Biology subjects

Kawano, R.

Publications and source records attributed to Kawano, R..

6 recordsLinked to original sources

bioRxiv2026

Selective Recognition, Covalent Linkage and Chemical Exchange of Artificial Cells through Engineered α-Hemolysin

Bottom-up synthetic biology aims not only to build individual artificial cells but also to connect them into higher-order systems that communicate with each other. Reported artificial cells communicate through partner recognition, physical connection, and chemical signaling that are typically implemented by separate molecular species. Here, we engineer the pore-forming protein -hemolysin (AH) with the SpyTag-SpyCatcher (ST-SC) pair to integrate partner recognition, covalent linkage between cells, and nanopore-mediated connection of their lumens into a single protein system. Fusion of ST or SC to AH preserved heptamer assembly and pore formation, and allowed the two heptamers (AH-ST and AH-SC) to be covalently joined. Cryo-electron microscopy resolved the resulting head-to-head 14-mer assembly at 2.20 [A]. Calcein leakage and single-channel recordings confirmed that the engineered pores remain functional. Mixing ST- and SC-displaying giant unilamellar vesicles (GUVs) resulted in partner-specific association between the two vesicle populations. Finally, photo-triggered release of a newly synthesized caged ethidium bromide (EtBr) in sender GUVs produced EtBr accumulation in receiver GUVs that required direct physical linkage between the two GUVs. This platform provides a modular route toward artificial-cell assemblies in which both connectivity and cargo exchange are programmable.

synthetic biology↗

bioRxiv2025

Self-Assembling Synthetic Peptides Remodel Mitochondrial Properties via Nanopore Formation

Pore-forming peptides (PFPs) are powerful tools for engineering biological membranes in vitro and in living cells. They can be derived from natural sources or designed de novo, with synthetic peptides offering distinct advantages in rational design unconstrained by natural sequences. While native PFPs have been targeted to specific organelles to modulate their functions, the potential of synthetic PFPs for organelle engineering remains largely unexplored. Here, we show that the 28-amino acid synthetic peptides, SVG28 and its variant SVG28D2, can be selectively targeted to mitochondria for functional alteration. More specifically, molecular dynamics simulations first confirmed the structural stability of the SVG28 nanopore even when fused to the TOM20 transmembrane domain, a mitochondrial targeting sequence. In isolated lipid bilayers, TOM20-SVG28 formed pores with a 0.3-nS, conductance, or 1.0-nm pore. The expression efficiency was further enhanced by two amino acid substitutions (SVG28D2). In cells, both TOM20-SVG28 and TOM20-SVG28D2 localized to the mitochondria, induced morphological alterations, and reduced both membrane potential and ATP production. These observations in silico, in vitro and in cellulo collectively indicate that the synthetic SVG28 peptides self-assemble into pores in the outer mitochondrial membrane, likely permitting proton permeation. Importantly, the matrix protein Su9 remained confined within mitochondria, suggesting preserved inner membrane integrity, and thus specific effect on the target. This work establishes a framework for the design and application of genetically-encoded synthetic PFPs in living cells, opening new avenues for cellular engineering.

synthetic biology↗

bioRxiv2023

Structural flexibility of apolipoprotein E-derived arginine-rich peptides improves their cell penetration capability

Amphipathic arginine-rich peptide, A2-17, exhibits moderate perturbation of lipid membranes and the highest cell penetration among its structural isomers. We investigated the direct cell-membrane penetration mechanism of the A2-17 peptide. We designed structurally constrained versions of A2-17, stapled (StpA2-17) and stitched (StchA2-17), whose -helical conformations were stabilized by chemical crosslinking. Circular dichroism confirmed that StpA2-17 and StchA2-17 had higher -helix content than A2-17 in aqueous solution. Upon liposome binding, only A2-17 exhibited a coil-to-helix transition. Confocal microscopy revealed that A2-17 had higher cell penetration efficiency than StpA2-17 in HeLa cells. Partitioning into lipid membranes was more prominent for StchA2-17 than for A2-17 or StpA2-17; StchA2-17 remained on the cell membrane without cell penetration. Tryptophan fluorescence analysis suggested that A2-17 and its analogs had similar membrane-insertion positions between the interface and hydrophobic core. Atomic force microscopy demonstrated that A2-17 reduced the mechanical rigidity of liposomes to a greater extent than StpA2-17 and StchA2-17. Finally, electrophysiological analysis showed that A2-17 induced a higher charge influx through transient pores in a planer lipid bilayer than StpA2-17 and StchA2-17. These findings indicate that structural flexibility, which enables diverse conformations of A2-17, leads to a membrane perturbation mode that contributes to cell membrane penetration.

biophysics↗

bioRxiv2022

Facilitation by a hERG blocker is induced by pore opening and operates by pore reopening

A drug that blocks the cardiac myocyte voltage-gated K+ channels encoded by the human Ether-a-go-go-Related Gene (hERG) carries a potential risk of long QT syndrome and life-threatening cardiac arrhythmia, including Torsade de Points. Interestingly, certain hERG blockers can also facilitate hERG activation to increase hERG currents, which may reduce proarrhythmic potential. However, the molecular mechanism involved in the facilitation effect of hERG blockers remains unclear. The hallmark feature of the facilitation effect by hERG blockers is that a depolarizing preconditioning pulse shifts voltage-dependence of hERG activation to more negative voltages. Here we utilize a D540K hERG mutant to study the mechanism of the facilitation effect. D540K hERG is activated by not only depolarization but also hyperpolarization. This unusual gating property of the D540K hERG channel enables testing of hypotheses about the mechanism by which voltage induces facilitation of hERG by blockers. With D540K hERG, we find that nifekalant, a hERG blocker and Class III antiarrhythmic agent, facilitates not only current activation by depolarization but also current activation by hyperpolarization. Our results indicate that induction of facilitation is coupled to pore opening, not voltage per se. We propose that a gated-access mechanism is involved in the voltage-dependence of induction of facilitation. This study provides a molecular mechanism for modulation of hERG channels by nifekalant, a clinically important antiarrhythmic agent. Significance statementNifekalant is a clinically important antiarrhythmic agent and a hERG blocker which can also facilitate voltage-dependent activation of hERG channels after a preconditioning pulse. Here we show that the mechanism of action of the preconditioning pulse is to open a conductance gate to enable drug access to a facilitation site. Moreover, we find that facilitation increases hERG currents by altering pore dynamics, rather than acting through voltage sensors.

pharmacology and toxicology↗

bioRxiv2021

Single-cell RNA-seq analysis reveals penaeid shrimp hemocyte subpopulations and cell differentiation process

Crustacean aquaculture is expected to be a major source of fishery commodities in the near future. Hemocytes are key players of the immune system in shrimps; however, their classification, maturation, and differentiation are still under debate. To date, only discrete and inconsistent information on the classification of shrimp hemocytes has been reported, showing that the morphological characteristics are not sufficient to resolve their actual roles. Our present study using single-cell RNA sequencing, revealed nine types of hemocytes of Marsupenaeus japonicus based on their transcriptional profiles. We identified markers of each subpopulation and the differentiation pathways involved in their maturation. We also discovered cell growth factors that might play crucial roles in hemocyte differentiation. Different immune roles among these subpopulations were suggested from the analysis of differentially expressed immune-related genes. These results provide a unified classification of shrimp hemocytes, which improves the understanding of its immune system.

cell biology↗

bioRxiv2020

Linker histone H1 determines cell stiffness and differentiation

ABSTRACTThe flexibility or stiffness, one mechanical property of cells, is a promising label-free biomarker for underlying cytoskeletal or nuclear changes associated with various disease processes and changes in cell state. However, the molecular changes responsible for the whole-cell mechanical stiffness remain to be clarified. Recently, it was shown that the deterministic lateral displacement (DLD) microfluidic device, originally developed for size fractionation of some particles, might be applied to distinguish cells according to their stiffness. In this experiment, using the DLD device and various cell lines differentially expressing histone H1, a positively-charged protein localized in the linker region of chromatin, we found linear relationships between histone H1 quantity and cell stiffness. We also found that the histone H1 quantity affected cell size and even cell differentiation.

cell biology↗