Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues

Abstract

Green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its homologs from diverse marine animals are widely used as universal genetically encoded fluorescent labels. Many laboratories have focused their efforts on identification and development of fluorescent proteins with novel characteristics and enhanced properties, resulting in a powerful toolkit for visualization of structural organization and dynamic processes in living cells and organisms. The diversity of currently available fluorescent proteins covers nearly the entire visible spectrum, providing numerous alternative possibilities for multicolor labeling and studies of protein interactions. Photoactivatable fluorescent proteins enable tracking of photolabeled molecules and cells in space and time and can also be used for super-resolution imaging. Genetically encoded sensors make it possible to monitor the activity of enzymes and the concentrations of various analytes. Fast-maturing fluorescent proteins, cell clocks, and timers further expand the options for real time studies in living tissues. Here we focus on the structure, evolution, and function of GFP-like proteins and their numerous applications for in vivo imaging, with particular attention to recent techniques.

Structure and properties of chimeric small heat shock proteins containing yellow fluorescent protein attached to their C-terminal ends

“…Fluorescent chimeric proteins are widely used for the investigation of intracellular localization and protein-protein interaction (Chudakov et al 2010). Chimeras of HspB1, HspB5, HspB6, and HspB8 containing different fluorescent proteins (green, cyan, yellow, and citrine) fused to the N-terminal ends or to the C-terminal ends of sHsp were designed and successfully used in a number of investigations Abraham 2011, 2013;Borrelli et al 2002;Shelden et al 2002;Qian et al 2006;Fontaine et al 2005Fontaine et al , 2006Sun et al 2004Sun et al , 2007Doshi et al 2009).…”

“…In order to analyze the mechanism of sHsp functioning, it is desirable to develop a method that can follow the formation of different oligomers of sHsp and their translocation in the cell. Utilization of fluorescent proteins seems to be a very promising tool for this purpose (Chudakov et al 2010). A number of different fluorescent chimeras of sHsp have been designed and used for investigation of the intracellular location (Doshi et al 2009;Qian et al 2006;Borrelli et al 2002;Shelden et al 2002;Abraham 2011, 2013;Irobi et al 2004;Sun et al 2007) and formation of homooligomeric and heterooligomeric complexes of sHsp (Fontaine et al 2005;Fontaine et al 2006;Sun et al 2004;Datskevich et al 2012a).…”

Novel approaches to in vitro transgenesis

“…cherry, tomato; Chudakov et al 2010). Fluorescent proteins display different properties, including variable brightness, ability to form monomers or multimers, photo-switching, photo-activation, photo-conversion, and pH and temperature sensitivity (Giepmans et al 2006, Chudakov et al 2010.…”

Fluorescent optical fiber sensors for cell viability monitoring

“…2,3 Another common way for making cells visible is using GFP-transfected cells, which usually provides more stable uorescent signals because the concentration of the stain within the cells remains almost constant. 4,5 However, transfected cells are sometimes avoided because of the unpredictable inuence of the modication to the results of the experiment. Cells that die aer dye activation can provide an unwanted uorescence background for rather a long time aer death.…”