A time-resolved small-angle X-ray scattering (SAXS) system for protein solution samples using an X-ray free-electron laser (XFEL) was established by developing a SAXS diffractometer by integrating a helium path into the DAPHNIS system initially designed for Serial Femtosecond Crystallography (SFX) experiments at BL2 of SACLA. This modification enabled us to successfully capture the SAXS profiles of ovalbumin under conditions without any reaction trigger, using both the newly developed system and the sample solution flow device that was originally designed for SFX experiments. Furthermore, we conducted acid denaturation experiments on cytochrome c, using a T-junction-type solution mixing flow system, and observed the denaturation-induced changes in the SAXS profiles.

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Temperature crucially affects molecular processes in living organisms and thus it is one of the vital physical parameters for life. To investigate how temperature is biologically maintained and regulated and its biological impact on organisms, it is essential to measure the spatial distribution and/or temporal changes of temperature across different biological scales, from whole organism to subcellular structures. Fluorescent nanothermometers have been developed as probes for temperature measurement by fluorescence microscopy for applications in microscopic scales where macroscopic temperature sensors are inaccessible, such as embryos, tissues, cells, and organelles. Although fluorescent nanothermometers have been developed from various materials, fluorescent protein-based ones are especially of interest because they can be introduced into cells as the transgenes for expression with or without specific localization, making them suitable for less-invasive temperature observation in living biological samples. In this article, we review protein-based fluorescent nanothermometers also known as genetically-encoded temperature indicators (GETIs), covering most published GETIs, for developers, users, and researchers in thermal biology as well as interested readers. We provide overviews of the temperature sensing mechanisms and measurement methods of these protein-based fluorescent nanothermometers. We then outline key information for GETI development, focusing on unique protein engineering techniques and building blocks distinct to GETIs, unlike other fluorescent nanothermometers. Furthermore, we propose several standards for the characterization of GETIs. Additionally, we explore various issues and offer perspectives in the field of thermal biology.

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Enzyme function is often regulated by weak metal-ion binding, which results from conformational changes while maintaining conformational fluctuations. We analyzed the structure and function of cutinase-like enzyme, Cut190, using biophysical methods such as X-ray crystallography and molecular dynamics (MD) simulations, showing that its structure and function are finely regulated by weak Ca²⁺ binding and release. We succeeded to stabilize the enzyme by introducing a disulfide-bond which can degrade polyethylene terephthalate (PET) to PET monomers at the glass transition temperature of PET, ≈70°C. In this study, using the stabilized Cut190 mutants, Cut190**SS and Cut190**SS_F77L, we evaluated the requirement of Ca²⁺ for catalytic activity at 70°C, showing that the enzyme expressed the activity even in the absence of Ca²⁺, in contrast to that at 37°C. These results were supported by multicanonical MD analysis, which showed that the respective forms of the enzyme, such as closed, open, and engaged forms, were exchangeable, possibly because the potential energy barriers between the respective forms were lowered. Taken together, the conformational equilibrium to express the catalytic activity was regulated by weak Ca²⁺ binding at 37°C, and was also regulated by increasing temperature. The respective conformational states of Cut190**SS and Cut190**SS_F77L correlated well with their different catalytic activities for PET.

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Micromanipulation techniques are essential in studies of cell function, both for single cells and for cell collectives. Various types of micromanipulators are now commercially available. Hydraulic micromanipulators have the advantage of analogue operation, allowing the user to move the glass microneedle in direct response to their own hand movements. However, they require regular maintenance to maintain their performance. On the other hand, some electric micromanipulators can operate in minute steps of several hundred nanometers, but they are expensive. This paper describes our assembly of a low-cost electric micromanipulator. The device consists of three commercially available stages, three linear DC motors to drive them, and a lab-made control circuit. Using this device, we were able to direct a glass microneedle to cut an MDCK cell sheet. We also manipulated an aspiration pipette to aspirate a portion of a Dictyostelium cell. In addition, we were able to gently touch the tip of an electroporation pipette to the surface of a single target cell in a sheet of fish epidermal keratocytes and load FITC into the cell. Our device can be assembled at one-fourth the cost of commercially available hydraulic micromanipulators. This could make it easier, both economically and technically, to add micromanipulators to all of a laboratory’s microscopes.

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Ligand–receptor docking simulation is difficult when the biomolecules have high intrinsic flexibility. If some knowledge on the ligand–receptor complex structure or inter-molecular contact sites are presented in advance, the difficulty of docking problem considerably decreases. This paper proposes a generalized-ensemble method “cartesian-space division mD-VcMD” (or CSD-mD-VcMD), which calculates stable complex structures without assist of experimental knowledge on the complex structure. This method is an extension of our previous method that requires the knowledge on the ligand–receptor complex structure in advance. Both the present and previous methods enhance the conformational sampling, and finally produce a binding free-energy landscape starting from a completely dissociated conformation, and provide a free-energy landscape. We applied the present method to same system studied by the previous method: A ligand (ribocil A or ribocil B) binding to an RNA (the aptamer domain of the FMN riboswitch). The two methods produced similar results, which explained experimental data. For instance, ribocil B bound to the aptamer’s deep binding pocket more strongly than ribocil A did. However, this does not mean that two methods have a similar performance. Note that the present method did not use the experimental knowledge of binding sites although the previous method was supported by the knowledge. The RNA-ligand binding site could be a cryptic site because RNA and ligand are highly flexible in general. The current study showed that CSD-mD-VcMD is actually useful to obtain a binding free-energy landscape of a flexible system, i.e., the RNA-ligand interacting system.

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Mitochondria isolated from cells are essential tools in biological research. However, many mitochondria are often damaged during the isolation process. Although cryopreservation can greatly improve the usability of isolated mitochondria, it typically leads to significant loss of activity following freezing and thawing. In this study, we present our own techniques for mitochondrial isolation and cryopreservation to overcome these challenges. Our isolation method begins by selectively weakening the plasma membrane through the incorporation of digitonin, under conditions that do not increase membrane permeability. The plasma membrane is then selectively ruptured to release mitochondria. Notably, mitochondria contract within the cell before the plasma membrane ruptures, a process that facilitates their extraction. The isolated mitochondria showed polarized inner membranes in approximately 90% of the population. Compared to mitochondria isolated by homogenization, they retained more intermembrane space proteins and exhibited greater outer membrane integrity. For cryopreservation, rapid thawing was critical to maintaining mitochondrial activity after freeze-thaw cycles. When thawing was completed in under 1.5 minutes, the proportion of polarized mitochondria decreased by only about 10%. These findings suggest that our isolation and cryopreservation protocols are promising for applications requiring intact, functional mitochondria.

This graphical abstract illustrates the iMIT (intact Mitochondria Isolation Technique), our own method for isolating intact mitochondria with minimal structural damage. The process consists of four steps: (1) selective weakening of the plasma membrane using digitonin; (2) mitochondrial contraction within the cell; (3) gentle disruption of the plasma membrane by pipetting to release mitochondria; and (4) collection of mitochondria by differential centrifugation. This approach minimizes mechanical stress and avoids detergent exposure to mitochondria, thereby preserving outer membrane integrity.

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