Optical analysis of cell membrane dynamics and recycling processes in cultured human ethmoidal cells using a fluorescent dye FM4-64.

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  • Takeno Sachio
    Department of Otolaryngology, Hiroshima University School of Medicione
  • Tatsukawa Takaharu
    Department of Otolaryngology, Hiroshima University School of Medicione
  • Kawamoto Hiroko
    Department of Otolaryngology, Hiroshima University School of Medicione
  • Fukushima Noriyuki
    Department of Otolaryngology, Hiroshima University School of Medicione
  • Yazin Koji
    Department of Otolaryngology, Hiroshima University School of Medicione

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  • FM4-64™を用いた培養節骨洞粘膜上皮細胞における細胞膜リサイクリング過程の観察

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The styrylpyridinium dyes, which are nontoxic and water-soluble membrane probes, have been used both for investigating the mechanisms of activity-dependent membrane recycling and for visualizing the endocytosis phase in a wide range of species. In this study, we visualized cell membrane-internalization and transport in cultured human ethmoidal cells using a fluorescent probe FM4-64 and laser scanning confocal microscopy. After treatment for 5min at 30°C, FM4-64 labeling became apparent in the plasma membrane of ciliated cells. The labeling was pronounced on the apical side corresponding to the area where numerous cilia assembled. A different pattern was observed in non-ciliated cells where the plasma membrane was weakly fluorescent on their basolateral side. Using time-lapse conf ocal microscopy, we were able to follow vesicle internalization and augmented membrane turnover in some of the ciliated cells triggered by various concentrations of histamine (10-3M to 10-5M). The fluorescent labeling of these cells began to rise until 10 minutes concomitant with distinctly visualized beating cilia. We have shown that this dye serves as a sensitive vital reporter for membrane dynamics, detecting such events as endosome to membrane transport during receptor-mediated endocytosis in vitro.

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