title: In vivo fluorescence correlation spectroscopy analyses of FMBP-1, a silkworm transcription factor.
abstract: Fibroin modulator-binding protein 1 (FMBP-1) is a silkworm transcription factor that has a unique DNA-binding domain called the one score and three amino acid peptide repeat (STPR). Here we used fluorescence correlation spectroscopy (FCS) to analyze the diffusion properties of an enhanced green fluorescent protein-tagged FMBP-1 protein (EGFP-FMBP-1) expressed in posterior silk gland (PSG) cells of Bombyx mori at the same developmental stage as natural FMBP-1 expression. EGFP-FMBP-1 clearly localized to cell nuclei. From the FCS analyses, we identified an immobile DNA-bound component and three discernible diffusion components. We also used FCS to observe the movements of wild-type and mutant EGFP-FMBP-1 proteins in HeLa cells, a simpler experimental system. Based on previous in vitro observation, we also introduced a single amino acid substitution in order to suppress stable FMBP-1-DNA binding; specifically, we replaced the ninth Arg in the third repeat within the STPR domain with Ala. This mutation completely disrupted the slowest diffusion component as well as the immobile component. The diffusion properties of other FMBP-1 mutants (e.g. mutants with N-terminal or C-terminal truncations) were also analyzed. Based on our observations, we suggest that the four identifiable movements might correspond to four distinct FMBP-1 states: (a) diffusion of free protein, (b) and
authors.affiliation: Faculty of Advanced Life Science Hokkaido University Sapporo Japan.
authors.forename: Motosuke
authors.initials: M
authors.lastname: Tsutsumi
authors.affiliation: Faculty of Advanced Life Science Hokkaido University Sapporo Japan; Biomedical Research Support Center Nagasaki University School of Medicine Nagasaki, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Muto
authors.affiliation: Faculty of Advanced Life Science Hokkaido University Sapporo Japan.
authors.forename: Shohei
authors.initials: S
authors.lastname: Myoba
authors.affiliation: Faculty of Science Hokkaido University Sapporo Japan.
authors.forename: Mai
authors.initials: M
authors.lastname: Kimoto
authors.affiliation: Faculty of Advanced Life Science Hokkaido University Sapporo Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Kitamura
authors.affiliation: Faculty of Advanced Life Science Hokkaido University Sapporo Japan.
authors.forename: Masakatsu
authors.initials: M
authors.lastname: Kamiya
authors.affiliation: Faculty of Advanced Life Science Hokkaido University Sapporo Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kikukawa
authors.affiliation: Faculty of Science Hokkaido University Sapporo Japan.
authors.forename: Shigeharu
authors.initials: S
authors.lastname: Takiya
authors.affiliation: Faculty of Advanced Life Science Hokkaido University Sapporo Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Demura
authors.affiliation: Faculty of Advanced Life Science Hokkaido University Sapporo Japan; Chitose Institute of Science and Technology Chitose, Japan.
authors.forename: Keiichi
authors.initials: K
authors.lastname: Kawano
authors.affiliation: Faculty of Advanced Life Science Hokkaido University Sapporo Japan.
authors.forename: Masataka
authors.initials: M
authors.lastname: Kinjo
authors.affiliation: Faculty of Advanced Life Science Hokkaido University Sapporo Japan.
authors.forename: Tomoyasu
authors.initials: T
authors.lastname: Aizawa
keywords: DNA‐binding protein; fluorescence correlation spectroscopy; one score and three peptide repeat domain; silkworm; transcription factor