Distinct components of nucleoside-modified messenger RNA vaccines cooperate to instruct efficient germinal center responses.

Nucleoside-modified mRNA vaccines elicit protective antibodies through their ability to promote T follicular helper (Tfh) cells. The lipid nanoparticle (LNP) component of mRNA vaccines possesses inherent adjuvant activity. However, to what extent the nucleoside-modified mRNA can be sensed and contribute to Tfh cell responses remains largely undefined. Herein, we deconvoluted the signals induced by LNP and mRNA that instruct dendritic cells (DCs) to promote Tfh cell differentiation. We demonstrated that the nucleoside-modified mRNA drives the production of type I interferons that act on DCs to induce their maturation and the induction of Th1-biased Tfh responses. Conversely, LNP favors the acquisition of a Tfh cell-inducing program in DCs, a stronger Th2 polarization in Tfh cells, and allows for rapid mRNA translation by DCs within the draining lymph node. Our work unravels distinct adjuvant features of mRNA and LNP necessary for the induction of Tfh cells, with implications for vaccine design.

Cellular Immunity of Drosophila willistoni Reveals Novel Complexity in Insect Anti-Parasitoid Defense.

Coevolution of hosts and their parasites has shaped heterogeneity of effector hemocyte types, providing immune defense reactions with variable effectiveness. In this work, we characterize hemocytes of Drosophila willistoni, a species that has evolved a cellular immune system with extensive variation and a high degree of plasticity. Monoclonal antibodies were raised and used in indirect immunofluorescence experiments to characterize hemocyte subpopulations, follow their functional features and differentiation. Pagocytosis and parasitization assays were used to determine the functional characteristics of hemocyte types. Samples were visualized using confocal and epifluorescence microscopy. We identified a new multinucleated giant hemocyte (MGH) type, which differentiates in the course of the cellular immune response to parasitoids. These cells differentiate in the circulation through nuclear division and cell fusion, and can also be derived from the central hematopoietic organ, the lymph gland. They have a binary function as they take up bacteria by phagocytosis and are involved in the encapsulation and elimination of the parasitoid. Here, we show that, in response to large foreign particles, such as parasitoids, MGHs differentiate, have a binary function and contribute to a highly effective cellular immune response, similar to the foreign body giant cells of vertebrates.

mRNA-LNP vaccine-induced CD8+ T cells protect mice from lethal SARS-CoV-2 infection in the absence of specific antibodies.

The role of CD8+ T cells in SARS-CoV-2 pathogenesis or mRNA-LNP vaccine-induced protection from lethal COVID-19 is unclear. Using mouse-adapted SARS-CoV-2 virus (MA30) in C57BL/6 mice, we show that CD8+ T cells are unnecessary for the intrinsic resistance of female or the susceptibility of male mice to lethal SARS-CoV-2 infection. Also, mice immunized with a di-proline prefusion-stabilized full-length SARS-CoV-2 Spike (S-2P) mRNA-LNP vaccine, which induces Spike-specific antibodies and CD8+ T cells specific for the Spike-derived VNFNFNGL peptide, are protected from SARS-CoV-2 infection-induced lethality and weight loss, while mice vaccinated with mRNA-LNPs encoding only VNFNFNGL are protected from lethality but not weight loss. CD8+ T cell depletion ablates protection in VNFNFNGL but not in S-2P mRNA-LNP-vaccinated mice. Therefore, mRNA-LNP vaccine-induced CD8+ T cells are dispensable when protective antibodies are present but essential for survival in their absence. Hence, vaccine-induced CD8+ T cells may be critical to protect against SARS-CoV-2 variants that mutate epitopes targeted by protective antibodies.

A bipartite NLS motif mediates the nuclear import of Drosophila moesin.

The ERM protein family, which consists of three closely related proteins in vertebrates, ezrin, radixin, and moesin (ERM), is an ancient and important group of cytoplasmic actin-binding and organizing proteins. With their FERM domain, ERMs bind various transmembrane proteins and anchor them to the actin cortex through their C-terminal F-actin binding domain, thus they are major regulators of actin dynamics in the cell. ERMs participate in many fundamental cellular processes, such as phagocytosis, microvilli formation, T-cell activation and tumor metastasis. We have previously shown that, besides its cytoplasmic activities, the single ERM protein of Drosophila melanogaster, moesin, is also present in the cell nucleus, where it participates in gene expression and mRNA export. Here we study the mechanism by which moesin enters the nucleus. We show that the nuclear import of moesin is an NLS-mediated, active process. The nuclear localization sequence of the moesin protein is an evolutionarily highly conserved, conventional bipartite motif located on the surface of the FERM domain. Our experiments also reveal that the nuclear import of moesin does not require PIP2 binding or protein activation, and occurs in monomeric form. We propose, that the balance between the phosphorylated and non-phosphorylated protein pools determines the degree of nuclear import of moesin.

Expression and purification of the receptor-binding domain of SARS-CoV-2 spike protein in mammalian cells for immunological assays.

The receptor-binding domain (RBD) of the spike glycoprotein of SARS-CoV-2 virus mediates the interaction with the host cell and is required for virus internalization. It is, therefore, the primary target of neutralizing antibodies. The receptor-binding domain soon became the major target for COVID-19 research and the development of diagnostic tools and new-generation vaccines. Here, we provide a detailed protocol for high-yield expression and one-step affinity purification of recombinant RBD from transiently transfected Expi293F cells. Expi293F mammalian cells can be grown to extremely high densities in a specially formulated serum-free medium in suspension cultures, which makes them an excellent tool for secreted protein production. The highly purified RBD is glycosylated, structurally intact, and forms homomeric complexes. With this quick and easy method, we are able to produce large quantities of RBD (80 mg·L-1 culture) that we have successfully used in immunological assays to examine antibody titers and seroconversion after mRNA-based vaccination of mice.

Protein Phosphatase 4 Is Required for Centrobin Function in DNA Damage Repair.

Genome stability in human cells relies on the efficient repair of double-stranded DNA breaks, which is mainly achieved by homologous recombination (HR). Among the regulators of various cellular functions, Protein phosphatase 4 (PP4) plays a pivotal role in coordinating cellular response to DNA damage. Meanwhile, Centrobin (CNTRB), initially recognized for its association with centrosomal function and microtubule dynamics, has sparked interest due to its potential contribution to DNA repair processes. In this study, we investigate the involvement of PP4 and its interaction with CNTRB in HR-mediated DNA repair in human cells. Employing a range of experimental strategies, we investigate the physical interaction between PP4 and CNTRB and shed light on the importance of two specific motifs in CNTRB, the PP4-binding FRVP and the ATR kinase recognition SQ sequences, in the DNA repair process. Moreover, we examine cells depleted of PP4 or CNTRB and cells harboring FRVP and SQ mutations in CNTRB, which result in similar abnormal chromosome morphologies. This phenomenon likely results from the impaired resolution of Holliday junctions, which serve as crucial intermediates in HR. Taken together, our results provide new insights into the intricate mechanisms of PP4 and CNTRB-regulated HR repair and their interrelation.

Evolution of insect innate immunity through domestication of bacterial toxins.

Toxin cargo genes are often horizontally transferred by phages between bacterial species and are known to play an important role in the evolution of bacterial pathogenesis. Here, we show how these same genes have been horizontally transferred from phage or bacteria to animals and have resulted in novel adaptations. We discovered that two widespread bacterial genes encoding toxins of animal cells, cytolethal distending toxin subunit B (cdtB) and apoptosis-inducing protein of 56 kDa (aip56), were captured by insect genomes through horizontal gene transfer from bacteria or phages. To study the function of these genes in insects, we focused on Drosophila ananassae as a model. In the D. ananassae subgroup species, cdtB and aip56 are present as singular (cdtB) or fused copies (cdtB::aip56) on the second chromosome. We found that cdtB and aip56 genes and encoded proteins were expressed by immune cells, some proteins were localized to the wasp embryo's serosa, and their expression increased following parasitoid wasp infection. Species of the ananassae subgroup are highly resistant to parasitoid wasps, and we observed that D. ananassae lines carrying null mutations in cdtB and aip56 toxin genes were more susceptible to parasitoids than the wild type. We conclude that toxin cargo genes were captured by these insects millions of years ago and integrated as novel modules into their innate immune system. These modules now represent components of a heretofore undescribed defense response and are important for resistance to parasitoid wasps. Phage or bacterially derived eukaryotic toxin genes serve as macromutations that can spur the instantaneous evolution of novelty in animals.

Plk4 Is a Novel Substrate of Protein Phosphatase 5.

The conserved Ser/Thr protein phosphatase 5 (PP5) is involved in the regulation of key cellular processes, including DNA damage repair and cell division in eukaryotes. As a co-chaperone of Hsp90, PP5 has been shown to modulate the maturation and activity of numerous oncogenic kinases. Here, we identify a novel substrate of PP5, the Polo-like kinase 4 (Plk4), which is the master regulator of centriole duplication in animal cells. We show that PP5 specifically interacts with Plk4, and is able to dephosphorylate the kinase in vitro and in vivo, which affects the interaction of Plk4 with its partner proteins. In addition, we provide evidence that PP5 and Plk4 co-localize to the centrosomes in Drosophila embryos and cultured cells. We demonstrate that PP5 is not essential; the null mutant flies are viable without a severe mitotic phenotype; however, its loss significantly reduces the fertility of the animals. Our results suggest that PP5 is a novel regulator of the Plk4 kinase in Drosophila.

Molecular Characterization of Novel Mycoviruses in Seven Umbelopsis Strains.

The presence of viruses is less explored in Mucoromycota as compared to other fungal groups such as Ascomycota and Basidiomycota. Recently, more and more mycoviruses are identified from the early-diverging lineages of fungi. We have determined the genome of 11 novel dsRNA viruses in seven different Umbelopsis strains with next-generation sequencing (NGS). The identified viruses were named Umbelopsis ramanniana virus 5 (UrV5), 6a (UrV6a); 6b (UrV6b); 7 (UrV7); 8a (UrV8a); 8b (UrV8b); Umbelopsis gibberispora virus 1 (UgV1); 2 (UgV2) and Umbelopsis dimorpha virus 1a (UdV1a), 1b (UdV1b) and 2 (UdV2). All the newly identified viruses contain two open reading frames (ORFs), which putatively encode the coat protein (CP) and the RNA-dependent RNA polymerase (RdRp), respectively. Based on the phylogeny inferred from the RdRp sequences, eight viruses (UrV7, UrV8a, UrV8b, UgV1, UgV2, UdV1a, UdV1b and UdV2) belong to the genus Totivirus, while UrV5, UrV6a and UrV6b are placed into a yet unclassified but well-defined Totiviridae-related group. In UrV5, UgV1, UgV2, UrV8b, UdV1a, UdV2 and UdV1b, ORF2 is predicted to be translated as a fusion protein via a rare +1 (or -2) ribosomal frameshift, which is not characteristic to most members of the Totivirus genus. Virus particles 31 to 32 nm in diameter could be detected in the examined fungal strains by transmission electron microscopy. Through the identification and characterization of new viruses of Mucoromycota fungi, we can gain insight into the diversity of mycoviruses, as well as into their phylogeny and genome organization.

GST-IVTT pull-down: a fast and versatile in vitro method for validating and mapping protein-protein interactions.

Over the past few decades, dozens of in vitro methods have been developed to map, investigate and validate protein-protein interactions. However, most of these approaches are time-consuming and labour-intensive or require specialised equipment or substantial amounts of purified proteins. Here, we describe a fast and versatile research protocol that is suitable for the in vitro analysis of the physical interaction between proteins or for mapping the binding surfaces. The principle of this method is based on the immobilisation of the protein/domain of interest to a carrier followed by its incubation with a labelled putative binding partner, which is generated by a coupled in vitro transcription/translation reaction. Interacting proteins are removed from the carrier, fractionated and visualised by SDS/PAGE autoradiography (or western blotting). This simple and cheap method can be easily carried out in every wet lab.

Parallel import mechanisms ensure the robust nuclear localization of actin in Drosophila.

Actin, as an ancient and fundamental protein, participates in various cytoplasmic as well as nuclear functions in eukaryotic cells. Based on its manifold tasks in the nucleus, it is a reasonable assumption that the nuclear presence of actin is essential for the cell, and consequently, its nuclear localization is ensured by a robust system. However, today only a single nuclear import and a single nuclear export pathway is known which maintain the dynamic balance between cytoplasmic and nuclear actin pools. In our work, we tested the robustness of the nuclear import of actin, and investigated whether the perturbations of nuclear localization affect the viability of the whole organism. For this aim, we generated a genetic system in Drosophila, in which we rescued the lethal phenotype of the null mutation of the Actin5C gene with transgenes that express different derivatives of actin, including a Nuclear Export Signal (NES)-tagged isoform which ensures forced nuclear export of the protein. We also disrupted the SUMOylation site of actin, suggested earlier to be responsible for nuclear retention, and eliminated the activity of the single nuclear import factor dedicated to actin. We found that, individually, none of the above mentioned manipulations led to a notable reduction in nuclear actin levels and thus, fully rescued lethality. However, the NES tagging of actin, together with the knock out of its importin, significantly reduced the amount of nuclear actin and induced lethality, confirming that the presence of actin in the nucleus is essential, and thereby, over-secured. Supporting this, we identified novel nuclear importins specific to actin, which sheds light on the mechanism behind the robustness of nuclear localization of actin, and supports the idea of essentiality of its nuclear functions.

STABILON, a Novel Sequence Motif That Enhances the Expression and Accumulation of Intracellular and Secreted Proteins.

The dynamic balance of transcriptional and translational regulation together with degron-controlled proteolysis shapes the ever-changing cellular proteome. While a large variety of degradation signals has been characterized, our knowledge of cis-acting protein motifs that can in vivo stabilize otherwise short-lived proteins is very limited. We have identified and characterized a conserved 13-mer protein segment derived from the p54/Rpn10 ubiquitin receptor subunit of the Drosophila 26S proteasome, which fulfills all the characteristics of a protein stabilization motif (STABILON). Attachment of STABILON to various intracellular as well as medically relevant secreted model proteins resulted in a significant increase in their cellular or extracellular concentration in mammalian cells. We demonstrate that STABILON acts as a universal and dual function motif that, on the one hand, increases the concentration of the corresponding mRNAs and, on the other hand, prevents the degradation of short-lived fusion proteins. Therefore, STABILON may lead to a breakthrough in biomedical recombinant protein production.

The Small Heat Shock Protein, HSPB1, Interacts with and Modulates the Physical Structure of Membranes.

Small heat shock proteins (sHSPs) have been demonstrated to interact with lipids and modulate the physical state of membranes across species. Through these interactions, sHSPs contribute to the maintenance of membrane integrity. HSPB1 is a major sHSP in mammals, but its lipid interaction profile has so far been unexplored. In this study, we characterized the interaction between HSPB1 and phospholipids. HSPB1 not only associated with membranes via membrane-forming lipids, but also showed a strong affinity towards highly fluid membranes. It participated in the modulation of the physical properties of the interacting membranes by altering rotational and lateral lipid mobility. In addition, the in vivo expression of HSPB1 greatly affected the phase behavior of the plasma membrane under membrane fluidizing stress conditions. In light of our current findings, we propose a new function for HSPB1 as a membrane chaperone.

Characterization of Four Novel dsRNA Viruses Isolated from Mucor hiemalis Strains.

We previously screened the total nucleic acid extracts of 123 Mucor strains for the presence of dsRNA molecules without further molecular analyses. Here, we characterized five novel dsRNA genomes isolated from four different Mucor hiemalis strains with next-generation sequencing (NGS), namely Mucor hiemalis virus 1a (MhV1a) from WRL CN(M) 122; Mucor hiemalis virus 1b (MhV1b) from NRRL 3624; Mucor hiemalis virus 2 (MhV2) from NRRL 3616; and Mucor hiemalis virus 3 (MhV3) and Mucor hiemalis virus (MhV4) from NRRL 3617 strains. Genomes contain two open reading frames (ORF), which encode the coat protein (CP) and the RNA dependent RNA polymerase (RdRp), respectively. In MhV1a and MhV1b, it is predicted to be translated as a fusion protein via -1 ribosomal frameshift, while in MhV4 via a rare +1 (or-2) ribosomal frameshift. In MhV2 and MhV3, the presence of specific UAAUG pentanucleotide motif points to the fact for coupled translation termination and reinitialization. MhV1a, MhV2, and MhV3 are part of the clade representing the genus Victorivirus, while MhV4 is seated in Totivirus genus clade. The detected VLPs in Mucor strains were from 33 to 36 nm in diameter. Hybridization analysis revealed that the dsRNA molecules of MhV1a-MhV4 hybridized to the corresponding molecules.

Microtubule Organizing Centers Contain Testis-Specific γ-TuRC Proteins in Spermatids of Drosophila.

Microtubule nucleation in eukaryotes is primarily promoted by γ-tubulin and the evolutionary conserved protein complex, γ-Tubulin Ring Complex (γ-TuRC). γ-TuRC is part of the centrosome and basal body, which are the best-known microtubule-organizing centers. Centrosomes undergo intensive and dynamic changes during spermatogenesis, as they turn into basal bodies, a prerequisite for axoneme formation during spermatogenesis. Here we describe the existence of a novel, tissue-specific γ-TuRC in Drosophila. We characterize three genes encoding testis-specific components of γ-TuRC (t-γ-TuRC) and find that presence of t-γ-TuRC is essential to male fertility. We show the diverse subcellular distribution of the t-γ-TuRC proteins during post-meiotic development, at first at the centriole adjunct and then also on the anterior tip of the nucleus, and finally, they appear in the tail region, close to the mitochondria. We also prove the physical interactions between the t-γ-TuRC members, γ-tubulin and Mozart1. Our results further indicate heterogeneity in γ-TuRC composition during spermatogenesis and suggest that the different post-meiotic microtubule organizing centers are orchestrated by testis-specific gene products, including t-γ-TuRC.

The nuclear activity of the actin-binding Moesin protein is necessary for gene expression in Drosophila.

Ezrin-Radixin-Moesin (ERM) proteins play an essential role in the cytoplasm by cross-linking actin filaments with plasma membrane proteins. Research has identified the nuclear localization of ERMs, as well as the involvement of a single Drosophila ERM protein, Moesin, in nuclear mRNA exports. However, the question of how important the nuclear activity of ERM proteins are for the life of an organism has so far not been explored. Here, we present the first attempt to reveal the in vivo relevance of nuclear localization of Moesin in Drosophila. With the help of a nuclear export signal, we decreased the amount of Moesin in the nuclei of the animals. Furthermore, we observed various developmental defects, demonstrating the importance of ERM function in the nucleus for the first time. Transcriptome analysis of the mutant flies revealed that the lack of nuclear Moesin function leads to expression changes in nearly 700 genes, among them heat-shock genes. This result together with additional findings revealed that in Drosophila the expression of protein chaperones requires the nuclear functions of Moesin. DATABASE: GEO accession number: GSE155778.

Novel perspectives of target-binding by the evolutionarily conserved PP4 phosphatase.

Protein phosphatase 4 (PP4) is an evolutionarily conserved and essential Ser/Thr phosphatase that regulates cell division, development and DNA repair in eukaryotes. The major form of PP4, present from yeast to human, is the PP4c-R2-R3 heterotrimeric complex. The R3 subunit is responsible for substrate-recognition via its EVH1 domain. In typical EVH1 domains, conserved phenylalanine, tyrosine and tryptophan residues form the specific recognition site for their target's proline-rich sequences. Here, we identify novel binding partners of the EVH1 domain of the Drosophila R3 subunit, Falafel, and demonstrate that instead of binding to proline-rich sequences this EVH1 variant specifically recognizes atypical ligands, namely the FxxP and MxPP short linear consensus motifs. This interaction is dependent on an exclusively conserved leucine that replaces the phenylalanine invariant of all canonical EVH1 domains. We propose that the EVH1 domain of PP4 represents a new class of the EVH1 family that can accommodate low proline content sequences, such as the FxxP motif. Finally, our data implicate the conserved Smk-1 domain of Falafel in target-binding. These findings greatly enhance our understanding of the substrate-recognition mechanisms and function of PP4.

A fraction of barrier-to-autointegration factor (BAF) associates with centromeres and controls mitosis progression.

Barrier-to-Autointegration Factor (BAF) is a conserved nuclear envelope (NE) component that binds chromatin and helps its anchoring to the NE. Cycles of phosphorylation and dephosphorylation control BAF function. Entering mitosis, phosphorylation releases BAF from chromatin and facilitates NE-disassembly. At mitotic exit, PP2A-mediated dephosphorylation restores chromatin binding and nucleates NE-reassembly. Here, we show that in Drosophila a small fraction of BAF (cenBAF) associates with centromeres. We also find that PP4 phosphatase, which is recruited to centromeres by CENP-C, prevents phosphorylation and release of cenBAF during mitosis. cenBAF is necessary for proper centromere assembly and accurate chromosome segregation, being critical for mitosis progression. Disrupting cenBAF localization prevents PP2A inactivation in mitosis compromising global BAF phosphorylation, which in turn leads to its persistent association with chromatin, delays anaphase onset and causes NE defects. These results suggest that, together with PP4 and CENP-C, cenBAF forms a centromere-based mechanism that controls chromosome segregation and mitosis progression.

Sperm-Leucylaminopeptidases are required for male fertility as structural components of mitochondrial paracrystalline material in Drosophila melanogaster sperm.

Drosophila melanogaster sperm reach an extraordinary long size, 1.8 mm, by the end of spermatogenesis. The mitochondrial derivatives run along the entire flagellum and provide structural rigidity for flagellar movement, but its precise function and organization is incompletely understood. The two mitochondrial derivatives differentiate and by the end of spermatogenesis the minor one reduces its size and the major one accumulates paracrystalline material inside it. The molecular constituents and precise function of the paracrystalline material have not yet been revealed. Here we purified the paracrystalline material from mature sperm and identified by mass spectrometry Sperm-Leucylaminopeptidase (S-Lap) family members as important constituents of it. To study the function of S-Lap proteins we show the characterization of classical mutants and RNAi lines affecting of the S-Lap genes and the analysis of their mutant phenotypes. We show that the male sterile phenotype of the S-Lap mutants is caused by defects in paracrystalline material accumulation and abnormal structure of the elongated major mitochondrial derivatives. Our work shows that S-Lap proteins localize and accumulate in the paracrystalline material of the major mitochondrial derivative. Therefore, we propose that S-Lap proteins are important constituents of the paracrystalline material of Drosophila melanogaster sperm.

Developmental and tissue specific changes of ubiquitin forms in Drosophila melanogaster.

In most Eukaryotes, ubiquitin either exists as free monoubiquitin or as a molecule that is covalently linked to other proteins. These two forms cycle between each other and due to the concerted antagonistic activity of ubiquitylating and deubiquitylating enzymes, an intracellular ubiquitin equilibrium is maintained that is essential for normal biological function. However, measuring the level and ratio of these forms of ubiquitin has been difficult and time consuming. In this paper, we have adapted a simple immunoblotting technique to monitor ubiquitin content and equilibrium dynamics in different developmental stages and tissues of Drosophila. Our data show that the level of total ubiquitin is distinct in different developmental stages, lowest at the larval-pupal transition and in three days old adult males, and highest in first instar larvae. Interestingly, the ratio of free mono-ubiquitin remains within 30-50% range of the total throughout larval development, but peaks to 70-80% at the larval-pupal and the pupal-adult transitions. It stays within the 70-80% range in adults. In developmentally and physiologically active tissues, the ratio of free ubiquitin is similarly high, most likely reflecting a high demand for ubiquitin availability. We also used this method to demonstrate the disruption of the finely tuned ubiquitin equilibrium by the abolition of proteasome function or the housekeeping deubiquitylase, Usp5. Our data support the notion that the ubiquitin equilibrium is regulated by tissue- and developmental stage-specific mechanisms.