キズ－細胞の死－をどうやって感じているのか

Epithelial cells are connected each other, cover our body and make compartments separated from surrounding environment. The tight junction, one of typical cell-to-cell junctions regulates flow of substances through cell gaps in epithelial cell sheets. This is called a barrier function of epithelial sheets. When this barrier function becomes weak due to wound, death of a part of cells constituting epithelial sheets, living cells surrounding the dead cells quickly accumulate actomyosin at the live/dead interphases within several minutes, forming an actomyosin ring surrounding dead cells. Contraction of this ring helps wound closure and extrusion of dead cells. It takes less than 1 hour to close the wound when only single cell is dead. After the completion of the closure, concentrated actomyosin quickly disappears along with formation of new tight junctions. Although cells can close the wound by migration of surrounding cells, this contraction of actomyosin ring is effective for closure and specific to epithelial cells with tight junctions. Signals stimulating the accumulation of actomyosin have been sought for decades, with no important candidate. Tight junction components, ZO-1 and ZO-2 are known to be essential for tight junction formation. Interestingly, loss of both ZO-1 and ZO-2 caused high accumulation of actomyosin at cell-cell interphases. It is possible that ZO-1/-2 in tight junctions reduce myosin activity near tight junctions. Loss of ZO-1/-2 may induce myosin activation and accumulation. Molecular analyses along this line would be essential for understanding the early signaling cascade leading to wound responses

Cell Biological Approaches

Our cell biological approaches are introduced in this article. First, we show development of a new fixation protocol using trichloroacetic acid (TCA) that is ideal for immunofluorescence microscopy detecting cellular distribution of phosphorylated ERM (ezrin/radixin/moesin) proteins. TCA denatures proteins differently from organic solvents or aldehydes and can be a good alternative to conventional fixatives when immunostaining is not successful. Second, using this new fixation protocol, the small GTPase, Rho was found to be successfully immunostained and this led to understanding of molecular mechanism for determination of the position of the cleavage furrow at cytokinesis through microtubules of the mitotic apparatus. Rho activator is translocated to the presumptive furrow region through microtubules, then, Rho is accumulated and activated there leading to myosin II contractility for furrowing. Third, we show the cell ablation using laser beam during microscopic observation. This enables us to analyze cell responses to the death of neighboring cells in epithelial sheets at early stages

Actin filament association at adherens junctions

The adherens junction (AJ) is a cadherin-based and actin filament associated cell-to-cell junction. AJs can contribute to tissue morphogenesis and homeostasis and their association with actin filaments is crucial for the functions. There are three types of AJs in terms of the mode of actin filament/AJ association. Among many actin-binding proteins associated with AJs, α-catenin is one of the most important actin filament/AJ linkers that functions in all types of AJs. Although α-catenin in cadherin-catenin complex appears to bind to actin filaments within cells, it fails to bind to actin filaments in vitro mysteriously. Recent report revealed that α-catenin in the complex can bind to actin filaments in vitro when forces are applied to the filament. In addition to force-sensitive vinculin binding, α-catenin has another force-sensitive property of actin filament-binding. Elucidation of its significance and the molecular mechanism is indispensable for understanding AJ formation and maintenance during tissue morphogenesis, function and repair

Basement membrane dynamics in epithelial morphogenesis

In homeostatic epithelial tissues, the basement membrane appears to be a quiet, motionless structure. However, during embryonic development and tissue regeneration, the basement membrane dramatically changes its distribution and shows a variety of dynamics such as compositional transition and physicochemical alterations. Recently, it has been pointed out that the shape and function of epithelial tissues is greatly influenced by the way of formation and arrangement of the basement membrane. Here, we outline the current understanding of the roles of the basement membrane dynamics in epithelial morphogenesis, and briefly introduce our approach to visualize the movement of basement membrane components

Cranial cartilages : Players in the evolution of the cranium during evolution of the chordates in general and of the vertebrates in particular

The present contribution is chiefly a review, augmented by some new results on amphioxus and lamprey anatomy, that draws on paleontological and developmental data to suggest a scenario for cranial cartilage evolution in the phylum chordata. Consideration is given to the cartilage‐related tissues of invertebrate chordates (amphioxus and some fossil groups like vetulicolians) as well as in the two major divisions of the subphylum Vertebrata (namely, agnathans, and gnathostomes). In the invertebrate chordates, which can be considered plausible proxy ancestors of the vertebrates, only a viscerocranium is present, whereas a neurocranium is absent. For this situation, we examine how cartilage‐related tissues of this head region prefigure the cellular cartilage types in the vertebrates. We then focus on the vertebrate neurocranium, where cyclostomes evidently lack neural‐crest derived trabecular cartilage (although this point needs to be established more firmly). In the more complex gnathostome, several neural‐crest derived cartilage types are present: namely, the trabecular cartilages of the prechordal region and the parachordal cartilage the chordal region. In sum, we present an evolutionary framework for cranial cartilage evolution in chordates and suggest aspects of the subject that should profit from additional study

Absence of Radial Spokes in Mouse Node Cilia Is Required for Rotational Movement but Confers Ultrastructural Instability as a Trade-Off

SummaryDetermination of left-right asymmetry in mouse embryos is established by a leftward fluid flow that is generated by clockwise rotation of node cilia. How node cilia achieve stable unidirectional rotation has remained unknown, however. Here we show that brief exposure to the microtubule-stabilizing drug paclitaxel (Taxol) induces randomly directed rotation and changes the ultrastructure of node cilia. In vivo observations and a computer simulation revealed that a regular 9+0 arrangement of doublet microtubules is essential for stable unidirectional rotation of node cilia. The 9+2 motile cilia of the airway, which manifest planar beating, are resistant to Taxol treatment. However, the airway cilia of mice lacking the radial spoke head protein Rsph4a undergo rotational movement instead of planar beating, are prone to microtubule rearrangement, and are sensitive to Taxol. Our results suggest that the absence of radial spokes allows node cilia to rotate unidirectionally but, as a trade-off, renders them ultrastructurally fragile

Real-time TIRF observation of vinculin recruitment to stretched α-catenin by AFM

Adherens junctions (AJs) adaptively change their intensities in response to intercellular tension; therefore, they integrate tension generated by individual cells to drive multicellular dynamics, such as morphogenetic change in embryos. Under intercellular tension, α-catenin, which is a component protein of AJs, acts as a mechano-chemical transducer to recruit vinculin to promote actin remodeling. Although in vivo and in vitro studies have suggested that α-catenin-mediated mechanotransduction is a dynamic molecular process, which involves a conformational change of α-catenin under tension to expose a cryptic vinculin binding site, there are no suitable experimental methods to directly explore the process. Therefore, in this study, we developed a novel system by combining atomic force microscopy (AFM) and total internal reflection fluorescence (TIRF). In this system, α-catenin molecules (residues 276–634; the mechano-sensitive M1-M3 domain), modified on coverslips, were stretched by AFM and their recruitment of Alexa-labeled full-length vinculin molecules, dissolved in solution, were observed simultaneously, in real time, using TIRF. We applied a physiologically possible range of tensions and extensions to α-catenin and directly observed its vinculin recruitment. Our new system could be used in the fields of mechanobiology and biophysics to explore functions of proteins under tension by coupling biomechanical and biochemical information