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Visualization of mechanical stress-mediated Ca2+ signaling in the gut using intravital imaging.

ABSTRACT: Mechanosensory systems have been implicated in the maintenance of gut homeostasis, but details on the related mechanisms are scarce. Recently, we generated a conditional Ca2+ biosensor yellow cameleon 3.60 (YC3.60)-expressing transgenic mouse model and established a five-dimensional (5D; x, y, z, time, and Ca2+) intravital imaging system for investigating lymphoid tissues and enteric epithelial cell responses. To validate this gut-sensing system, we visualized responses of enteric nervous system (ENS) cells in Nestin-Cre/YC3.60flox mice with specific YC3.60 expression. The ENS, including the myenteric (Auerbach's) and submucous (Meissner's) plexuses, could be visualized without staining in this mouse line, indicating that the probe produced sufficient fluorescent intensity. Furthermore, the myenteric plexus exhibited Ca2+ signaling during peristalsis without stimulation. Nerve endings on the surface of enteric epithelia also exhibited Ca2+ signaling without stimulation. Mechanical stress induced transient salient Ca2+ flux in the myenteric plexus and in enteric epithelial cells in the Nestin-Cre/YC3.60 and the CAG-Cre/YC3.60 lines, respectively. Furthermore, the potential TRPM7 inhibitors were shown to attenuate mechanical stress-mediated Ca2+ signaling. These data indicate that the present intravital imaging system can be used to visualize mechanosensory Ca2+ signaling in ENS cells and enteric epithelial cells.

SUBMITTER: Aihara Y

PROVIDER: S-EPMC7573108 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Visualization of mechanical stress-mediated Ca2+ signaling in the gut using intravital imaging.

Aihara Yoshiko Y Fukuda Yota Y Takizawa Akiyoshi A Osakabe Naomi N Aida Tomomi T Tanaka Kohichi K Yoshikawa Soichiro S Karasuyama Hajime H Adachi Takahiro T

Bioscience of microbiota, food and health 20200611 4

Mechanosensory systems have been implicated in the maintenance of gut homeostasis, but details on the related mechanisms are scarce. Recently, we generated a conditional Ca2+ biosensor yellow cameleon 3.60 (YC3.60)-expressing transgenic mouse model and established a five-dimensional (5D; x, y, z, time, and Ca2+) intravital imaging system for investigating lymphoid tissues and enteric epithelial cell responses. To validate this gut-sensing system, we visualized responses of ...[more]

PMID: 33117619

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Project description:Kidney organoids cultured on adherent matrices in the presence of superfusate flow generate vascular networks and exhibit more mature podocyte and tubular compartments compared with static controls (Homan KA, Gupta N, Kroll KT, Kolesky DB, Skylar-Scott M, Miyoshi T, Mau D, Valerius MT, Ferrante T, Bonventre JV, Lewis JA, Morizane R. Nat Methods 16: 255-262, 2019; Takasato M, Er PX, Chiu HS, Maier B, Baillie GJ, Ferguson C, Parton RG, Wolvetang EJ, Roost MS, Chuva de Sousa Lopes SM, Little MH. Nature 526: 564-568, 2015.). However, their physiological function has yet to be systematically investigated. Here, we measured mechano-induced changes in intracellular Ca2+ concentration ([Ca2+]i) in tubules isolated from organoids cultured for 21-64 days, microperfused in vitro or affixed to the base of a specimen chamber, and loaded with fura-2 to measure [Ca2+]i. A rapid >2.5-fold increase in [Ca2+]i from a baseline of 195.0 ± 22.1 nM (n = 9; P ≤ 0.001) was observed when microperfused tubules from organoids >40 days in culture were subjected to luminal flow. In contrast, no response was detected in tubules isolated from organoids <30 days in culture. Nonperfused tubules (41 days) subjected to a 10-fold increase in bath flow rate also exhibited a threefold increase in [Ca2+]i from baseline (P < 0.001). Mechanosensitive PIEZO1 channels contribute to the flow-induced [Ca2+]i response in mouse distal tubule (Carrisoza-Gaytan R, Dalghi MG, Apodaca GL, Kleyman TR, Satlin LM. The FASEB J 33: 824.25, 2019.). Immunodetectable apical and basolateral PIEZO1 was identified in tubular structures by 21 days in culture. Basolateral PIEZO1 appeared to be functional as basolateral exposure of nonperfused tubules to the PIEZO1 activator Yoda 1 increased [Ca2+]i (P ≤ 0.001) in segments from organoids cultured for >30 days, with peak [Ca2+]i increasing with advancing days in culture. These results are consistent with a maturational increase in number and/or activity of flow/stretch-sensitive Ca2+ channels, including PIEZO1, in tubules of static organoids in culture.

Dataset Information

Visualization of mechanical stress-mediated Ca2+ signaling in the gut using intravital imaging.

Publications

Visualization of mechanical stress-mediated Ca<sup>2+</sup> signaling in the gut using intravital imaging.

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