Taketoshi Kiya

Genetically encoded indicators that can detect concentrations of metabolites and signalling molecules through fluorescence lifetime changes are gaining attention, because they expand the potential for quantitative imaging. These indicators offer advantages over conventional fluorescence intensity-based indicators by minimizing artifacts such as variations in indicator concentration, cellular morphological changes, and focus drift. However, the availability of fluorescence lifetime-based genetically encoded indicators remains limited, particularly those compatible with the widely used conventional 488 nm laser in microscopy. Here, we introduce qMaLioffG, a single green fluorescent protein-based ATP indicator that exhibits a substantial fluorescence lifetime shift (1.1 ns) within physiologically relevant ATP concentrations. This enables quantitative imaging of ATP levels in the cytoplasm and mitochondria under steady-state conditions across various cell types, providing insights into ATP distribution. We demonstrate that qMaLioffG can be used in multicellular systems, applying it to Drosophila brain and HeLa cell spheroids to reveal spatially heterogeneous ATP levels.

Optical heating coupled with near-infrared (NIR) light and photothermal materials enables local heating within biospecimens, minimizing undesirable thermal damage. Here, we demonstrated that photothermally heating lipid bilayers embedded with a phthalocyanine dye (VPc) efficiently perturbs the bilayers, resulting in increased permeability. Notably, microscopic studies revealed that the changes in membrane permeability may not follow the conventional mechanism of temperature-sensitive liposomes, which involve a bulk temperature increase that induces a phase transition across the entire lipid bilayer. Furthermore, the heat generated by NIR laser illumination rarely diffused into the surrounding environment, and the dye was located within the bilayers at the molecular level, where it effectively transferred heat to the lipid bilayer. We prepared VPc-embedded liposomes encapsulating acetylcholine (ACh) and demonstrated the NIR laser-triggered release of ACh, creating a concentration jump across a few cells or within a limited single cell region. This method induced Ca²⁺ flux through ACh receptor stimulation in thermally delicate biospecimens such as C2C12 myotubes and the Drosophila brain.

The precise control of cell activity is crucial for understanding and potentially treating many disorders. Focusing on neurons and myotubes, recent advancements in nanotechnology have introduced photoresponsive nanoparticles as an alternative tool for modulating cell function with high spatial and temporal resolution. This approach offers a noninvasive alternative to traditional stimulation techniques, reducing potential tissue damage and improving the specificity of cell activation. Here, we introduce an approach envisioning fully organic polydopamine nanoparticles (PDNPs) to remotely modulate the activity of differentiated SH-SY5Y cells and differentiated C2C12 cells, via near-infrared (NIR) laser stimulation. Confocal microscopy imaging revealed the possibility of thermally activating individual neuron-like cells, eliciting a significant cellular response characterized by the generation of calcium transients and the subsequent release of the neurotransmitter acetylcholine. Similarly, we demonstrated the possibility of precisely triggering the muscle contraction of single myotubes. Additionally, we investigated the antioxidant properties of PDNPs, demonstrating their capacity to prevent an increase in oxidative stress levels related to an increase in intracellular temperature. Moreover, proteomic analysis revealed that a PDNP treatment could positively affect neuronal plasticity and nervous system maturation, besides promoting muscle growth and preserving its functional integrity, underscoring its potential to support both neural and musculoskeletal development. Eventually, the effect of the NIR laser irradiation in the presence of PDNPs in neuron-like cells was successfully evaluated ex vivo on brains of Drosophila melanogaster, genetically modified to express the fluorescent calcium indicator jGCaMP7c.

Although genome editing techniques have made significant progress, introducing exogenous genes into the genome through knock-in remains a challenge in many organisms. In silkworm Bombyx mori, TALEN-mediated knock-in methods have been established. However, difficulties in construction and limitations of the target sequence have hindered the application of these methods. In the present study, we verified several CRISPR/Cas9-mediated knock-in methods to expand the application of gene knock-in techniques and found that the short single-stranded oligodeoxynucleotide (ssODN)-mediated method is the most effective in silkworms. Using ssODN-mediated methods, we established knock-in silkworm strains that harbor an attP sequence, a 50 bp phiC31 integrase recognition site, at either the BmHr38 (Hormone receptor 38) or Bmdsx (doublesex) locus. Additionally, we found that the long ssODN (lsODN)-mediated method successfully introduced the GAL4 gene at the doublesex locus in embryos. The present study provides valuable information on CRISPR/Cas9-mediated knock-in methods in silkworms, expanding the utility of genome editing techniques in insects and paving the way for analyzing gene and genome function in silkworms.

Optical heating coupled with near-infrared (NIR) light and photothermal materials enables thermal confinement within biospecimens, minimizing undesirable thermal damage. Here, we demonstrated that photothermally heating lipid bilayers embedded with a unique phthalocyanine dye (VPc) efficiently perturbs the bilayers, resulting in increased permeability. Notably, microscopic studies revealed that the mechanism causing changes in membrane permeability may not follow the conventional temperature-sensitive liposome model. Furthermore, the heat generated by NIR laser illumination rarely diffused into the surrounding environment, and the dye was located within the bilayers at the molecular level, where it effectively transferred heat to the lipid bilayer. We prepared VPc-embedded liposomes encapsulating acetylcholine (ACh) and demonstrated the NIR laser-triggered release of ACh, creating a concentration jump across a few cells or within a limited single cell region. This method induced Ca ²⁺ flux through ACh receptor stimulation in thermally delicate biospecimens such as C2C12 myotubes and the Drosophila brain.

The widespread use of fluorescence lifetime imaging microscopy (FLIM) for quantitative imaging is hindered by the limited availability of FLIM-based genetically encoded indicators compatible with conventional 488 nm lasers. Here, we present qMaLioffG, a single green fluorescent protein FLIM indicator that shows fluorescence lifetime changes in ATP concentrations within the physiological range. This allows the quantitative imaging of endogenous ATP to investigate the cellular energy status of different cell types.

The widespread use of fluorescence lifetime imaging microscopy (FLIM) for quantitative imaging is hindered by the limited availability of a FLIM-based genetically encoded indicator using a conventional 488 nm laser. Here, we present qMaLioffG, a single green fluorescent protein FLIM indicator showing a fluorescence lifetime change in ATP concentration within the physiological range. This allows quantitative imaging of endogenous ATP to investigate cellular energy status of different cell types.

Innate behavior, such as courtship behavior, is controlled by a genetically defined set of neurons. To date, it remains challenging to visualize and artificially control the neural population that is active during innate behavior in a whole-brain scale. Immediate early genes (IEGs), whose expression is induced by neural activity, can serve as powerful tools to map neural activity in the animal brain. We screened for IEGs in vinegar fly Drosophila melanogaster and identified stripe/egr-1 as a potent neural activity marker. Focusing on male courtship as a model of innate behavior, we demonstrate that stripe - GAL4- mediated reporter expression can label fruitless ( fru )-expressing neurons involved in courtship in an activity (experience)-dependent manner. Optogenetic reactivation of the labeled neurons elicited sexual behavior in males, whereas silencing of the labeled neurons suppressed courtship and copulation. Further, by combining stripe-GAL4- mediated reporter expression and detection of endogenous Stripe expression, we established methods that can label neurons activated under different contexts in separate time windows in the same animal. The cell assembly analysis of fru neural population in males revealed that distinct groups of neurons are activated during interactions with a female or another male. These methods will contribute to building a deeper understanding of neural circuit mechanisms underlying innate insect behavior.

Photo-caged methodologies have been indispensable for elucidating the functional mechanisms of pharmacologically active molecules at the cellular level. A photo-triggered removable unit enables control of the photo-induced expression of pharmacologically active molecular function, resulting in a rapid increase in the concentration of the bioactive compound near the target cell. However, caging the target bioactive compound generally requires specific heteroatom-based functional groups, limiting the types of molecular structures that can be caged. We have developed an unprecedented methodology for caging/uncaging on carbon atoms using a unit with a photo-cleavable carbon-boron bond. The caging/uncaging process requires installation of the CH₂-B group on the nitrogen atom that formally assembles an N-methyl group protected with a photoremovable unit. N-Methylation proceeds by photoirradiation via carbon-centered radical generation. Using this radical caging strategy to cage previously uncageable bioactive molecules, we have photocaged molecules with no general labeling sites, including acetylcholine, an endogenous neurotransmitter. Caged acetylcholine provides an unconventional tool for optopharmacology to clarify neuronal mechanisms on the basis of photo-regulating acetylcholine localization. We demonstrated the utility of this probe by monitoring uncaging in HEK cells expressing a biosensor to detect ACh on the cell surface, as well as Ca²⁺ imaging in Drosophila brain cells (ex vivo).