Kaoru Katoh

Carbon fiber technique for the investigation of single-cell mechanics in intact cardiac myocytes

This protocol describes a method for attaching single isolated cardiac myocytes to carbon fibers for mechanical manipulation and measurement. This method relies on cell-adhesive carbon fibers that attach easily to the cell membrane without causing damage, and is thus applicable to intact myocytes. To connect the carbon fiber to micromanipulators, a fiber holder with glass capillaries must first be fabricated. After connection of the fibers to the micromanipulators, firm attachment is easily established by gently pressing the fiber tip onto the cell membrane. Unlike other methods, this technique does not require vast technical expertise, and therefore greatly facilitates experiments. This method enables detection of the effect of drugs, genetic defects or the expression of exogenous proteins on both active and passive properties of cardiac myocytes. In combination with other experimental procedures, this technique can also be applied to the study of mechano-transduction. This protocol can be completed in 3.5 h.

Microtubule-Cyclodextrin Conjugate: Functionalization of Motile Filament with Molecular Inclusion Ability

A microtubule-β-cyclodextrin conjugate was prepared on a kinesin-adsorbed glass surface by chemical and biochemical means. Fluorescence microscope observation and a motility assay indicated that the conjugate simultaneously expressed an inherent motor function and an inclusion property.

Fluorescence microscopy reveals molecular localisation at line defects in nematic liquid crystals

Topological defects easily form in liquid crystals (LCs) as a result of frustrations in spatially dependent anisotropic molecular ordering, and have been regarded as promising tools for facilitating manipulation of relatively large non-LC materials such as colloids. However, it remains unclear whether low-molecular-weight (LMW) impurities that do not aggregate or self-assemble in bulk LCs because of the dominance of entropy can localise at LC defects. Here, by fluorescence microscopy, we directly show the localisation of LMW molecules at the topological line defects of a nematic LC. It is theoretically explained that excess free energy density of nematic ordering at the defect core allows LMW solutes to accumulate at a non-negligible level overcoming the entropy leading to their uniform distributions. Our results demonstrate the usefulness of LC defects as a bottom-up field that enables micromanipulation of LMW molecules and realisation of transformable three-dimensional micro-architectures composed of versatile small functional molecules.

Isolation of mitochondria by gentle cell membrane disruption, and their subsequent characterization.

Mitochondria play a key role in several physiological processes as in integrating signals in the cell. However, understanding of the mechanism by which mitochondria sense and respond to signals has been limited due to the lack of an appropriate model system. In this study, we developed a method to isolate and characterize mitochondria without cell homogenization. By gently pipetting cells treated with streptolysin-O, a pore-forming membrane protein, we disrupted the cell membrane and were able to isolate both elongated and spherical mitochondria. Fluorescence imaging combined with super resolution microscopy showed that both the outer and inner membranes of the elongated mitochondria isolated using the newly developed method were intact. In addition, a FRET-based ATP sensor expressed in the mitochondrial matrix demonstrated that ATP generation by FoF1-ATPase in the isolated elongated mitochondria was as high as that in intracellular mitochondria. On the other hand, some of the spherical mitochondria isolated with this method had the outer membrane that no longer encapsulated the inner membrane. In addition, all mitochondria isolated using conventional procedures involving homogenization were spherical, many of them had damaged membranes, and low levels of ATP generation. Our results suggest that elongated mitochondria isolated from cells through gentle cell membrane disruption using a pore-forming protein tend to be more similar to intracellular mitochondria, having an intact membrane system and higher activity than spherical mitochondria.

Ribosome Incorporation into Somatic Cells Promotes Lineage Transdifferentiation towards Multipotency

Recently, we reported that bacterial incorporation induces cellular transdifferentiation of human fibroblasts. However, the bacterium-intrinsic cellular- transdifferentiation factor remained unknown. Here, we found that cellular transdifferentiation is caused by ribosomes. Ribosomes, isolated from both prokaryotic and eukaryotic cells, induce the formation of embryoid body-like cell clusters. Numerous ribosomes are incorporated into both the cytoplasm and nucleus through trypsin-activated endocytosis, which leads to cell-cluster formation. Although ribosome-induced cell clusters (RICs) express several stemness markers and differentiate into derivatives of all three germ layers in heterogeneous cell populations, RICs fail to proliferate, alter the methylation states of pluripotent genes, or contribute to teratoma or chimera formation. However, RICs express markers of epithelial–mesenchymal transition without altering the cell cycle, despite their proliferation obstruction. These findings demonstrate that incorporation of ribosomes into host cells induces cell transdifferentiation and alters cellular plasticity.

Uncovering different states of topological defects in schlieren textures of a nematic liquid crystal

Topological defects are ubiquitously found in physical systems and therefore have been an important research subject of not only condensed matter physics but also cosmology. However, their fine structures remain elusive because of the microscopic scales involved. In the case of a liquid crystal, optical microscopy, although routinely used for the identification of liquid crystal phases and associated defects, does not have resolution high enough to distinguish fine structures of topological defects. Here we show that polarised and fluorescence microscopy, with the aid of numerical calculations on the orientational order and resulting image distortions, can uncover the structural states of topological defects with strength m =  ±1 in a thin cell of a nematic liquid crystal. Particularly, defects with m = +1 exhibit four different states arising from chiral symmetry breaking and up-down symmetry breaking. Our results demonstrate that optical microscopy is still a powerful tool to identify fine states of liquid crystalline defects.