Fumiya Kozawa, Tomokazu Tamura, Naoki Takahashi, Taishi Kakizuka, Taro Ichimura, Rumi Shimada, Yasuyuki Hashimoto, Hironoshin Onizuska, Sayaka Kashiwagi, Tomoko Kamasaki, Maho Amano, Takeharu Nagai, Takasuke Fukuhara, Yoichiro Fujioka and Yusuke Ohba : The crucial role of intercellular calcium wave propagation triggered by influenza A virus in promoting infection, Cell Communication & Signaling, 23, 1, 361, 2025.
(要約)
Influenza A viruses (IAVs) initially infect a few host cells before spreading to neighboring cells. However, the molecular mechanisms underlying this dissemination remain unclear. We have previously demonstrated that intracellular Ca2+ plays a crucial role in facilitating IAV infection. This study aims to clarify the connections between intracellular Ca2+ dynamics and spread of IAV infection. Madin-Darby canine kidney (MDCK) cells stably expressing a Ca2+ indicator for optical imaging were established. Cells were cultured in Matrigel to form monolayers, and cell-to-cell Ca2+ dynamics within IAV-infected cells were analyzed using fluorescence microscopy. IAV infection upregulated the frequency of intercellular calcium wave propagations (iCWPs), facilitating viral spread. ADP released from initially infected cells mediated iCWPs via the P2Y1 receptor. P2Y1 antagonist suppressed both the generation of iCWPs and spread of viral infection. Enhanced endocytosis by the surrounding cells that received ADP signaling upregulated viral entry. Expression of IAV matrix protein 2 (M2) in initially infected cells triggered iCWPs through ADP diffusion, thereby increasing infection. Conversely, an ion permeability-deficient mutation of M2 or inhibition of its ion channel activity suppressed iCWPs. Intercellular calcium signaling plays a crucial role in the early expansion and establishment of IAV infection, presenting a potential target for IAV prophylaxis.
Taishi Kakizuka, Hidenori Nakaoka, Yusuke Hara, Aya Ichiraku, Yoshiyuki Arai, Hiroya Itoga, Shuichi Onami, Taro Ichimura, Takeharu Nagai and Kazuki Horikawa : Mesoscale heterogeneity is a critical determinant for spiral pattern formation in developing social amoeba, Scientific Reports, 15, 1, 1422, 2025.
(要約)
Heterogeneity is a critical determinant for multicellular pattern formation. Although the importance of microscale and macroscale heterogeneity at the single-cell and whole-system levels, respectively, has been well accepted, the presence and functions of mesoscale heterogeneity, such as cell clusters with distinct properties, have been poorly recognized. We investigated the biological importance of mesoscale heterogeneity in signal-relaying abilities (excitability) in the self-organization of spiral waves of intercellular communications by studying the self-organized pattern formation in a population of Dictyostelium discoideum cells, a classical signal-relaying system model. By utilizing pulse-count analysis to evaluate cellular excitability, we successfully visualized the development of mesoscale heterogeneity in excitability, whose spatial scale was comparably large to that of the traveling waves of intercellular communication. Together with perturbation experiments, our detailed analysis of the structural change in mesoscale heterogeneity and associated wave dynamics demonstrated the functional importance of mesoscale heterogeneity in generating the spiral wave pattern, whose experimental observations were first realized. We propose that mesoscale heterogeneity, in addition to microscale and macroscale heterogeneities, is a critical determinant of diverse multicellular pattern formations.
(キーワード)
Dictyostelium / Cell Communication / Models, Biological / Signal Transduction
Takayuki Haruki, Shota Yonezawa, Keiichi Koizumi, Yasuhiko Yoshida, M. Tomonobu Watanabe, Hideaki Fujita, Yusuke Oshima, Makito Oku, Akinori Taketani, Moe Yamazaki, Taro Ichimura, Makoto Kadowaki, Isao Kitajima and Shigeru Saito : Application of the Dynamical Network Biomarker Theory to Raman Spectra, Biomolecules, 12, 12, 2022.
(要約)
The dynamical network biomarker (DNB) theory detects the early warning signals of state transitions utilizing fluctuations in and correlations between variables in complex systems. Although the DNB theory has been applied to gene expression in several diseases, destructive testing by microarrays is a critical issue. Therefore, other biological information obtained by non-destructive testing is desirable; one such piece of information is Raman spectra measured by Raman spectroscopy. Raman spectroscopy is a powerful tool in life sciences and many other fields that enable the label-free non-invasive imaging of live cells and tissues along with detailed molecular fingerprints. Naïve and activated T cells have recently been successfully distinguished from each other using Raman spectroscopy without labeling. In the present study, we applied the DNB theory to Raman spectra of T cell activation as a model case. The dataset consisted of Raman spectra of the T cell activation process observed at 0 (naïve T cells), 2, 6, 12, 24 and 48 h (fully activated T cells). In the DNB analysis, the F-test and hierarchical clustering were used to detect the transition state and identify DNB Raman shifts. We successfully detected the transition state at 6 h and related DNB Raman shifts during the T cell activation process. The present results suggest novel applications of the DNB theory to Raman spectra ranging from fundamental research on cellular mechanisms to clinical examinations.
(キーワード)
dynamical network biomarker (DNB) theory / Raman spectra / Raman spectroscopy / T cell activation / transition state
Taro Ichimura, T. Kakizuka, Kazuki Horikawa, K. Seiriki, A. Kasai, H. Hashimoto, K. Fujita, M. T. Watanabe and T. Nagai : Exploring rare cellular activity in more than one million cells by a transscale scope, Scientific Reports, 11, 1, 16539, 2021.
(要約)
In many phenomena of biological systems, not a majority, but a minority of cells act on the entire multicellular system causing drastic changes in the system properties. To understand the mechanisms underlying such phenomena, it is essential to observe the spatiotemporal dynamics of a huge population of cells at sub-cellular resolution, which is difficult with conventional tools such as microscopy and flow cytometry. Here, we describe an imaging system named AMATERAS that enables optical imaging with an over-one-centimeter field-of-view and a-few-micrometer spatial resolution. This trans-scale-scope has a simple configuration, composed of a low-power lens for machine vision and a hundred-megapixel image sensor. We demonstrated its high cell-throughput, capable of simultaneously observing more than one million cells. We applied it to dynamic imaging of calcium ions in HeLa cells and cyclic-adenosine-monophosphate in Dictyostelium discoideum, and successfully detected less than 0.01% of rare cells and observed multicellular events induced by these cells.
Gracy Brit David, Hideaki Fujita, Kyota Yasuda, Kazuko Okamoto, Yulia Panina, Junya Ichinose, Osamu Sato, Masanobu Horie, Taro Ichimura, Yasushi Okada and M. Tomonobu Watanabe : Linking substrate and nucleus via actin cytoskeleton in pluripotency maintenance of mouse embryonic stem cells, Stem Cell Research, 41, 101614, 2019.
(要約)
Pluripotency of mouse embryonic stem cells is regulated by transcription factor regulatory networks as well as mechanical stimuli sensed by the cells. It has been unclear how the mechanical strain applied to the plasma membrane is transferred to the nucleus in mouse embryonic stem cells (mESCs). We here investigated the machinery of the mechanotransduction based on the finding that spontaneous differentiation of mESCs was inhibited with the downregulation of ROCK2 in cells attached to soft substrates. On examining the effects of actin bindings to both focal adhesions and cell junctions in cells on soft substrates, co-localization of actin filaments and α-catenin, which links actin to E-cadherin, decreased after differentiation induction. Also, disrupting actin-nucleus mechanical link through dominant negative assay of Nesprins helps to sustain the pluripotency genes; thus, revealing that mechanical strain relayed by actin-Nesprin connection is required for the initiation of the differentiation process.