Current chemical strategies for modifying the surface of extracellular vesicles (sEVs) often struggle to balance efficient functionalization with preserving structural integrity. Here, we present a modular approach for the surface modification of sEVs using a chimeric adaptor protein (CAP). The CAP was designed with three key features: a SNAP-tag for stable and modular binding, long and rigid linker to enhance spatial accessibility and conjugation efficiency, and the N-terminal sorting domain derived from syntenin to improve CAP expression on the sEV. We established a postsynthetic method to introduce diverse functional molecules onto sEVs, creating a versatile system termed "sEV-X" (where X represents an organic molecule, protein, or nanoparticle). Quantitative analyses at the single-molecule level revealed a linear relationship between CAP expression and the number of conjugated functional molecules, underscoring the importance of steric hindrance mitigation in sEV surface engineering. Moreover, antibody-conjugated sEVs as drug carriers, demonstrated significant tumor-specific delivery and therapeutic efficacy in a tumor-bearing mouse model, underscoring the potential of CAP-expressing sEVs as a customizable therapeutic vesicle. Overall, the CAP technology may serve as a universal platform for advancing the development of sEV-based therapeutics.

Investigation of the fundamental microscopic processes occurring in organic reactions is essential for optimising both organocatalysts and synthetic strategies. In this study, single-molecule fluorescence microscopy was employed to study the Diels–Alder reaction catalysed by a first-generation MacMillan catalyst, providing direct insights into its kinetic dynamics. This reaction proceeds via a series of reversible processes under equilibrium conditions ( S ⇄ IM 1 ⇄ IM 2 → P , IM 1 and IM 2 : N,O-acetal and iminium ion intermediates, respectively). The individual reaction trajectories of single molecules were directly observed in real-time, and the kinetic transitions between the different states were quantitatively analysed using a hidden Markov model, thereby enabling precise determination of the kinetic rate constants and transition probabilities at the single-molecule level. In particular, the unique structural features of the MacMillan catalyst were probed to reveal how specific interactions stabilise the reaction intermediates and influence their kinetic behaviours. These findings highlight the importance of single-molecule fluorescence microscopy in understanding the fundamental mechanisms of organic reactions and guiding the rational design of more effective catalysts.

Epidermal growth factor receptor (EGFR) dimerization plays a pivotal role in cellular signaling, influencing proliferation and disease progression, particularly in cancer. Despite extensive studies, the quantitative relationship between EGFR expression levels and dimerization efficiency remains incompletely understood. In this study, we investigated EGFR dimerization kinetics using ensemble-level biochemical assays and single-molecule tracking (SMT) in living cells. Our findings revealed noncanonical negative cooperative dimerization, where the monomer-to-dimer transition rate decreased as EGFR expression increased, challenging the assumptions of a simplistic reaction model. Furthermore, we identified a dimer-specific degradation pathway highlighting the open-system nature of the plasma membrane environment. These findings establish a quantitative framework for understanding EGFR dimerization dynamics, offering insights into the complex regulatory principles governing membrane protein interactions. This model not only improves our understanding of EGFR-mediated signaling but also suggests broader applicability for the therapeutic targeting of membrane protein systems.

Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disorder with a complex pathogenesis that requires combination therapies rather than monotherapies. Extracellular vesicles (EVs) exhibit inherently efficient delivery to the liver and can be engineered to carry various therapeutic substances, making them promising agents. In this study, EVs were engineered to display fibroblast growth factor 21 (FGF21) on their surface and encapsulate miR-223 (223/F-EVs), aiming to improve steatosis and alleviate inflammation and fibrosis, respectively. Introducing the 223/F-EVs into human liver cell lines significantly reduced both basal and induced levels of lipid storage, inflammation, and fibrosis markers. Furthermore, using an FGF21-blocking antibody or miR-223 inhibitor effectively diminished the efficacy of the 223/F-EVs, confirming the essential roles of FGF21 and miR-223 in these processes. In a Choline-Deficient, l-Amino acid-defined, High-Fat Diet (CDAHFD)-fed mouse model, intravenously administered 223/F-EVs demonstrated liver-preferential delivery and a marked reduction in the MASH phenotype without compromising bone density, unlike conventional FGF21 treatment. Collectively, 223/F-EVs convey FGF21 and miR-223 exclusively to the liver, offering strategic advantages by mitigating MASH progression via multiple pathways. This study lays a solid foundation for further investigation of engineered EVs as a transformative therapeutic approach for treating MASH.

Death receptor (DR) is a unique transmembrane receptor mediating extrinsic factor-induced programmed cell death pathways. For most DRs, the regulatory mechanism has been revealed to involve agonistic death ligand (e.g., TRAIL, TNF-alpha)-driven receptor clustering and activation, offering an in-depth understanding of the mechanistic basis for cell death regulation and therapeutic strategies. However, only death receptor 6 (DR6) remains orphan, and its molecular mechanism is poorly understood. Here, we identified retinol binding protein 4 (RBP4), a major vitamin-A carrier protein, as an antagonistic ligand of DR6. Single-molecule photobleaching and single-receptor tracking analyses revealed that RBP4 drives DR6 dimerization on the plasma membrane to prevent DR6 prodegenerative hetero-interactions and consequent neurodegenerative processes. DR6 homodimerization predominantly depends on an extracellular intrinsically disordered region, allowing the agonistic propensity of increasing Ca2+ levels. Collectively, our study establishes the distinct molecular mechanism of DR6 and highlights the non-canonical role of RBP4 as a cell death regulator. ### Competing Interest Statement The authors have declared no competing interest.

Understanding the spatial organization of membrane proteins is crucial for unraveling key principles in cell biology. The reaction-diffusion model is commonly used to understand biochemical patterning; however, applying reaction-diffusion models to subcellular phenomena is challenging because of the difficulty in measuring protein diffusivity and interaction kinetics in the living cell. In this work, we investigated the self-organization of the plasmalemma vesicle-associated protein (PLVAP), which creates regular arrangements of fenestrated ultrastructures, using single-molecule tracking. We demonstrated that the spatial organization of the ultrastructures is associated with a decrease in the association rate by actin destabilization. We also constructed a reaction-diffusion model that accurately generates a hexagonal array with the same 130 nm spacing as the actual scale and informs the stoichiometry of the ultrastructure, which can be discerned only through electron microscopy. Through this study, we integrated single-molecule experiments and reaction-diffusion modeling to surpass the limitations of static imaging tools and proposed emergent properties of the PLVAP ultrastructure.

The biological process of aging is thought to result in part from accumulation of senescent cells in organs. However, the present study identified a subset of fibroblasts and smooth muscle cells which are the major constituents of organ stroma neither proliferative nor senescent in tissues of the elderly, which we termed "mid-old status" cells. Upregulation of pro-inflammatory genes (IL1B and SAA1) and downregulation of anti-inflammatory genes (SLIT2 and CXCL12) were detected in mid-old cells. In the stroma, SAA1 promotes development of the inflammatory microenvironment via upregulation of MMP9, which decreases the stability of epithelial cells present on the basement membrane, decreasing epithelial cell function. Remarkably, the microenvironmental change and the functional decline of mid-old cells could be reversed by a young cell-originated protein, SLIT2. Our data identify functional reversion of mid-old cells as a potential method to prevent or ameliorate aspects of aging-related tissue dysfunction.

Abstract The biological process of aging is thought to result in part from accumulation of senescent cells in organs. However, the present study identified that the numbers of full-senescent cells were not increased in normal elderly tissue. Instead, fibroblasts and smooth muscle cells that were neither proliferative nor fully senescent were prevalent in tissues of the elderly, which we termed “mid-old” cells. Upregulation of pro-inflammatory genes (IL1β, SAA1) and downregulation of anti-inflammatory genes (SLIT2, CXCL12) were detected in mid-old cells. In the stroma, SAA1 promotes development of the inflammatory microenvironment via upregulation of MMP9, which decreases the stability of epithelial cells present on the basement membrane, decreasing epithelial cell function. Strikingly, the microenvironmental change and the functional decline of mid-old cells could be rejuvenated by a young cell-originated protein, SLIT2. We provided the functional reverse of mid-old cells rather than elimination of senescent cells as a new concept about rejuvenation.

Transporting substances such as gases, nutrients, waste, and cells is the primary function of blood vessels. Vascular cells use membrane proteins to perform crucial endothelial functions, including molecular transport, immune cell infiltration, and angiogenesis. A thorough understanding of these membrane receptors from a clinical perspective is warranted to gain insights into the pathogenesis of vascular diseases and to develop effective methods for drug delivery through the vascular endothelium. This review summarizes state-of-the-art single-molecule imaging techniques, such as super-resolution microscopy, single-molecule tracking, and protein-protein interaction analysis, for observing and studying membrane proteins. Furthermore, recent single-molecule studies of membrane proteins such as cadherins, integrins, caveolins, transferrin receptors, vesicle-associated protein-1, and vascular endothelial growth factor receptor are discussed.