title: In vivo imaging and quantitative monitoring of autophagic flux in tobacco BY-2 cells.
abstract: Autophagy has been shown to play essential roles in the growth, development and survival of eukaryotic cells. However, simple methods for quantification and visualization of autophagic flux remain to be developed in living plant cells. Here, we analyzed the autophagic flux in transgenic tobacco BY-2 cell lines expressing fluorescence-tagged NtATG8a as a marker for autophagosome formation. Under sucrose-starved conditions, the number of punctate signals of YFP-NtATG8a increased, and the fluorescence intensity of the cytoplasm and nucleoplasm decreased. Conversely, these changes were not observed in BY-2 cells expressing a C-terminal glycine deletion mutant of the NtATG8a protein (NtATG8aΔG). To monitor the autophagic flux more easily, we generated a transgenic BY-2 cell line expressing NtATG8a fused to a pH-sensitive fluorescent tag, a tandem fusion of the acid-insensitive RFP and the acid-sensitive YFP. In sucrose-rich conditions, both fluorescent signals were detected in the cytoplasm and only weakly in the vacuole. In contrast, under sucrose-starved conditions, the fluorescence intensity of the cytoplasm decreased, and the RFP signal clearly increased in the vacuole, corresponding to the fusion of the autophagosome to the vacuole and translocation of ATG8 from the cytoplasm to the vacuole. Moreover, we introduce a novel simple easy way to monitor the autophagic flux non-invasively by only measuring the ratio of fluorescence of RFP and YFP in the cell suspension using a fluorescent image analyzer without microscopy. The present in vivo quantitative monitoring system for the autophagic flux offers a powerful tool for determining the physiological functions and molecular mechanisms of plant autophagy induced by environmental stimuli.
authors.affiliation: Department of Applied Biological Science; Tokyo University of Science; Noda, Chiba, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Hanamata
authors.affiliation: Department of Applied Biological Science; Tokyo University of Science; Noda, Chiba, Japan; Research Institute for Science and Technology; Tokyo University of Science; Noda, Chiba, Japan.
authors.forename: Takamitsu
authors.initials: T
authors.lastname: Kurusu
authors.affiliation: Department of Applied Biological Science; Tokyo University of Science; Noda, Chiba, Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Okada
authors.affiliation: Department of Applied Biological Science; Tokyo University of Science; Noda, Chiba, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Suda
authors.affiliation: Department of Applied Biological Science; Tokyo University of Science; Noda, Chiba, Japan.
authors.forename: Koki
authors.initials: K
authors.lastname: Kawamura
authors.affiliation: Department of Applied Biological Science; Tokyo University of Science; Noda, Chiba, Japan.
authors.forename: Emi
authors.initials: E
authors.lastname: Tsukada
authors.affiliation: Department of Applied Biological Science; Tokyo University of Science; Noda, Chiba, Japan; Research Institute for Science and Technology; Tokyo University of Science; Noda, Chiba, Japan.
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Kuchitsu
mesh_terms: D001343:Autophagy; D002460:Cell Line; D003593:Cytoplasm; D005453:Fluorescence; D010588:Phagosomes; D059828:Plant Cells / Q000502:physiology*; D010940:Plant Proteins / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011993:Recombinant Fusion Proteins; D014026:Nicotiana / Q000502:physiology*; D014617:Vacuoles
chemical_list: D010940:Plant Proteins; D011993:Recombinant Fusion Proteins
keywords: autophagosome; autophagy; autophagy-related protein 8 (ATG8); in vivo quantitative imaging; tobacco bright yellow-2 (BY-2) suspension-cultured cells