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First published online December 5, 2007
doi: 10.1242/10.1242/jcs.005801

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Advances in fluorescent protein technology

¹ The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
² Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development (NICHD), Bethesda, MD 20892, USA
³ National High Magnetic Field Laboratory and Department of Biological Science, The Florida State University, Tallahassee, FL 32310, USA

View larger version (57K): [in this window] [in a new window]   Fig. 1. (A) FP β-barrel architecture and approximate dimensions, and chromophore structures of common Aequorea FP derivatives. (1) BFP, (2) CFP, (3) EGFP, (4) YFP. The tryptophan residue (Trp66) in (2) is illustrated in the cis conformation as occurs for Cerulean derivatives (Malo et al., 2007) rather than the trans isomer that is common to CFP and related variants. Portions of the chromophores that are conjugated and give rise to fluorescence are shaded with colors corresponding to the emission spectral profile. (B) Aequorea GFP mutation map showing common mutations superimposed on a topological layout of the peptide structure. β-sheets are numbered and depicted as thin, green cylinders with an arrow pointing towards the C-terminus, whereas -helices are depicted as gray cylinders. Mutations are color-coded to represent the variants to which they apply: BFPs (blue), CFPs (cyan), GFPs (green), YFPs (yellow), Sapphire (violet), folding, shared and monomerizing (gray). Note that almost 75% of the mutations are located in the central helix and in β-sheet strands 7, 8 and 10. In general, wavelength-specific mutations occur near the central helix containing the chromophore, whereas folding mutations occur throughout the sequence. Many of the cyan and yellow FP mutations introduced near the termini of the proteins resulted during the CyPet and YPet mutagenesis efforts (Nguyen and Daugherty, 2005). Several of the sfGFP folding mutations (S30R, Y39N, F99S and N105T) also occur away from the chromophore. The monomerizing mutation A206K is useful for all known GFP derivatives, but is replaced by A206V in sfGFP and EBFP2.

View larger version (131K): [in this window] [in a new window]   Fig. 2. (A) Chromophore structural variation in yellow, orange, and red FPs. (1) FPs derived from DsRed and other reef coral organisms thought to have a cis-chromophore. The residue at position 66 can be Met, Gln, Thr, Cys or Glu. (2) eqFP611, a red variant derived from Entacmaea quadricolor, is the only known FP featuring a trans-chromophore (Petersen et al., 2003). (3) ZsYellow (also zFP538), derived from the button polyp Zoanthus, features a novel three-ring chromophore that is created when the lysine residue at position 66 cyclizes with its own -carbon to form a tetrahydropyridine ring conjugated to the chromophore (Remington et al., 2005). (4) mOrange, one of the mFruit proteins (Shaner et al., 2004), also features a three-ring chromophore where Thr66 cyclizes with the preceding carbonyl carbon to yield a partially conjugated oxazole ring (Shu et al., 2006). (B) DsRed mutation map showing mutations of useful variants superimposed on a topological layout of the peptide structure. β-sheets are numbered and depicted as thin, red cylinders with an arrow pointing towards the C-terminus, whereas -helices are depicted by gray cylinders. Extensions of the N- and C-termini due to the addition of amino acids derived from GFP to improve fusion performance are shaded in light green. Mutations are color-coded to represent the variants to which they apply: mRFP1 (red), mCherry (cyan), mPlum (violet), dTomato (yellow), monomerizing mutations (green) and shared mutations (gray). Note that unlike the cluster of mutations surrounding the chromophore for Aequorea GFP variants (Fig. 1), red FP mutations are distributed throughout the sequence.

View larger version (103K): [in this window] [in a new window]   Fig. 3. (A-L) Subcellular localization of selected monomeric FP fusions (listed in Table 1) with targeting proteins imaged in widefield fluorescence. Images are pseudocolored to match the FP emission profile. The FP fusion terminus and number of linker amino acids is indicated after the name of the targeted organelle or fusion protein. (A) EBFP2-mito-N-7 (human cytochrome C oxidase subunit VIII; mitochondria); (B)mCerulean-paxillin-N-22 (chicken; focal adhesions); (C) mTFP1-actin-C-7 (human β-actin; filamentous actin); (D)mEmerald-keratin-N-17 (human cytokeratin 18; intermediate filaments); (E) superfolder GFP-lamin B1-C-10 (human lamin B1; nuclear envelope); (F)mVenus-Cx43-N-7 (rat -1 connexin-43; gap junctions); (G) YPet-EB3-N-7 (human microtubule-associated protein; RP/EB family); (H) mKO-Golgi-N-7 (N-terminal 81 amino acids of human β-1,4-glactosyltransferase; Golgi complex); (I) tdTomato-zyxin-N-7 (human zyxin; focal adhesions); (J) TagRFP-tubulin-C-6 (human -tubulin; microtubules); (K)mCherry-vimentin-N-7 (human vimentin; intermediate filaments); (L)mPlum--actinin-N-19 (human non-muscle; cytoskeleton). (M-Q) Fusion of mEGFP with human histone H2B (mEGFP-H2B-N-6). (M) interphase; (N)prophase; (O) prometaphase; (P)metaphase; (Q) anaphase.

View larger version (40K): [in this window] [in a new window]   Fig. 4. Photoactivation, photoconversion and photoswitching mechanisms for optical highlighter FPs. (A) Photoactivation of PA-GFP (illustrated) and PS-CFP2 is believed to occur due to decarboxylation of Glu222 followed by conversion of the chromophore from a neutral to anionic state. (B) Green-to-red photoconversion for Kaede, KikGR, Dendra2 and Eos, all of which contain the HYG chromophore, occurs when the FP is illuminated with ultraviolet or violet radiation to induce cleavage between the amide nitrogen and -carbon atoms in the His62 residue leading to subsequent formation of a conjugated dual imidazole ring system. (C) Photoswitching of Dronpa involves cis-trans photoisomerization induced by alternating radiation between 405 nm and 488 nm. A similar isomerization mechanism is suggested to operate in mTFP0.7 and KFP1.

View larger version (89K): [in this window] [in a new window]   Fig. 5. Optical highlighter FPs in action imaged with laser scanning confocal microscopy. (A-C) Photoactivation of mPA-GFP-actin-C-7 in opossum kidney (OK cell line) epithelial cells. (A) Circular region of interest selected with an Olympus FV1000 tornado scanner is illuminated at 405 nm for 5 seconds, t=0 minutes. (B) The photoactivated actin chimera first translocates to the ruffles at the cellular margins as fluorescence intensity decreases in the activated region, t=5 minutes. (C) Ruffles, cytoplasmic actin pools and the filamentous actin network gain more intensity at t=60 minutes. (D-F) Tracking of mitochondria labeled with tdEos-mito-N-7 in rabbit kidney (RK-13 cell line) epithelial cells. (D) Photoconversion of a single mitochondrion (red) in a selected region at 405 nm illumination, t=0 minutes. (E) Close approach of a non-converted (green) mitochondrion (arrow), t=10 minutes. (F) Cargo exchange between mitochondria (arrow), t=20 minutes. (G-I) Examination of lamellipodia with Dendra2-actin-C-7 in OK cells. (G) Photoconversion (red) of the selected region (box) with a 405 nm laser, t=0 minutes. (H) The photoconverted channel illustrates podosome formation by photoconverted actin and changes in leading edges, t=20 minutes. (I) Photoconverted lamellipod retracts amid increased podosome formation and generation of a new leading edge, t=45 minutes. (J-L) Photoswitching of the actin cytoskeleton with Dronpa-actin-C-7 in rat thoracic aorta (A7r5 cell line) myoblasts. (J) Actin network imaged with a 488-nm laser, t=0 minutes. (K) After completely photoswitching the labeled actin `off' at 488 nm, the region spelling FV10 was activated with a 405 nm laser, t=3 minutes. (L) FV10 region photobleached while imaging the actin network at 488 nm.

View larger version (49K): [in this window] [in a new window]   Fig. 6. Photoactivated localization microscopy (PALM) imaging of focal adhesions near the coverslip surface in paraformaldehyde-fixed Gray fox lung fibroblast cells. (A) Widefield fluorescence image of multiple focal adhesions labeled with tdEos fused to human vinculin. (B) Total internal reflection fluorescence (TIRF) summed-molecule image of the focal adhesion within the region indicated by the box in A. (C) PALM view of the focal adhesion structure shown in B, which includes the apparent assembly of vinculin into a partial network (arrows).

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