title: An enhanced mutant of red fluorescent protein DsRed for double labeling and developmental timer of neural fiber bundle formation.
abstract: We developed a new variant of coral-derived red fluorescent protein, DsRed S197Y, which is brighter and essentially free from secondary fluorescence peak. This makes it an ideal reporter for double labeling with green fluorescent protein (GFP). Though purified protein shows only 20% stronger fluorescence emission, culture cells that express DsRed S197Y exhibit a 3-3.5 times higher level of fluorescence than the cells that express wild-type DsRed. The much slower fluorescence maturation of DsRed than that of GFP is a beneficial feature for a fluorescent developmental timer application. When GFP and DsRed S197Y are expressed simultaneously, emissions start at different latency. This provides information about the time after the onset of expression. It reflects the order of cell differentiation if the expression is activated upon differentiation of certain types of cells. We applied this system to the developing brain of Drosophila and visualized, for the first time, the formation order of neural fibers within a large bundle. Our results showed that newly extending fibers of the mushroom body neurons mainly run into the core rather than to the periphery of the existing bundle. DsRed-based timer thus presents an indispensable tool for developmental biology and genetics of model organisms.
authors.affiliation: Tsukita Cell Axis Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Corporation (JST), Kyoto Research Park, Kyoto 600-8813, Japan. vverkhus@cell.tsukita.jst.go.jp
authors.forename: V V
authors.initials: VV
authors.lastname: Verkhusha
authors.forename: H
authors.initials: H
authors.lastname: Otsuna
authors.forename: T
authors.initials: T
authors.lastname: Awasaki
authors.forename: H
authors.initials: H
authors.lastname: Oda
authors.forename: S
authors.initials: S
authors.lastname: Tsukita
authors.forename: K
authors.initials: K
authors.lastname: Ito
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002460:Cell Line; D004330:Drosophila; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins; D007814:Larva / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism*; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*; D009154:Mutation; D009474:Neurons / Q000378:metabolism*; D017956:Photoreceptor Cells, Invertebrate / Q000196:embryology; D010957:Plasmids / Q000378:metabolism; D013053:Spectrophotometry; D013696:Temperature; D013997:Time Factors
chemical_list: D008164:Luminescent Proteins; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins