A bright and photostable photoconvertible fluorescent protein

Abstract

Photoconvertible fluorescent proteins are potential tools for investigating dynamic processes in living cells and for emerging super-resolution microscopy techniques. Unfortunately, most probes in this class are hampered by oligomerization, small photon budgets or poor photostability. Here we report an EosFP variant that functions well in a broad range of protein fusions for dynamic investigations, exhibits high photostability and preserves the approximately 10-nm localization precision of its parent.

Figure 1. Tracking dynamics in live cells with mEos2 fusions

(a)–(c): Mitochondrial dynamics observed by photoconversion of a single mitochondrion (red) in adherent HeLa epithelial cells expressing mEos2–7AA linker-mitochondria. (a) Region of interest (white polygon) is illuminated at 405 nm for 5 sec, t = 0; (b) the photoconverted mitochondrion fragments and exchanges cargo with an adjacent mitochondrion, t = 5 min; (c) a portion of the fragmented mitochondrion condenses and migrates away from neighbors, t = 15 min. (d)–(f): Gap junction plaque (GJs) dynamics in HeLa cells expressing the connexin fusion mEos2–7AA linker-Cx43. (d) The central GJ plaque in a trio is photoconverted to red (white box), t = 0; (e) Fusion of a native plaque (green) to the photoconverted plaque, as well as new plaque growth are observed, t = 70 min; (f) Rotated plaque reveals photoconverted area surrounded by unconverted fusion and new growth, t = 120 min. (g)–(j): Observing mitosis in pig kidney cells (LLC-PK1 line) with the histone fusion mEos2–6AA linker-H2B. (g) Approximately half of the condensed chromatin in a prophase cell is photoconverted (white polygon) for 3 sec, t = 0; (h) Alignment at the spindle shows clear segregation between the native and photoconverted chromatin. Note the unaligned chromosome that serves as a reference point, t = 40 min; (i) In early anaphase, the unaligned chromosome is still present, t = 70 min; (j) in telophase, the daughter nuclei begin to form, t = 170 min. Scale bars represent 10 μm.

Figure 2. PALM Images of mEos2 fusions

(a–c) mEos2 fusions to microtubule end-binding protein EB3 in a highly expressing cell decorate microtubule filaments. (a) Widefield TIRF image showing the edge in a HeLa cell and area of interest. (b) TIRF-resolution image in area of interest (scale bar 500 nm). (c) The same region of interest in PALM; note the emergence of fibers that are irresolvable in the standard TIRF image. (d–f) mEos2 fused to vimentin. (d) Widefield TIRF image of the network close to the surface and region of interest. (e) TIRF resolution image in region of interest (scale bar 1 μm). (f) PALM image of same region. Note what appears to be an intersection of three fibers in the bottom right of the TIRF image (e) is shown to not be. (g–l) mEos2 fusions to keratin. (g, j) Whole cell TIRF images with areas of interest, (h–i) and (k–l), respectively. (h, k) Diffraction limited images of the adjacent regions of interest (scale bars of 1 μm and 200 nm, respectively). (i) The corresponding PALM image for (h) details the intricate network of keratin fibers. (l) The simple blob that appears in (k) is resolved into an intersection of fibers, with a large dark area in the center of the hub that appears as a bright blur in the diffraction limited image. Features of ~50nm are resolved despite substantially higher probe localization, a similar lower limit on feature sizes has been exhibited elsewhere with other probes,.