Monomeric fluorescent timers that change color from blue to red report on cellular trafficking

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Nature Chemical Biology (Impact Factor: 13). 02/2009; 5(2):118-26. DOI: 10.1038/nchembio.138
Source: PubMed


Based on the mechanism for chromophore formation in red fluorescent proteins, we developed three mCherry-derived monomeric variants, called fluorescent timers (FTs), that change their fluorescence from the blue to red over time. These variants exhibit distinctive fast, medium and slow blue-to-red chromophore maturation rates that depend on the temperature. At 37 degrees C, the maxima of the blue fluorescence are observed at 0.25, 1.2 and 9.8 h for the purified fast-FT, medium-FT and slow-FT, respectively. The half-maxima of the red fluorescence are reached at 7.1, 3.9 and 28 h, respectively. The FTs show similar timing behavior in bacteria, insect and mammalian cells. Medium-FT allowed for tracking of the intracellular dynamics of the lysosome-associated membrane protein type 2A (LAMP-2A) and determination of its age in the targeted compartments. The results indicate that LAMP-2A transport through the plasma membrane and early or recycling endosomes to lysosomes is a major pathway for LAMP-2A trafficking.


Available from: Kateryna Morozova
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    • "Blots were developed using Western Lightning ECL or ECL+ (PerkinElmer). UAS-FT-nSyb was generated by chimeric PCR using the primers in Table S1 and making use of the UAS-nSyb-Ub-pUAST vector (Uytterhoeven et al., 2011) and a vector with the slow FT (Subach et al., 2009) as on November 25, 2014 Downloaded from Published November 24, 2014 Images were captured using a confocal microscope (A1R) for immunohistochemistry , live imaging of shi ts1 mutants expressing UAS-FT-nSyb, and the colocalization of FM 4–64 (excitation of 488 nm; emission of 700/75 nm) accumulations with Rab5-GFP (excitation of 488 nm; emission of 525/50 nm), through a 60×, 1.2 NA water immersion lens (immunohistochemistry ) or near-infrared Apochromat 60×, 1.0 NA water dipping lens (live imaging and colocalization) at room temperature. "
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    • "Some GFP variants, especially red-shifted variants, undergo additional chromophore modification. The maturation time for most fluorescent proteins ranges from minutes to hours, but can be days in the case of the " Timer " type of fluorescent proteins (Terskikh et al. 2000; Subach et al. 2009). In general, relatively fast maturation rates are desired. "
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    • "Methods employing SNAP and FT have advantages: they (1) do not need antibodies , radioisotopes, CHX, or photoactivation; (2) can examine protein degradation in a single living cell; and (3) can distinguish old from new proteins by fluorescence wavelength . Indeed, a recent study (Subach et al., 2009) showed the temporal mobilization of LAMP-2A using FT. Thus the SNAP and FT-based methods would seem to have a variety of applications. "
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