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

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 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.

Figure 1

Structural basis of the amino acid substitutions converting mCherry into the FTs. (a) Alignment of the amino acid sequences of FTs with GFP and mCherry. Residues buried in β-can are shaded. Stars indicate residues that are forming the chromophore. β-sheet-forming regions and α-helixes are denoted as arrows and ribbons, respectively. Mutations in FTs relative to mCherry are shown in black. Alignment numbering follows that of GFP. (b-d) Immediate environment of the chromophore in parental mCherry with mutations found in fast-FT (b), medium-FT (c) and slow-FT (d). The surrounding residues are shown within 9.5 Å of the chromophore. The chromophore is shown in black, conserved amino acid residues are in light gray, and mutated residues are in gray. Water molecules are represented as gray spheres. Hydrogen bonds are indicated with dashed gray lines. Substitution of amino acid residues was performed using Swiss PDBViewer v.3.7 (http://www.expasy.org/spdbv/). The best fits for the introduced mutated residues were achieved by selection of the rotamers with the lowest scores.

Figure 2

Fluorescence properties of the purified FTs. (a) Time changes of the excitation spectra for the blue (peak at 403 nm) and red (peak at 583 nm) forms of the fast-FT at 37 °C. The emission was measured at 466 and 625 nm, respectively. (b) Time changes of the emission spectra for the blue (peak at 466 nm) and red (peak at 606 nm) forms of the fast-FT at 37 °C. The blue and red forms were excited at 402 and 540 nm, respectively. (c) Maturation kinetics of the blue (solid lines) and red (dotted lines) forms for the fast-FT (triangles), medium-FT (squares) and slow-FT (circles) at 37 °C. The experimental data were fitted using the kinetic scheme shown in Scheme 1. (d-f) Time changes of the red to blue ratio for the fast-FT (d), medium-FT (e) and slow-FT (f) determined at different temperatures. The blue and red fluorescence emissions were detected at 466 nm and 606 nm with excitation at 402 nm and 580 nm, respectively. (g-i) The pH dependences of the fluorescence intensities of the blue (g) and red (h) forms and the ratio between the red and blue forms (i) for fast-FT (triangles), medium-FT (squares), slow-FT (circles) and EBFP2 (g) or mCherry (h) (diamonds). The fluorescence for blue and red forms was registered at 466 nm and 606 nm with excitation at 400 nm and 580 nm, respectively. Experimental error is less than 5%.

Figure 3

Behavior of the FTs in D. melanogaster S2 cells. (a-c) Time changes of the blue (solid symbols) and red (open symbols) mean fluorescence intensities of the S2 cells stably expressing fast-FT (a), medium-FT (b) and slow-FT (c) at 25 °C determined using flow cytometry. The maxima of the blue fluorescence were achieved at 13 h with fast-FT (a), 21 h with medium-FT (b) and 80 h with slow-FT (c). The maxima of the red fluorescence were achieved at 200 h with fast-FT (a) and 197 h with medium-FT (b). The experimental data were fitted using the kinetic scheme shown in Scheme 1. For all curves the coefficients of determination, R², are larger than 0.91. Error bars, s.d.

Figure 4

Behavior of the FTs and LAMP-2A-medium-FT fusion protein in mammalian cells. (a) HeLa Tet-Off cells transiently expressing slow-FT in cytoplasm were analyzed at different times using flow cytometry. Dot histograms for the selected times such as 1.5 (green), 7 (light green), 17 (yellow), 41 (orange) and 56 (red) hours after the addition of doxycycline are shown. The histogram for the control cells transfected with an empty vector is shown in dark blue. Doxycycline was added 7 h after the transfection. (b) Time changes of the blue (blue circles) and red (red circles) mean fluorescence intensities of HeLa Tet-Off cells expressing the slow-FT at 37 °C determined using flow cytometry. The maxima of the blue and red fluorescence intensities were achieved at 17 and 41 h, respectively. The experimental data were fitted using the kinetic scheme shown in Scheme 1. The coefficients of determination, R², are larger than 0.92. Error bars, s.d. (c) Intracellular localization of the LAMP-2A-medium-FT fusion protein in HeLa Tet-Off cells at different time points after the shutting down of transcription with doxycycline. The blue and red forms of LAMP-2A-medium-FT are shown with green and red pseudocolors, respectively. Bar is 10 μm.

Scheme 1

Suggested transformations of the chemical structures of the chromophore along the red chromophore formation pathway in the FTs.