title: Simultaneous time-lamination imaging of protein association using a split fluorescent timer protein.
abstract: Studies of temporal behaviors of protein association in living cells are crucially important for elucidating the fundamental roles and the mechanism of interactive coordination for cell activities. We developed a method for investigating the temporal alternation of a particular protein assembly using monomeric fluorescent proteins, fluorescent timers (FTs), of which the fluorescent color changes from blue to red over time. We identified a dissection site of the FTs, which allows complementation of the split FT fragments. The split fragments of each FT variant recovered their fluorescence and maintained inherent rates of the color changes upon the reassembly of the fragments in vitro. We applied this method to visualize the aggregation process of α-synuclein in living cells. The size of the aggregates with the temporal information was analyzed from ratio values of the blue and red fluorescence of the reconstituted FTs, from which the aggregation rates were evaluated. This method using the split FT fragments enables tracing and visualizing temporal alternations of various protein associations by single fluorescence measurements at a given time point.
authors: †Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-0033, Japan.
authors: Ayari
authors: A
authors: Takamura
authors: †Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-0033, Japan.
authors: Mitsuru
authors: M
authors: Hattori
authors: †Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-0033, Japan.
authors: Hideaki
authors: H
authors: Yoshimura
authors: †Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-0033, Japan.
authors: Takeaki
authors: T
authors: Ozawa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry*; D008958:Models, Molecular; D057054:Molecular Imaging / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D013997:Time Factors; D051844:alpha-Synuclein / Q000737:chemistry
chemical_list: D008164:Luminescent Proteins; D010446:Peptide Fragments; D051844:alpha-Synuclein