Extracellular vesicles (EVs) play critical roles in intercellular communications. Recent studies suggested that tumor–derived EVs induce phenotypic changes in target cells, creating a premetastatic niche that promotes cancer metastasis. Here, I introduce studies on the interaction between EVs and target cells by single–particle tracking and super–resolution movie observation. EVs could be categorized into several subtypes, which have different tetraspanin marker proteins. Integrin α6β4 and α6β1, with the aid of CD151 in all the EV subtypes, bound to laminin more frequently than fibronectin on the target cell surface. EV subtypes containing CD63 have lower membrane fluidity and induce the accumulation of raft–like structures underneath the EV in the target cell membrane. Upon the EV binding, integrin β1 molecules are accumulated underneath the EVs in the target cell membrane, which trigger intracellular signal transduction, Ca2+ mobilization, activation of dynamin, and promote internalization of the EVs by target cells. Thus, this review introduces the studies on dynamics of EVs and their association with target cells as revealed by high–resolution imaging.

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Giant unilamellar vesicles (GUVs) mimic cell–sized membranes, allowing direct optical microscopic observation with lipid compositions arbitrarily controlled. Although extensively studied, GUV preparation remains challenging under physiological conditions. To address this, simplified methods such as gentle hydration with osmolytes, reverse-phase/centrifugation, and related protocols, have been developed, enabling efficient GUV formation. These advances broaden the applications from fundamental membrane studies to use of natural lipid sources. In this article, I mainly review the experimental approaches developed in our laboratory.

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Optical manipulation has emerged as an indispensable tool for the non–invasive and non–destructive interrogation of biological systems at the molecular and cellular levels. Here, we present a novel application of laser–induced perturbation for manipulating molecular dynamics in neuronal cells. This approach provides a promising framework for investigating membrane-associated phenomena at high spatial and temporal resolutions.

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Substrate supported lipid bilayers (SLBs) are versatile model of the biological membrane. We have developed a patterned SLB composed of polymerized and natural (fluid) lipid bilayers. The polymeric bilayer acts as a framework to define the geometry of the fluid bilayers and enhance their stability. The fluid bilayer can contain natural molecules including membrane proteins and can reproduce the functions of the biological membrane. A nanometric space can be generated on the surface of patterned SLBs and enable to detect membrane–bound molecules with a significantly lowered background noise. Patterned SLBs in combination with a nanometric confinement provide a promising platform for biophysical studies as well as biomedical applications.

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Macrophages are known to play important role in early immune reaction in our body. Recent research have elucidated that macrophages existing surround the tumor tissues underwent quiescence in immune response against tumor cells. An appropriate stimulation of macrophages by using drug containing nanoparticles may be one of the key challenges. Macrophages are known to phagocytize particulate materials by means of particle size, surface hydrophobicity, and particle shapes. We have been working on the preparation of thermoresponsive core–corona type nanoparticles and their application to biomaterials. Here, preparation of thermoresponsive and transformable nanoparticles and their interaction with macrophages are overviewed. The appropriate control of the particle properties, i.e., core glass transition temperature and corona transition temperature, we can modulate phagocytosis of nanoparticles by macrophages.

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Mitochondria are essential for energy metabolism and apoptosis. Dysfunction, especially mitochondrial DNA (mtDNA) mutations, causes intractable diseases. We developed the MITO–Porter, a nanocarrier that delivers molecules directly into mitochondria. With this system, RNA supplementation therapy reduced mutation levels and improved function in patient–derived cells. We further applied the MITO–Porter to mitochondrial genome editing by delivering CRISPR/Cas9 ribonucleoproteins, achieving sequence–specific mtDNA cleavage and heteroplasmy correction. Ongoing studies address scale–up and social implementation, including industry–academia collaboration. While challenges remain in specificity, safety, and regulation, mitochondrial–targeted Drug Delivery System (DDS) offers a promising platform for novel therapeutic strategies.

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Mitochondria are double–membrane organelles that originated from bacterial endosymbiosis. They perform multiple functions, including not only energy production via aerobic respiration but also the metabolism of various substances and participation in cellular responses. Mitochondria frequently undergo fusion and fission, and they significantly alter their morphology in response to cellular signals and differentiation. However, the detailed molecular mechanisms controlling mitochondrial fusion and fission remain unclear. We have found that during mitochondrial dysfunction, the L–OPA1 protein–the gene product responsible for optic atrophy–is proteolytically cleaved in the inner membrane, thereby suppressing its fusion activity. This, in turn, leads to the selective degradation of dysfunctional mitochondria as part of mitochondrial quality control. Furthermore, using a silkworm expression system, we successfully expressed and purified membrane-bound L–OPA1, and reconstituted GTP–dependent inner membrane fusion reactions in vitro. In this review, we outline the molecular details of mitochondrial fusion, focusing on our recent research progress.

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We identified Golgi membrane–associated degradation (GOMED) as a novel intracellular degradation pathway. GOMED has been implicated in diverse physiological contexts, including the regulation of insulin secretion in pancreatic β–cells and the suppression of iron accumulation in the brain. In this review, we focus on the role of GOMED in erythrocyte differentiation during mouse embryogenesis. Specifically, we highlight how residual Golgi membranes in reticulocytes serve as isolation membranes to sequester and eliminate mitochondria, based on ultrastructural evidence obtained from electron microscopy. Furthermore, we discuss the unique features of GOMED in comparison with canonical Atg5/Atg7–dependent autophagy and emphasize its indispensable role in ensuring proper erythrocyte maturation.

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Active molecules derived from various materials for cellular function and disease control have been developed. However, developed active molecules exhibit poor intracellular translocation, rendering them unsitable for use as therapeutic reagents. To enhance intracellular translocation efficiency, the development of intracellular delivery methods utilizing cell–penetrating peptides (CPPs) is advancing globally. Nevertheless, even if the intracellular translocation efficiency of active molecules increases, they cannot fully exert their intended physiological activity unless they reach the specific “location” within the cell where they should function. The author has previously conducted research on developing CPPs with high targeting affinity for mitochondria and on the application of DDS for developing active molecules to mitochondria using CPPs as carriers. In this review, I here show the development of CPPs with mitochondrial targeting function, the techniques employed using peptide chemistry, and examples of intracellular delivery of active molecules.

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Contamination of portable water source with organic micropollutants have increasingly recognized as a potential risk for the human health. Such contaminants include pesticide, herbicide, medicines, PFAS (per– and polyfluoroalkyl substance) and so on. These organic micropollutants could be removed by conventional spiral wound nanofiltration (NF) technology, however, it often requires extensive pretreatment, thus had to increase greenhouse gas emission in the entire treatment process while solving human health issue. In order to solve this problem, direct nanofiltration (dNF) technology has been developed. The hollow fiber dNF membrane, with the patented polyelectrolytes based layer by layer technology, was tested with actual surface water that contains micropollutants. For most of the micropollutant including PFAS, the dNF permeate showed below detection limits, which was not possible with conventional hollow fiber UF/MF technology due to their larger pore size. The technology was applied to the surface water in The Netherlands and Treated Municipal Waster Water in Mexico with successful results.

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