mice 1,2 . A major set of pathogenic CD4 T cells specifically recognizes the 12-20 segment of the insulin B-chain (B:12-20), an epitope that is generated from direct presentation of insulin peptides by antigen-presenting cells 3,4 . These T cells do not respond to antigen-presenting cells that have taken up insulin that, after processing, leads to presentation of a different segment representing a one-residue shift, B:13-21 4 . CD4 T cells that recognize B:12-20 escape negative selection in the thymus and cause diabetes, whereas those that recognize B:13-21 have only a minor role in autoimmunity 3-5 . Although presentation of B:12-20 is evident in the islets 3,6 , insulin-specific germinal centres can be formed in various lymphoid tissues, suggesting that insulin presentation is widespread 7,8 . Here we use live imaging to document the distribution of insulin recognition by CD4 T cells throughout various lymph nodes. Furthermore, we identify catabolized insulin peptide fragments containing defined pathogenic epitopes in β-cell granules from mice and humans. Upon glucose challenge, these fragments are released into the circulation and are recognized by CD4 T cells, leading to an activation state that results in transcriptional reprogramming and enhanced diabetogenicity. Therefore, a tissue such as pancreatic islets, by releasing catabolized products, imposes a constant threat to self-tolerance. These findings reveal a self-recognition pathway underlying a primary autoantigen and provide a foundation for assessing antigenic targets that precipitate pathogenic outcomes by systemically sensitizing lymphoid tissues.

Chikungunya virus (CHIKV), a mosquito-borne human pathogen, causes a disabling disease characterized by severe joint pain that can persist for weeks, months, or even years in patients. The nonstructural protein 3 (nsP3) plays essential roles during acute infection, but little is known about the function of nsP3 during chronic disease. Here, we used subdiffraction multicolor microscopy for spatial and temporal analysis of CHIKV nsP3 within human cells that persistently replicate replicon RNA. Round cytoplasmic granules of various sizes (i) contained nsP3 and stress granule assembly factors 1 and 2 (G3BP1/2), (ii) were next to double-stranded RNA foci and nsP1-positive structures, and (iii) were close to the nuclear membrane and the nuclear pore complex protein Nup98. Analysis of protein turnover and mobility by live-cell microscopy revealed that the granules could persist for hours to days, accumulated newly synthesized protein, and moved through the cytoplasm at various speeds. The granules also had a static internal architecture and were stable in cell lysates. Refractory cells that had cleared the noncytotoxic replicon regained the ability to respond to arsenite-induced stress. In summary, nsP3 can form uniquely stable granular structures that persist long-term within the host cell. This continued presence of viral and cellular protein complexes has implications for the study of the pathogenic consequences of lingering CHIKV infection and the development of strategies to mitigate the burden of chronic musculoskeletal disease brought about by a medically important arthropod-borne virus (arbovirus). IMPORTANCE Chikungunya virus (CHIKV) is a reemerging alphavirus transmitted by mosquitos and causes transient sickness but also chronic disease affecting muscles and joints. No approved vaccines or antivirals are available. Thus, a better understanding of the viral life cycle and the role of viral proteins can aid in identifying new therapeutic targets. Advances in microscopy and development of noncytotoxic replicons (A. Utt, P. K. Das, M. Varjak, V. Lulla, A. Lulla, A. Merits, J Virol 89:3145-3162, 2015, https://doi.org/10.1128/JVI.03213-14) have allowed researchers to study viral proteins within controlled laboratory environments over extended durations. Here we established human cells that stably replicate replicon RNA and express tagged nonstructural protein 3 (nsP3). The ability to track nsP3 within the host cell and during persistent replication can benefit fundamental research efforts to better understand long-term consequences of the persistence of viral protein complexes and thereby provide the foundation for new therapeutic targets to control CHIKV infection and treat chronic disease symptoms., (Copyright © 2018 Remenyi et al.)