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Biology subjects

Toda, S.

Publications and source records attributed to Toda, S..

7 recordsLinked to original sources

Coupling Mechanical Regulation with Biochemical Reaction-Diffusion Circuits Yields Robust Self-Organized Pattern Formation.

Cell-cell signaling circuits that combine local self-activation with long-range inhibition have long been proposed as a theoretical mechanism sufficient to generate cellular patterns, such as spots and stripes. Here we construct synthetic pattern-forming circuits, implementing local self-activation (positive feedback) using juxtacrine synNotch receptor interactions, and implementing long-range inhibition using diffusible competitor molecules. While combining local positive feedback with long-range inhibition leads to more spatially heterogeneous cell states, these synthetic circuits do not robustly lead to well defined patterns. We find, however, that if we couple these reaction-diffusion circuits with induction of genes that regulate cell mechanics - such Cadherin molecules that promote local cell adhesion and sorting - we observed the emergence of much more well-defined cellular patterns. Theoretical analysis indicates while reaction-diffusion circuits can be sufficient to generate patterns under precisely balanced parameter conditions, the close coupling of cell mechanical/sorting significantly increases the robustness of pattern formation (the parameter space yielding patterns). Thus, circuits that closely integrate signaling and mechanical changes may underlie many evolved morphogenic pattern formation systems.

synthetic biology↗

Reconstituting organotypic 2D microtissue co-cultures via sequential stenciling

In mammalian organisms, native tissue function depends on precise spatial organization down to the cellular level. Reconstituting tissue architectures in 2D in vitro platforms can provide a means to study direct and indirect cell-cell interactions in a variety of tissue contexts while remaining compatible with high-throughput assays and high-resolution live imaging. We combine cost-effective stereolithography leveraging 3D printing with replica molding to stencil spatially defined, multicellular culture systems with sub-millimeter resolution onto planar substrates. The system is designed for ease of use, requires no complex fabrication setups and scales readily to 96-well plates. Sequential stencil application and removal under a biosafety cabinet enables controlled positioning of multiple cell types and supported the maturation of tissue assemblies. We demonstrate the utility of this stencil-based patterning strategy in three applications. First, we employ a combination of two circular stencils to recreate a structural feature characteristic for the tumor microenvironment of solid tumors: the encapsulation of colorectal cancer cells by cancer-associated fibroblasts. Resulting cell patternings reproduce native tissue dynamics of the densely packed tumor tissues, in which cancer-associated fibroblast cells actively compress the cancer cells and confer targeted therapy resistance. Second, we probe the synthetic, diffusible morphogen system synNotch in patterned cell patches, where GFP-releasing cells generate a ligand-dependent gradient. Third, we recapitulate the characteristic crypt-villus architecture of the mammalian intestine by patterning intestinal organoids within a stencil-restricted crypt region and allowing differentiating cells to collectively migrate along a designed villus axis. The presented strategy allows for rebuilding multicellular tissue architectures in vitro with biologically relevant spatial precision for high-throughput drug screenings and dissection of tissue-specific cellular interactions.

bioengineering↗

A synNotch-based morphogen detection system reveals sFRP2 enhances Wnt3a signaling

Morphogen gradients provide essential positional information during tissue development, yet the extracellular mechanisms that regulate morphogen transport and presentation remain poorly understood. Here, we introduce a mechanosensitive detection system based on synthetic Notch (synNotch) receptors that selectively detects surface-bound, but not soluble, morphogen complexes. Applying this platform to Wnt signaling, we demonstrate that secreted Frizzled-related protein 2 (sFRP2) promotes the recruitment of Wnt3a to the cell surface via heparan sulfate proteoglycans, enabling coordinated endocytosis and robust activation of canonical Wnt/{beta}-catenin signaling. Notably, sFRP2 extends the effective signaling range of Wnt3a and amplifies Wnt responses under ligand-limiting conditions. In intestinal organoid cultures, sFRP2 enhances Wnt3a-driven growth and induces high-Wnt morphological states with low-level Wnt concentrations. These findings identify sFRP2 as an extracellular carrier that stabilizes surface-bound Wnt3a and regulates both the strength and spatial range of Wnt signaling. More broadly, this work demonstrates the utility of synNotch mechanosensing for dissecting extracellular morphogen dynamics and highlights morphogen carrier proteins as a platform for optimizing organoid culture.

cell biology↗

Extended poly(A) tails are a shared feature of herpesvirus mRNAs

Poly(A) tails are present on most cellular and viral mRNAs, providing a platform for poly(A)-binding proteins that stimulate translation and regulate the deadenylation and stability of transcripts in the cytoplasm. Here we leverage nanopore direct RNA sequencing to analyse the distribution of poly(A) tail lengths on cellular and viral mRNAs across Herpesviridae and other DNA and RNA virus infections. We find that herpesvirus mRNA poly(A) tails are consistently longer than those on cellular and other viral transcripts, presenting a previously unrecognized yet widespread mechanism to advantage herpesviral gene expression. This contrasts with the templated poly(A) tails on coronavirus RNAs and those on cytoplasmically transcribed poxviral mRNAs, which are more similar in length to those on host mRNAs. Herpesviral noncoding RNAs display differential poly(A) tailing patterns which do not correlate with nuclear localisation while individual herpesviral mRNAs also show variation in the extent to which their poly(A) tail lengths change during the virus lifecycle, suggestive of additional uncharacterised layers of poly(A) tail length regulation. Importantly, while we detect non-adenosine nucleotides within herpesviral poly(A) tails, which are known to oppose deadenylase activity, this "mixed tailing" is not at sufficient frequency to explain the widespread extended tails of herpesvirus mRNAs.

microbiology↗

Engineering Inducible Cell Fate Transitions by Harnessing Epigenetic Silencing

During development, cell-cell communication induces state transitions that are maintained by epigenetic regulation. Here, we harness endogenous epigenetic silencing machinery to engineer synthetic circuits that induce stable gene expression changes. Using synthetic Notch receptors that control the chromatin regulators KRAB and Dnmt3L, we developed input-sensing switches that induce self-sustained silencing of target loci. We used these modules to construct circuits in which combinatorial inputs direct a choice among multiple alternative states. These epigenetic switches can also be inverted to yield sustained activation of target genes. We demonstrate that these epigenetic switches can be used to drive morphological changes triggered by transient cell-cell interactions, but that remain stable over many cell divisions, as is observed in development. Additionally, these circuits can be used to drive the reprogramming of fibroblasts to a neuron-like state. These synthetic epigenetic circuits represent an important step towards engineering cell populations capable of coordinated multi-cell state decisions.

synthetic biology↗

Elasto-Plastic Transition in Epithelial Folding

During morphogenesis, epithelial sheets undergo sequential folding to form three-dimensional organ structures. The resulting folds are irreversible, ensuring that morphogenesis progresses in one direction. However, the mechanism establishing the irreversibility of folding remains unclear. Here, we report a novel mechanical property of epithelia that is responsible for folding irreversibility. Using a newly developed mechanical indentation assay, we demonstrate that short-term or low-curvature folding induces an elastic, shape-restoring response. In contrast, combined long-term, high-curvature folding results in plastic, irreversible deformation. This elastic-to-plastic transition occurs in a switch-like manner, with critical thresholds for the folding curvature and duration. Specific cells at the fold initiate this transition, sensing the curvature and duration of folding on their apical side via mechanosensitive signaling pathways, including transient receptor potential canonical (TRPC) 3/6-mediated calcium influx and ligand-independent epidermal growth factor receptor activation. These pathways induce F-actin accumulation into a bracket-like structure across the fold, establishing the transition. The duration threshold is determined and tunable by the actin polymerization rate. These results demonstrate that cells control the irreversibility of epithelial folding by detecting folding characteristics and adaptively switching between elastic and plastic responses. This finding resolves a long-standing question about the directionality of morphogenesis.

developmental biology↗

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↗