Biology subjects

Fukunaga, H.

Publications and source records attributed to Fukunaga, H..

4 recordsLinked to original sources

bioRxiv2026

Active Nuclear Shuttling Enables Efficient Virus-Free CAR Gene Integration Using Ready-to-Use Lipid Nanoparticles

Non-viral engineering of chimeric antigen receptor T (CAR-T) cells is highly desirable to overcome the cost, safety, and scalability limitations associated with viral vectors and electroporation. However, efficient nuclear delivery and stable genomic integration of DNA in primary human T cells remain major challenges. Here, we established a virus-free CAR-T manufacturing platform using lipid nanoparticles (LNPs) combined with a nuclear localization signal (NLS) shuttle strategy. We developed proprietary ready-to-use LNPs that enable on-demand encapsulation of nucleic acids. To overcome nuclear transport barriers, NLS-fused transposase or genome-editing nuclease was used to bind donor DNA in the cytoplasm and promote active nuclear import. This approach enabled highly efficient and low-toxicity delivery of mRNA and plasmid DNA into primary human T cells. NLS-assisted transposase delivery markedly enhanced genomic integration of the CAR gene, resulting in high expression levels and improved cell viability compared with electroporation-based methods. In addition, TRAC locus-specific targeted integration was achieved more efficiently through end-joining-based repair pathways than through homology-directed repair following LNP delivery. The resulting engineered CAR-T cells exhibited potent and antigen-specific cytotoxic activity. Together, these results demonstrate that NLS-assisted LNP delivery overcomes a key bottleneck in non-viral gene integration and provides a robust strategy for the generation of functional CAR-T cells.

bioengineering↗

bioRxiv2025

Distinct dermal fibroblasts direct mechano-chemical signaling to the epidermis during pregnancy

Dermal fibroblasts alter extracellular matrix (ECM) and tissue mechanics dynamically in wound healing and fibrosis, however, are understudied in healthy skin remodeling across life stages. Here, by conducting spatio-temporal transcriptomics, we identified Cdh4+ dermal fibroblast subpopulation that remodels ECM and converts mechano-to-chemical signals to promote epidermal stem cell proliferation in expanding abdominal skin of pregnant mice. Mechanistically, Cdh4+ fibroblasts produce a pregnancy-responsive matrisome, resulting in denser and stiffer dermal fibrils. These fibroblasts then sense the stiffened substrate and convert the mechanical input into a chemical output via the YAP1-TGF{beta}2 axis, leading to the proliferation of epidermal stem cells via TGF{beta} receptor in the expanding skin. Thus, Cdh4+ fibroblasts fine-tune the dermal mechanofield and act as a mechanical-to-chemical signal conversion hub in healthy skin expansion.

developmental biology↗

bioRxiv2023

Dynamic coordination of the lever-arm swing of human myosin II in thick filaments on actin

Muscle myosins work in motor ensembles and must adapt their power stroke in response to mechanical actions by surrounding motors. Understanding the coordination of power strokes is essential for bridging microscopic molecular functions and macroscopic muscle contractions, but the details of this phenomenon remain elusive. Here we used high-speed atomic force microscopy to visualize the individual dynamics (lever-arm swing) of the myosin head bound to actin in DNA origami-based synthetic thick filaments. We observed spatially local lever-arm coordination, and our three-dimensional numerical model explained how mechanical communication between myosins achieved coordination. In a sarcomere model, the local coordination was spatially periodic and propagated toward the contraction direction. We confirmed that a structural mismatch between myosin head spacing (42.8 nm) and the actin helical pitch (37 nm) caused the coordination while improving contraction speed and energy efficiency. Our findings reveal a key physical basis of efficient muscle contraction.

biophysics↗

bioRxiv2021

The synergic role of actomyosin architecture and biased detachment in muscle energetics: insights in cross bridge mechanism beyond the lever-arm swing

Muscle energetics reflects the ability of myosin motors to convert chemical energy into mechanical energy. How this process takes place remains one of the most elusive questions in the field. Here we combined experimental measurements of in vitro sliding velocity based on DNA-origami built filaments carrying myosins with different lever arm length and simulations based on a Monte-Carlo model which accounts for three basic components: (i) the geometrical hindrance, (ii) the mechano-sensing mechanism, and (iii) the biased kinetics for stretched or compressed motors. The model simulations showed that the geometrical hindrance due to acto-myosin spatial mismatching and the preferential detachment of compressed motors are synergic in generating the rapid increase in the ATP-ase rate from isometric to moderate velocities of contraction, thus acting as an energy-conservation strategy in muscle contraction. The velocity measurements on a DNA-origami filament that preserves the motors distribution showed that geometrical hindrance and biased detachment generate a non-zero sliding velocity even without rotation of the myosin lever-arm, which is widely recognized as the basic event in muscle contraction. Because biased detachment is a mechanism for the rectification of thermal fluctuations, in the Brownian-ratchet framework, we predict that it requires a non-negligible amount of energy to preserve the second law of thermodynamics. Taken together, our theoretical and experimental results elucidate non-conventional components in the chemo-mechanical energy transduction in muscle.

biophysics↗