Laboratory for Cell Biology, Philipps University Marburg, Karl-von-Frisch-Str. 8, 35032 Marburg, Germany.
Molecular Plant Physiology and Photobiology, Philipps University Marburg, Karl-von-Frisch-Str. 8, 35032 Marburg, Germany.
Protist. 2020 Feb;171(1):125715. doi: 10.1016/j.protis.2020.125715. Epub 2020 Jan 30.
Phaeodactylum tricornutum is an ecologically and evolutionarily relevant microalga that has developed into an important model for molecular biological studies on organisms with complex plastids. The diatom is particularly suitable for in vivo protein localization analyses via fluorescence microscopy in which the green fluorescent protein (GFP) and its derivatives are dominantly used. Whereas GFP fluorescence emission is usually measured between 500 and 520nm in confocal microscopy, the autofluorescence of the P. tricornutum plastid is detected above 625nm. Here we established the fluorescent protein mRuby3 as tag for efficient in vivo protein localization studies by expressing a codon-optimized gene in P. tricornutum. mRuby3 was directed to seven different subcellular localizations by means of full-length marker protein or N-/C-terminal targeting signal fusions; its emission was detected efficiently between 580 and 605nm, being unequivocally distinguishable from the plastid autofluorescence in vivo. Moreover, mRuby3 proved to be highly suitable for co-localization experiments using confocal laser scanning microscopy in which mRuby3 fusion proteins were expressed in parallel with GFP-tagged proteins. Our results show the potential of mRuby3 for its application in studying protein targeting and localization in P. tricornutum, particularly underlining its compatibility with GFP and the plastid autofluorescence in signal detection.
三角褐指藻是一种具有生态学和进化意义的微藻，已发展成为具有复杂质体的生物体分子生物学研究的重要模式生物。该硅藻特别适合通过荧光显微镜进行体内蛋白质定位分析，其中绿色荧光蛋白（GFP）及其衍生物被广泛使用。尽管 GFP 荧光发射在共聚焦显微镜中通常在 500nm 至 520nm 之间进行测量，但三角褐指藻质体的自发荧光在 625nm 以上被检测到。在这里，我们通过在三角褐指藻中表达优化密码子的基因，将荧光蛋白 mRuby3 确立为用于高效体内蛋白质定位研究的标签。通过全长标记蛋白或 N-/C-末端靶向信号融合，将 mRuby3 定向到七个不同的亚细胞定位；其发射在 580nm 至 605nm 之间被有效检测到，与体内质体自发荧光具有明确的可分辨性。此外，mRuby3 被证明非常适合使用共聚焦激光扫描显微镜进行共定位实验，其中 mRuby3 融合蛋白与 GFP 标记蛋白平行表达。我们的结果表明，mRuby3 在研究三角褐指藻中蛋白质靶向和定位方面具有应用潜力，特别是在信号检测方面，其与 GFP 和质体自发荧光的兼容性尤为突出。
