Biology subjects

Matsuzaki, K.

Publications and source records attributed to Matsuzaki, K..

7 recordsLinked to original sources

bioRxiv2026

Analytical methodology for unveiling the physical effect of detergents on the function of full-length membrane proteins using a single-molecule tracking system

G protein-coupled receptors (GPCRs) are important targets for drug discovery because they are the largest and most diverse family of membrane proteins in the human body. As exemplified by {beta}2-adrenergic receptor ({beta}2AR), which is a typical class-A GPCRs, they are prone to denaturation and inactivation after solubilization. Although diverse techniques have been developed, current structural and functional analyses are still limited to proteins with relatively stable and high expression. However, some mutant variants and misfolded proteins involved in diseases have extremely low expression levels and may be difficult to analyze. To overcome these limitations, we established a novel analytical platform for evaluating the ligand-binding ability of full-length {beta}2AR as a model at the single-molecule level without purification and addressing challenges such as membrane proteins with low expression levels and structural instability. This method enables the direct use of unpurified receptors immediately after solubilization and allows us to use only a small amount of sample ([~]10 ng) for observation and to distinguish between specific and nonspecific ligand binding by fitting. Furthermore, we unveiled the physical properties of detergents on the structural stability of solubilized receptors and found that the lateral pressure within detergent micelles affects ligand-binding ability. Detergents that provided a fluid microenvironment were able to maintain ligand-binding ability for several days even after solubilization; conversely, detergents that provided a rigid microenvironment caused the protein to lose its activity earlier. Our method could be a promising tool for the structural and functional analysis of membrane proteins untargeted until now. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/740883v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@3d274corg.highwire.dtl.DTLVardef@defd0forg.highwire.dtl.DTLVardef@1b81eb4org.highwire.dtl.DTLVardef@e7c275_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Curvature-sensing peptide functions as a membrane interfactant that glues small extracellular vesicles to cell membranes and enhances vesicle cellular uptake

Small extracellular vesicles (sEVs) are lipid nanoparticles secreted from mammalian cells that are involved in the transfer of informational or therapeutically effective substances between cells. Although the scope of research on sEVs as biocompatible carriers for drug delivery to diseased tissues and cells is expanding, several challenges remain, such as low cellular uptake efficiency and difficulty in loading drugs. It is desirable to alleviate the energy barriers associated with the cellular uptake of sEVs and minimize perturbation of sEVs when loading drugs onto them. In this study, we developed a simple drug-loading system for sEVs using a dimeric curvature-sensing peptide, which enhances sEV accumulation on the cell surface by acting as an adhesive, subsequently inducing endocytic uptake of sEVs through a clathrin-mediated pathway. The dimeric curvature-sensing peptide selectively binds to the sEV surface within 10 min, even in the presence of serum proteins, and functions as a membrane interfactant to reduce the energy barriers for the cellular uptake of sEVs. The cellular uptake of sEVs and the dimeric curvature-sensing peptide under coexisting conditions increased to over fivefold and 20-fold, respectively, compared with those administered alone. Furthermore, the dimeric curvature-sensing peptide can efficiently load anticancer drugs onto the surface of sEVs, and the system effectively induces apoptosis in two types of cancer cells. Dimeric curvature-sensing peptide is a novel technique with potential applications in drug delivery.

biochemistry↗

bioRxiv2025

Kinetic asymmetry drives kinesin-1's unidirectional and processive movement

Kinesin-1 is a dimeric motor protein that uses ATP hydrolysis energy to move along microtubules in a hand-over-hand manner1. The unidirectional movement of kinesin-1 has traditionally been explained by an ATP-dependent power stroke action of the neck linker2-4, connecting its two catalytic domains (heads), that biases the diffusional motion forward (biased-diffusion). However, recent studies on synthetic molecular motors have supported a Brownian ratchet mechanism based on kinetic asymmetry between two locations (biased- binding)5-9, and which of these mechanisms applies to biological motors remains debated10-12. Here, we engineered a two-headed kinesin that alternately uses these mechanisms to step forward, allowing us to investigate how they contribute to unidirectional movement. The tethered head that uses biased-diffusion frequently rebound to the rear-binding site but eventually stepped forward, as the front head remained securely bound to the microtubule. The biased-binding mechanism proved more efficient by preventing rebinding of the detached head and was independent of ATP binding. Instead, ATP hydrolysis energy is primarily consumed to ensure preferential detachment of the rear head. These findings demonstrate that kinesin-1 functions as an information ratchet based on kinetic asymmetry in microtubule-binding and detachment of the heads, while power strokes serve to enhance movements under load.

biophysics↗

bioRxiv2025

Generation of iPSC-derived CD4+ Th1 cells enhancing chimeric antigen receptor-T cell cytotoxicity

CD4+ T cells are anticipated to enhance the overall immune response, including the anti-tumor activity of chimeric antigen receptor (CAR)-T cell therapy. In the past, we established a culture system to generate CD8+ T cells from iPS cells (iPSCs); however, it was challenging to generate CD4+ T cells. Drawing inspiration from the observation that adult T cell leukemia (ATL) cells are consistently CD4+ and possess Treg characteristics, we successfully generated CD4+ Treg cells by reprogramming ATL cells into iPSCs and then differentiating them into T cells. Gene expression analysis of this generation system suggested that RUNX3 serves as a key regulator in T cell differentiation within the ex vivo generation system. By knocking out RUNX3, we demonstrated the generation of antigen-specific CD4+ Th1 cells via the iPSC route, thereby enhancing the activity of CD8+ CAR-T cells against GD2-expressing lymphoma. These technologies hold significant promise for contributing to "off-the-shelf" immunotherapies against malignant tumors, including solid tumors.

immunology↗

bioRxiv2024

Kinetic regulation of kinesin's two motor domains coordinates its stepping along microtubules

The two identical motor domains (heads) of dimeric kinesin-1 move in a hand-over-hand process along a microtubule, coordinating their ATPase cycles such that each ATP hydrolysis is tightly coupled to a step and enabling the motor to take many steps without dissociating. The neck linker, a structural element that connects the two heads, has been shown to be essential for the head-head coordination; however, which kinetic step(s) in the chemomechanical cycle is "gated" by the neck linker remains unresolved. Here, we employed pre-steady state kinetics and single molecule assays to investigate how the neck linker conformation affects kinesins motility cycle. We show that the backward-pointing configuration of the neck linker in the front kinesin head confers higher affinity for microtubules, but does not change ATP binding and dissociation rates. In contrast, the forward-pointing configuration of the neck linker in the rear kinesin head decreases the ATP dissociation rate but has little effect on microtubule dissociation. In combination, these conformation-specific effects of the neck linker favor ATP hydrolysis and dissociation of the rear head prior to microtubule detachment of the front head, thereby providing a kinetic explanation for the coordinated walking mechanism of dimeric kinesin.

biophysics↗

bioRxiv2023

FIGNL1 AAA+ ATPase remodels RAD51 and DMC1 filaments in meiotic DNA replication and recombination

The formation of RAD51/DMC1 filaments on single-stranded (ss)DNAs essential for homology search and strand exchange in DNA double-strand break (DSB) repair is tightly regulated. FIGNL1 AAA+++ ATPase controls RAD51-mediated recombination in human cells. However, its role in gametogenesis remains unsolved. Here, we characterized a germ-line-specific conditional knockout (cKO) mouse of FIGNL1. Fignl1 cKO male mice showed defective chromosome synapsis and impaired meiotic DSB repair with the accumulation of RAD51/DMC1 on meiotic chromosomes, supporting a positive role of FIGNL1 in homologous recombination at a post-assembly stage of RAD51/DMC1 filaments. Fignl1 cKO spermatocytes also accumulate RAD51/DMC1 on chromosomes in pre-meiotic S-phase. These RAD51/DMC1 assemblies are independent of meiotic DSB formation. We also showed that purified FIGNL1 dismantles RAD51 filament on double-stranded (ds)DNA as well as ssDNA. These results suggest an additional role of FIGNL1 in limiting the non-productive assembly of RAD51/DMC1 on native dsDNAs during pre-meiotic S-phase and meiotic prophase I.

molecular biology↗

bioRxiv2018

Human RAD51 paralogue, SWSAP1, fosters RAD51 filament by regulating the anti-recombinase, FIGNL1 AAA+ ATPase

RAD51 assembly on single-stranded (ss)DNAs is a crucial step in the homology-dependent repair of DNA damage for genomic stability. The formation of the RAD51 filament is tightly regulated by various RAD51-interacting proteins. The familial breast cancer gene, BRCA2, as well as RAD52 and six RAD51 paralogues (SWSAP1, RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) promote RAD51 assembly on ssDNAs in humans. However, the mechanisms underlying the differential control of RAD51 filament dynamics by these factors remain largely unknown. Here, we report a role for the human RAD51 paralogue, SWSAP1, as a novel regulator of RAD51 assembly. Swsap1-deficient cells show defects in DNA damage-induced RAD51 assembly during both mitosis and meiosis. Defective RAD51 assembly in SWSAP1-depleted cells is suppressed by the depletion of FIGNL1, an AAA+ ATPase, which binds to RAD51 as well as SWSAP1. Purified FIGNL1 promotes the dissociation of RAD51 from ssDNAs. The in vitro dismantling activity of FIGNL1 does not require its ATPase but depends on RAD51-binding, suggesting a novel mechanism of RAD51 filament remodelling. Purified SWSAP1 inhibits the RAD51-dismantling activity of FIGNL1. Taken together, our data suggest that SWSAP1 protects RAD51 filaments by antagonizing the anti-recombinase, FIGNL1.

molecular biology↗