Dendra2—Photoswitchable Fluorescent Protein

Dendra2 is a monomeric, green-to-red photoswitchable fluorescent protein derived from octocoral Dendronephthya sp. (1). Dendra2 has been optimized for maturation and a bright fluorescence both before and after photoswitching. Its codon usage is optimized for high expression in mammalian cells (2), but Dendra2 can be expressed in many other systems as well.

Dendra2 is an ideal tool for real-time tracking protein dynamics (movement, degradation, etc.) and monitoring selective cell fate (1, 3–4). Once it is irreversibly switched to its red form, Dendra2 is highly photostable.

Dendra2 matures efficiently both at 20 and 37°C, which makes it applicable to a wide range of experimental systems, from cultured mammalian cells to cold-blooded animals. Mammalian cells transiently transfected with Dendra2 display an evenly distributed green signal (e. g., without aggregation) within 10–12 hr posttransfection. No cell toxicity is observed.

Visualizing Dendra2 and Dendra2-Tagged Proteins

1. Visualizing Green (Unswitched) Dendra2
   Unconverted Dendra2 has excitation and emission maxima at 490 and 507 nm, similarly to other green fluorescent proteins. Thus, a FITC (or analogous) filter set is ideal for visualizing green Dendra2.
2. Switching Dendra2 From Green to Red
   You can photoconvert Dendra2 from green to red by light irradiation in either the UV-violet (360–420 nm) or blue region (460–500 nm). We recommend that you use either a commonly-available, 488 nm Ar laser line or a 405 nm diode laser (more efficient, but UV-violet light can harm cells).
3. Visualizing Red (Switched) Dendra2
   Converted Dendra2 protein has excitation and emission maxima at 553 and 573 nm. Thus, a TRITC (or analogous) filter set can be used to visualize red Dendra2.

Excellent Performance in Fusion Applications

Dendra2 is a monomeric fluorescent protein which has been successfully fused to a variety of proteins including cytoplasmic beta-actin, BH3 interacting domain death agonist (BID), nucleolar protein fibrillarin, vimentin, and alpha-tubulin.

Green-to-red photoconversion of Dendra2-tagged proteins. HeLa cells were transiently transfected with vectors encoding Dendra2-tagged fusion proteins. Dendra2 was converted to the red state in selected cells by brief illumination with a 488 nm laser. Confocal images were taken in the green and red channels after photoconversion.

High-Contrast Visualization

After complete photoconversion, the red fluorescence of Dendra2 increases greater than or equal to 150–300 times, whereas the level of green fluorescence becomes at least 10–15 times lower. Thus, the increase in the red-to-green fluorescence ratio results in ~4000-fold contrast. This provides a molecular tool to simultaneously track both the movement of the activated protein and its replacement with the non-activated form.

References

1. Gurskaya, N. G. et al. (2006) Nat. Biotechnol. 24(4):461–465.
2. Haas, J., Park, E. C., and Seed, B. (1996) Curr. Biol. 6(3):315–324.
3. Chudakov, D. M., Lukyanov, S., and Lukyanov, K. A. (2007) Nat. Protoc. 2(8):2024–2032.
4. Zhang, L. et al. (2007) Biotechniques 42(4):446, 448, 450.