Biophysics and Physicobiology最新文献

Two-photon excitation laser scanning microscopy (TPLSM) has provided many insights into the life sciences, especially for thick biological specimens, because of its superior penetration depth and less invasiveness owing to the near-infrared wavelength of its excitation laser light. This paper introduces our four kinds of studies to improve TPLSM by utilizing several optical technologies as follows: (1) A high numerical aperture objective lens significantly deteriorates the focal spot size in deeper regions of specimens. Thus, approaches to adaptive optics were proposed to compensate for optical aberrations for deeper and sharper intravital brain imaging. (2) TPLSM spatial resolution has been improved by applying super-resolution microscopic techniques. We also developed a compact stimulated emission depletion (STED) TPLSM that utilizes electrically controllable components, transmissive liquid crystal devices, and laser diode-based light sources. The spatial resolution of the developed system was five times higher than conventional TPLSM. (3) Most TPLSM systems adopt moving mirrors for single-point laser beam scanning, resulting in the temporal resolution caused by the limited physical speed of these mirrors. For high-speed TPLSM imaging, a confocal spinning-disk scanner and newly-developed high-peak-power laser light sources enabled approximately 200 foci scanning. (4) Several researchers have proposed various volumetric imaging technologies. However, most technologies require large-scale and complicated optical setups based on deep expertise for microscopic technologies, resulting in a high threshold for biologists. Recently, an easy-to-use light-needle-creating device was proposed for conventional TPLSM systems to achieve one-touch volumetric imaging.

Spider silk is considered a promising next-generation biomaterial due to its exceptional toughness, coupled with its renewability and biodegradability. Contrary to the conventional view that spider silk is mainly composed of two types of silk proteins (spidroins), MaSp1 and MaSp2, multi-omics strategies are increasingly revealing that the inclusion of complex components confers the higher mechanical properties to the material. In this review, we focus on several recent findings that report essential components and mechanisms that are necessary to reproduce the properties of natural spider silk. First, we discuss the discovery of MaSp3, a newly identified spidroin that is a major component in the composition of spider silk, in addition to the previously understood MaSp1 and MaSp2. Moreover, the role of the Spider-silk Constituting Element (SpiCE), which is present in trace amounts but has been found to significantly increase the tensile strength of artificial spider silk, is explored. We also delve into the process of spidroin fibril formation through liquid-liquid phase separation (LLPS) that forms the hierarchical structure of spider silk. In addition, we review the correlation between amino acid sequences and mechanical properties such as toughness and supercontraction, as revealed by an analysis of 1,000 spiders. In conclusion, these recent findings contribute to the comprehensive understanding of the mechanisms that give spider silk its high mechanical properties and help to improve artificial spider silk production.

Neuropsin is one of serine proteases mainly found at the hippocampus and the amygdala, where it contributes to the long-term potentiation and memory acquisition by rebuilding of synaptic connections. Despite of the importance of neuropsin, the substrate specificity and regulation mechanisms of neuropsin have been unclear. Thus, we investigated the substrate specificity and the catalytic activity of neuropsin by the protein-ligand docking and molecular dynamics (MD) simulations and succeeded to reproduce the trend of the experimental results. Our study revealed that the substrate specificity and the activity of neuropsin depended on multiple factors: the substrate charge, the substrate orientation, the hydrogen bond network within the catalytic triad and the substrate, and the formation of the oxyanion hole. The apo neuropsin was not reactive without proper alignment of catalytic triad. The substrate binding induced the reactive alignment of catalytic triad. Then the substrate-neuropsin interaction forms the oxyanion hole that stabilizes the transition state and reduces the free-energy barrier of the following scission reaction.

Epidermal cells, such as keratinocytes, are regarded as the first sensory cells to transmit nociception and mechanoreception to free nerve endings extended from the dorsal root ganglion (DRG). Previous studies suggested that this transmission occurs as Ca²⁺ propagation via ATP receptors. Conversely, the influence of gap junctions on this Ca²⁺ propagation is largely unknown. Thus, we examined the localization and the role of connexin 43 among keratinocytes and DRG neurons. We co-cultured keratinocytes and DRG neurons and investigated the effect of pharmacological blockade of gap junctions on Ca²⁺ propagation upon stimulation of a single keratinocyte. Immunocytochemical experiments showed that connexin 43 is localized between keratinocytes and between keratinocytes and DRG neurons. Octanol, a gap junction inhibitor, significantly suppressed the concentrical Ca²⁺ propagation. Therefore, we conclude that the Ca²⁺ propagation mechanism via gap junctions from stimulated keratinocytes to free nerve endings should be taken into account.

In living tissues where cells migrate, the spatial distribution of mechanical properties, especially matrix stiffness, is generally heterogeneous, with cell scales ranging from 10 to 1000 μm. Since cell migration in the body plays a critical role in morphogenesis, wound healing, and cancer metastasis, it is essential to understand the migratory dynamics on the matrix with cell-scale stiffness heterogeneity. In general, cell migration is driven by the extension and contraction of the cell body owing to the force from actin polymerization and myosin motors in the actomyosin cytoskeleton. When a cell is placed on a matrix with a simple stiffness gradient, directional migration called durotaxis emerges because of the asymmetric extension and contraction of the pseudopodia, which is accompanied by the asymmetric distribution of focal adhesions. Similarly, to determine cell migration on a matrix with cell-scale stiffness heterogeneity, the interaction between cell-scale stiffness heterogeneity and cellular responses, such as the dynamics of the cell-matrix adhesion site, intracellular prestress, and cell shape, should play a key role. In this review, we summarize systematic studies on the dynamics of cell migration, shaping, and traction force on a matrix with cell-scale stiffness heterogeneity using micro-elastically patterned hydrogels. We also outline the cell migration model based on cell-shaping dynamics that explains the general durotaxis induced by cell-scale stiffness heterogeneity. This review article is an extended version of the Japanese article, Dynamics of Cell Shaping and Migration on the Matrix with Cell-scale Stiffness-heterogeneity, published in SEIBUTSU BUTSURI Vol. 61, p. 152-156 (2021).