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

Nature Chemical Biology volume 5, pages 118–126 (2009)Cite this article

Abstract

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 °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.

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Figure 1: Structural basis of the amino acid substitutions converting mCherry into the FTs.
Figure 2: Fluorescence properties of the purified FTs.
Figure 3: Behavior of the FTs in D. melanogaster S2 cells.
Figure 4: Behavior of the FTs and LAMP-2A–medium-FT fusion protein in mammalian cells.

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Acknowledgements

We thank J. Zhang (Albert Einstein College of Medicine) for assistance with flow cytometry. We are grateful to R. Tsien (University of California at San Diego) for the complementary DNA of mCherry and D.Reeves (Albert Einstein College of Medicine) for the pcDNA-3.1-LAMP-2A-TSapphire-GFP vector. This work was supported by grants from the US National Institutes of Health (GM070358 and GM073913 to V.V.V. and AG021904 to A.M.C.).

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Author notes

  1. Fedor V Subach and Oksana M Subach: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Anatomy and Structural Biology, and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, 10461, New York, USA

    Fedor V Subach, Oksana M Subach, Illia S Gundorov, Kateryna S Morozova, Kiryl D Piatkevich, Ana Maria Cuervo & Vladislav V Verkhusha

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Contributions

F.V.S. and I.S.G. developed the proteins. F.V.S., K.S.M. and K.D.P. characterized the proteins in vitro. O.M.S. and F.V.S. characterized the proteins in mammalian cells. V.V.V. designed and planned the project and, together with A.M.C., F.V.S. and O.M.S., wrote the manuscript.

Corresponding author

Correspondence to Vladislav V Verkhusha.

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Supplementary Figures 1–7, Supplementary Tables 1 and 2, and Supplementary Methods (PDF 400 kb)

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Subach, F., Subach, O., Gundorov, I. et al. Monomeric fluorescent timers that change color from blue to red report on cellular trafficking. Nat Chem Biol 5, 118–126 (2009). https://doi.org/10.1038/nchembio.138

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