ABSTRACT
Fluorescent proteins with emission wavelengths in the near-infrared and infrared range are in high demand for whole-body imaging techniques. Here we report near-infrared dimeric fluorescent proteins eqFP650 and eqFP670. To our knowledge, eqFP650 is the brightest fluorescent protein with emission maximum above 635 nm, and eqFP670 displays the most red-shifted emission maximum and high photostability.
Subject(s)
Biotechnology/methods , Luminescent Proteins , Whole Body Imaging/methods , Amino Acid Sequence , Animals , Biotechnology/instrumentation , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/metabolism , Escherichia coli/genetics , Escherichia coli/growth & development , HeLa Cells , Humans , Infrared Rays , Luminescent Proteins/genetics , Luminescent Proteins/toxicity , Mice , Molecular Sequence Data , Protein Multimerization , Protein Stability , Sequence Alignment , Transfection , Xenopus laevis/genetics , Xenopus laevis/metabolism , Zebrafish/genetics , Zebrafish/metabolismABSTRACT
Proteins of the GFP (green fluorescent protein) family are widely used as passive reporters for live cell imaging. In the present study we used H2B (histone H2B)-tKR (tandem KillerRed) as an active tool to affect cell division with light. We demonstrated that H2B-tKR-expressing cells behave normally in the dark, but transiently cease proliferation following green-light illumination. Complete light-induced blockage of cell division for approx. 24 h was observed in cultured mammalian cells that were either transiently or stably transfected with H2B-tKR. Illuminated cells then returned to normal division rate. XRCC1 (X-ray cross complementing factor 1) showed immediate redistribution in the illuminated nuclei of H2B-tKR-expressing cells, indicating massive light-induced damage of genomic DNA. Notably, nondisjunction of chromosomes was observed for cells that were illuminated during metaphase. In transgenic Xenopus embryos expressing H2B-tKR under the control of tissue-specific promoters, we observed clear retardation of the development of these tissues in green-light-illuminated tadpoles. We believe that H2B-tKR represents a novel optogenetic tool, which can be used to study mitosis and meiosis progression per se, as well as to investigate the roles of specific cell populations in development, regeneration and carcinogenesis in vivo.
Subject(s)
Cell Division/radiation effects , Chromatin/metabolism , Green Fluorescent Proteins/metabolism , Luminescent Proteins/metabolism , Molecular Probes/metabolism , Animals , Animals, Genetically Modified/embryology , Animals, Genetically Modified/metabolism , Cell Nucleus/metabolism , Chromatin/radiation effects , DNA Damage/radiation effects , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/metabolism , Embryo, Nonmammalian/radiation effects , Green Fluorescent Proteins/genetics , HeLa Cells , Histones/genetics , Histones/metabolism , Humans , Light , Luminescent Proteins/genetics , Molecular Probes/genetics , Neoplasms/metabolism , Neoplasms/pathology , Neoplasms/ultrastructure , Protein Transport/radiation effects , RNA, Messenger/metabolism , Recombinant Fusion Proteins/metabolism , X-ray Repair Cross Complementing Protein 1 , Xenopus laevisSubject(s)
Culture Media , Green Fluorescent Proteins/chemistry , Photochemistry , Cell Line , HumansABSTRACT
We report a new technique to detect enzyme activity inside cells. The method based on Fluorescence Lifetime Imaging (FLIM) technology allows one to follow sensor cleavage by proteolytic enzyme caspase-3. Specifically, we use the FLIM FRET of living cells via the confocal fluorescence microscopy. A specially designed lentivector pLVT with the DNA fragment of TagRFP-23-KFP was applied for transduction of A549 cell lines. Computer simulations are carried out to estimate FRET efficiency and to analyze possible steric restrictions of the reaction between the substrate TagRFP-23-KFP and caspase-3 dimer. Successful use of the fuse protein TagRFP-23-KFP to register the caspase-3 activation based on average life-time measurements is demonstrated. We show that the average life-time distribution is dramatically changed for cells with the modified morphology that is typical for apoptosis. Namely, the short-lived component at 1.8-2.1 ns completely disappears and the long-lived component appears at 2.4-2.6 ns. The latter is a fingerprint of the TagRFP molecule released after cleavage of the TagRFP-23-KFP complex by caspase-3. Analysis of life-time distributions for population of cells allows us to discriminate apoptotic and surviving cells within single frame and to peform statistical analysis of drug efficiency. This system can be adjusted for HTS by using special readers oriented on measurements of fluorescence life-time.