Kindling fluorescent proteins for precise in vivo photolabeling.
Photobleaching of green fluorescent protein (GFP) is a widely used approach for tracking the movement of subcellular structures and intracellular proteins. Although photobleaching is a powerful technique, it does not allow direct tracking of an object's movement and velocity within a living cell. Direct tracking becomes possible only with the introduction of a photoactivated fluorescent marker. A number of previous studies have reported optically induced changes in the emission spectra of fluorescent proteins. However, the ideal photoactivated fluorescent marker should be a nonfluorescent tag capable of "switching on" (i.e., becoming fluorescent) in response to irradiation by light of a particular wavelength, intensity, and duration. In this report, we generated a mutant of Anemonia sulcata chromoprotein asCP. The mutant protein is capable of unique irreversible photoconversion from the nonfluorescent to a stable bright-red fluorescent form ("kindling"). This "kindling fluorescent protein" (KFP1) can be used for precise in vivo photolabeling to track the movements of cells, organelles, and proteins. We used KFP1 for in vivo cell labeling in mRNA microinjection assays to monitor Xenopus laevis embryo development and to track mitochondrial movement in mammalian cells.
Chudakov
Dmitriy M
DM
Shemiakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, 117997 Moscow, Russia.
Belousov
Vsevolod V
VV
Zaraisky
Andrey G
AG
Novoselov
Vladimir V
VV
Staroverov
Dmitry B
DB
Zorov
Dmitry B
DB
Lukyanov
Sergey
S
Lukyanov
Konstantin A
KA
D000818:Animals; D002465:Cell Movement; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005453:Fluorescence; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects*; D008856:Microscopy, Fluorescence / Q000379:methods; D008928:Mitochondria / Q000502:physiology / Q000648:ultrastructure; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D012615:Sea Anemones / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013194:Staining and Labeling / Q000379:methods*; D014982:Xenopus laevis / Q000033:anatomy & histology / Q000196:embryology / Q000254:growth & development
D008164:Luminescent Proteins; D011994:Recombinant Proteins