Biology subjects

Morita, S.

Publications and source records attributed to Morita, S..

29 records · Page 2Linked to original sources

bioRxiv2022

Breast cancer progression and metastasis to lymph nodes reveals cancer cell plasticity and MHC class II-mediated immune regulation

Tumor-draining lymph nodes are critical sites for generating tumor antigen-specific T cells and are associated with durable immune responses. However, lymph nodes are often the first site of metastasis and lymph node metastases portend worse outcomes. Through cross-species single cell gene expression analysis of breast cancer progression and metastasis to lymph nodes, we uncovered features that define the heterogeneity, plasticity, and immune evasion of cancer cells. Notably, a subpopulation of metastatic cancer cells in the lymph node were marked by high levels of MHC class II (MHC-II) gene expression both in mice and humans. Mechanistically, the IFN-{gamma} and JAK/STAT signaling pathways mediate MHC-II expression in cancer cells. Ablation of IFNGR1/2 or CIITA, the transactivator of MHC-II, in cancer cells prevented tumor progression. Interestingly, MHC-II+ cancer cells lacked co-stimulatory molecule expression, engendered the expansion of regulatory T cells and blunted CD4+ effector T cells in the tumor-draining lymph nodes and favor tumor progression. Overall, our data suggests that cancer cell plasticity during breast cancer progression and metastasis to lymph nodes endows metastatic cells with the ability to avoid immune surveillance. These data provide the basis for new opportunities to therapeutically stimulate anti-cancer immune responses against local and systemic metastases.

cancer biology↗

bioRxiv2022

Allosteric Inhibition of the T Cell Receptor by a Designed Membrane Ligand

The T cell receptor (TCR) is a complex molecular machine that directs the activation of T cells, allowing the immune system to fight pathogens and cancer cells. Despite decades of investigation, the molecular mechanism of TCR activation is still controversial. One of the leading activation hypotheses is the allosteric model. This model posits that binding of pMHC at the extracellular domain triggers a dynamic change in the transmembrane (TM) domain of the TCR subunits, which leads to signaling at the cytoplasmic side. We sought to test this hypothesis by creating a TM ligand for TCR. Previously we described a method to create a soluble peptide capable of inserting into membranes and bind to the TM domain of the receptor tyrosine kinase EphA2 (Alves et al., eLife 2018). Here we show that the approach is generalizable to complex membrane receptors, by designing a membrane ligand for TCR. We observed that the designed peptide caused a reduction of Lck phosphorylation of TCR at the CD3{zeta} subunit. As a result, in the presence of this Peptide Inhibitor of TCR (PITCR), the proximal signaling cascade downstream of TCR activation was significantly dampened in T cells. Co-localization and co-immunoprecipitation results in DIBMA native nanodiscs confirmed that PITCR was able to bind to the TCR. We propose that PITCR binds into a crevice present between the TM helices of the CD3{zeta} and CD3{varepsilon}({delta}) subunits. Our results additionally indicate that PITCR disrupts the allosteric changes in the compactness of the TM bundle that occur upon TCR activation, lending support to the allosteric TCR activation model. The TCR inhibition achieved by PITCR might be useful to treat inflammatory and autoimmune diseases and to prevent organ transplant rejection, as in these conditions aberrant activation of TCR contributes to disease.

biophysics↗

bioRxiv2022

Cancer tissue of origin constrains the growth and metabolism of metastases

Metastases arise from a subset of cancer cells that disseminate from the primary tumor; however, the factors that contribute to proliferation of cancer cells in a secondary site are incompletely understood. The ability of cancer cells to thrive in a new tissue site is influenced by genetic and epigenetic changes that are important for disease initiation and progression, but these factors alone do not predict if and where cancers metastasize. Specific cancer types metastasize to consistent subsets of tissues, suggesting that factors associated with the primary tumor influence the tissue environments where cancers can grow. Using pancreatic cancer as a model, we find that primary and metastatic tumors share many metabolic similarities and that the tumor initiating capacity and proliferation of both primary- and metastasis-derived cells is favored in the primary site relative to the metastatic site. Moreover, propagating lung or liver metastatic cells in vivo to enrich for tumor cells adapted to grow in the lung or the liver does not enhance their relative ability to form large tumors in those sites, change their preference to grow in the primary site, nor stably alter some aspects of their metabolism relative to primary tumors. We also analyzed primary liver and lung cancer cells and find that these cells also exhibit a preference to grow in their primary site relative to metastatic sites. Together, these data suggest that the cancer tissue-of-origin influences the metabolism of both primary and metastatic tumors and may impact whether cancer cells can thrive in a metastatic site.

cancer biology↗

bioRxiv2022

Elements of divergence in germline determination in closely related species

Evolutionary transitions enable the wide diversity in life histories of plants and animals. This is particularly germane in the development of the germ line in which fitness is a direct readout of evolutionary change. Here, we focused on the gem line of two distinct sea urchin species who shared a common ancestor 50 million years ago. Even though they both rely on inherited mechanisms to specify their germ line, the integration of stage-matched single cell RNA-seq (scRNA-seq) datasets from these two sea urchins revealed a variety of differences in gene expression, including a broader expression of the germ line factor Nanos2 in Lytechinus variegatus (Lv) compared to Strongylocentrotus purpuratus (Sp). In Sp, Nanos2 mRNA expression is highly restricted to the primordial germ cells (PGCs) by a lability element in its 3UTR. This element is lacking in the mRNA of Lv Nanos2, explaining how this mRNA more broadly accumulates in the Lv embryos. We discovered that the Lv Nanos2 3UTR instead leads to a germline specific translation of the protein. The results emphasize that regulatory mechanisms resulting in germline diversity rely less on transcriptional regulation and more on post-transcriptional and post-translational restrictions of key gene products, such as Nanos2. Highlights- The first integration of scRNA-seq datasets comparing two echinoderm species. - We find Nanos2 positive cells in the embryonic soma of Lytechinus variegatus, an unusual occurrence, but not in Strongylocentrous purpuratus. - We discovered that this somatic Nanos2 mRNA is lacking an important regulatory element (GNARLE) in its 3UTR - Instead, in Lv, the 3UTR of Nanos2 leads to its specific translation in the germ cells.

developmental biology↗

bioRxiv2022

Delayed antigen-specific CD4+ T cell induction correlates with impaired immune responses to SARS-COV-2 mRNA vaccination in the elderly

Despite the clinical efficacy of coronavirus disease 2019 mRNA vaccines, the elderly demonstrate lower IgG levels and neutralizing titers and a higher risk of severe diseases. CD4+ T cells play a central role in regulating antigen-specific antibody and CD8+ T-cell responses; however, because their composition and functionality change significantly with age, relationships between age-associated defects in T cells and the immunogenicity of or reactogenicity to mRNA vaccines are unclear. Using a vaccine cohort (n = 216), we found that the elderly (aged [≥]65 years) showed delayed induction and early contraction of vaccine-specific CD4+ T cells, and that the compromised C-X-C motif chemokine receptor 3+ circulating T follicular helper cell response after the first dose was associated with the lower IgG levels. Additionally, the elderly experienced significantly fewer systemic adverse effects (AEs) after the second dose, with those exhibiting few AEs showing lower cytokine+ CD4+ T cells after the first dose and lower antibody levels after the second dose. Furthermore, T helper 1 cells in the elderly expressed higher levels of programmed cell death protein-1, a negative regulator of the T-cell response, which was associated with less production of vaccine-specific CD4+ T cells and impaired CD8+ T-cell expansion. Thus, efficient induction of vaccine-specific effector/memory CD4+ T cells after the first dose may trigger robust cytokine production after the second dose, leading to effective vaccine responses and higher systemic reactogenicity. These results suggested that an enhanced CD4+ T-cell response after the first dose is key to improved vaccination efficacy in the elderly. One Sentence SummaryWe compared immunogenicity and reactogenicity to COVID-19 mRNA vaccine in 107 adults (aged <65 years) and 109 elderly (aged [≥]65) individuals.

immunology↗

bioRxiv2022

The draft genome sequence of Japanese rhinoceros beetle Trypoxylus dichotomus

Beetles are the largest insect order and one of the most successful animal groups in terms of number of species. The Japanese rhinoceros beetle Trypoxylus dichotomus (Coleoptera, Scarabaeidae, Dynastini) is a giant beetle with distinctive exaggerated horns present on the head and prothoracic regions of the male. T. dichotomus has been used as research model in various fields such as evolutionary developmental biology, ecology, ethology, biomimetics, and drug discovery. In this study, de novo assembly of 615 Mb, representing 80% of the genome estimated by flow cytometry, was obtained using the 10x Chromium platform. The scaffold N50 length of the genome assembly was 8.02 Mb, with repetitive elements predicted to comprise 49.5% of the assembly. In total, 23,987 protein-coding genes were predicted in the genome. In addition, de novo assembly of the mitochondrial genome yielded a contig of 20,217 bp. We also analyzed the transcriptome by generating 16 RNA-seq libraries from a variety of tissues of both sexes and developmental stages, which allowed us to identify 13 co-expressed gene modules. The detailed genomic and transcriptomic information of T. dichotomus is the most comprehensive among those reported for any species of Dynastinae. This genomic information will be an excellent resource for further functional and evolutionary analyses, including the evolutionary origin and genetic regulation of beetle horns and the molecular mechanisms underlying sexual dimorphism.

genomics↗

bioRxiv2021

Discrete LAT condensates encode antigen information from single pMHC:TCR binding events

LAT assembly into a two-dimensional protein condensate is a prominent feature of antigen discrimination by T cells. Here, we use single-molecule imaging techniques to resolve the spatial position and temporal duration of each pMHC:TCR molecular binding event while simultaneously monitoring LAT condensation at the membrane. An individual binding event is sufficient to trigger a LAT condensate, which is self-limiting, and neither its size nor lifetime is correlated with the duration of the originating pMHC:TCR binding event. Only the probability of the LAT condensate forming is related to the pMHC:TCR binding dwell time. LAT condenses abruptly, but after an extended delay from the originating binding event. A LAT mutation that facilitates phosphorylation at the PLC-{gamma}1 recruitment site shortens the delay time to LAT condensation and alters T cell antigen specificity. These results identify a role for the LAT protein condensation phase transition in setting antigen discrimination thresholds in T cells.

biophysics↗

bioRxiv2021

Height, but not binding epitope, affects the potency of synthetic TCR agonists

Under physiological conditions, peptide-MHC (pMHC) molecules can trigger T-cell receptors (TCRs) as monovalent ligands that are sparsely distributed on the plasma membrane of an antigen-presenting cell. TCR can also be activated by artificial clustering, such as with pMHC tetramers or antibodies; however, these strategies circumvent many of the natural ligand discrimination mechanisms of the T cell and can elicit non-physiological signaling activity. We have recently introduced a synthetic TCR agonist composed of an anti-TCR{beta} Fab antibody fragment covalently bound to a DNA oligonucleotide, which serves as a membrane anchor. This Fab-DNA ligand efficiently activates TCR as a monomer when membrane-associated and exhibits a potency and activation profile resembling agonist pMHC. In this report, we explore the geometric requirements for effective TCR triggering and cellular activation by Fab-DNA ligands. We find that T cells are insensitive to the ligand binding epitope on the TCR complex, but that length of the DNA tether is important. Increasing the intermembrane distance spanned by Fab-DNA:TCR complexes decreases TCR triggering efficiency and T cell activation potency, consistent with the kinetic-segregation model of TCR triggering. These results establish design parameters for construction of synthetic TCR agonists that are able to activate polyclonal T cell populations, such as T cells from a human patient, in a similar manner as the native pMHC ligand. STATEMENT OF SIGNIFICANCEWe report geometric requirements for potent T cell activation by synthetic TCR ligands that mimic biophysical properties of the native pMHC ligand, but have the additional ability to activate polyclonal T cell populations. We find that increasing the space between apposed membranes at TCR binding events decreases ligand potency, but that changing the ligands binding epitope on the TCR has essentially no effect. The observed decrease in potency with increased ligand height is attributed to the longer ligands attenuated ability to trigger TCR at binding events.

biophysics↗

bioRxiv2019

2-Cl-C.OXT-A Stimulates Contraction through the Suppression of Phosphodiesterase Activity in Human Induced Pluripotent Stem Cell-derived Cardiac Organoids

Background2-Cl-C.OXT-A (COA-Cl) is a novel synthesized adenosine analog that activates S1P1 receptor (S1P1R) and combines with adenosine A1 receptor (A1R) in G proteins and was shown to enhance angiogenesis and improve the brain function in rat stroke models. However, the role of COA-Cl in hearts remains unclear. COA-Cl, which has a similar structure to xanthine derivatives, has the potential to suppress phosphodiesterase (PDE), which is an important factor involved in the beating of heart muscle. Methods and resultsCardiac organoids with fibroblasts, human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs), and hiPSC-derived endothelial cells (hiPSC-ECs) were cultured until they started beating. The beating and contraction of organoids were observed before and after the application of COA-Cl. COA-Cl significantly increased the beating rate and fractional area change in organoids. To elucidate the mechanism underlying these effects of COA-Cl on cardiac myocytes, pure hiPSC-CM spheroids were evaluated in the presence/absence of Suramin (antagonist of A1R). The effects of COA-Cl, SEW2871 (direct stimulator of S1P1R), two positive inotropes (Isoproterenol [ISO] and Forskolin [FSK]), and negative inotrope (Propranolol [PRP]) on spheroids were assessed based on the beating rates and cAMP levels. COA-Cl stimulated the beating rates about 1.5-fold compared with ISO and FSK, while PRP suppressed the beating rate. However, no marked changes were observed with SEW2871. COA-Cl, ISO, and FSK increased the cAMP level. In contrast, the level of cAMP did not change with PRP or SEW2871 treatment. The results were the same in the presence of Suramin as absence. Furthermore, an enzyme analysis showed that COA-Cl suppressed the PDE activity by half. ConclusionsCOA-Cl, which has neovascularization effects, suppressed PDE and increased the contraction of cardiac organoids, independent of S1P1R and A1R. These findings suggest that COA-Cl may be useful as an inotropic agent for promoting angiogenesis in the future.

pharmacology and toxicology↗

bioRxiv2018

doublesex regulates sexually dimorphic beetle horn formation by integrating spatial and temporal developmental contexts in the Japanese rhinoceros beetle Trypoxylus dichotomus

AbstractMany scarab beetles have sexually dimorphic exaggerated horns that are an evolutionary novelty. Since the shape, number, size, and location of horns are highly diverged within Scarabaeidae, beetle horns are an attractive model for studying the evolution of sexually dimorphic and novel traits. In beetles including the Japanese rhinoceros beetle Trypoxylus dichotomus, the sex determination gene doublesex (dsx) plays a crucial role in sexually dimorphic horn formation during larval-pupal development. However, knowledge of when and how dsx drives the gene regulatory network (GRN) for horn formation to form sexually dimorphic horns during development remains elusive. To address this issue, we identified a Trypoxylus-ortholog of the sex determination gene, transformer (tra), that regulates sex-specific splicing of the dsx pre-mRNA, and whose loss of function results in sex transformation. By knocking down tra function at multiple developmental timepoints during larval-pupal development, we estimated the onset when the sex-specific GRN for horn formation is driven. In addition, we also revealed that dsx regulates different aspects of morphogenetic activities during the prepupal and pupal developmental stages to form appropriate morphologies of pupal head and thoracic horn primordia as well as those of adult horns. Based on these findings, we discuss the evolutionary developmental background of sexually dimorphic trait growth in horned beetles.\n\nAuthor Summary:\n\nBeetle horns are highly enriched in a particular family Scarabaeidae, although the shape, size and number of horns are diversified within the group. In addition, many scarab beetle horns are sexually dimorphic. It has been questioned how a particular group of beetles has originated and diversified evolutionary novel horns. Here we found the exact time when morphological sexual dimorphism of horn primordia appeared, estimated the onset of the developmental program for sexually dimorphic horn formation driven by Doublesex, and revealed that Doublesex regulates different aspects of cell activities of horn primordia depending on the spatiotemporal contexts. Our study provides our understanding regarding regulatory shifts in these mechanisms during the evolution of sexually dimorphic traits in horned beetles.

developmental biology↗

bioRxiv2017

Multiple strategies for heat adaptation in rice endosperms revealed by on-site cell-specific analysis

Plant cells have multiple strategies to adapt to environmental stresses. Rice endosperms form chalkiness in a part of the tissue under heat conditions during the grain-filling stage, although nitrogen supply reduces chalky rice. Air spaces formed in the cells cause an irregular light reflection and create chalkiness, yet what exactly occurs remains unclear at cell level. Through on-site cell-specific analysis, we show that heat-treated cells adjust osmotically and retard protein synthesis to preserve protein storage vacuoles in the cytosol, resulting in air space formation. Application of nitrogen enhances heat tolerance to sustain protein body and amyloplast development during strong osmotic adjustment, which diminishes air spaces to avoid chalkiness. Hence, we conclude that rice endosperm cells could alter organelle compartments spatially during the heat adaptation, depending on the available nitrogen level. Our findings provide new insight into the cellular mechanism of rice chalky formation as a strategy for heat acclimation.

plant biology↗