Animations showing the various applications of fluorescent proteins for monitoring signaling processes in living cells. Please use the "Play" button to start the animations. Animations were created by Cameron Slayden, with scientific oversight by Klaus Hahn (Department of Cell Biology, Scripps Research Institute, CA USA).

Animation A. Fluorescence resonance energy transfer (FRET) reports the level and localization of a protein ligand. Two variants of green fluorescent protein (GFP) are attached to a protein or peptide that undergoes a change in conformation upon ligand binding. This conformational change alters intramolecular FRET.

Animation B. Intermolecular FRET reports protein activation. Variants of GFP are attached both to the target protein and a peptide or protein that can bind to only the activated state of the target protein. When the activated state occurs the two fluorophores are brought close enough to undergo FRET.

Animation C. Biosensors report the accumulation of a signal in a specific cellular region. When a signal is localized in a particular subcellular compartment, a fluorescent molecule which binds specifically to that signal accumulates in the compartment, producing a local increase in fluorescence intensity. In the animation, the increase in a lipid second messenger recruits a biodetector to a region of the plasma membrane.

Animation D. Single-chain biosensors report protein activation through intramolecular FRET. Fusion of the detector (the protein or domain that binds to the activated state of the target protein), the target protein, and the fluorophores produces a single-chain biosensor. The detector binds to the activated target protein bringing the two fluorophores close enough to undergo intramolecular FRET.

Animation E. Detecting protein conformation changes with an attached fluorescent dye. Conformational changes in the protein can result in a change in either the wavelength or intensity of a dye attached covalently to the protein. These changes in fluorescence are often associated with a transition from a hydrophilic to a hydrophobic environment for the fluorophore when the attached protein changes conformation.

Animation F. FRET or a dye report activation of an endogenous, untagged protein. Fusion of two GFP variants to the detector protein or peptide can report activation of endogenous, untagged proteins through intramolecular FRET. Alternatively, a single environmentally sensitive fluorescent dye can undergo a change in fluorescence in response to interaction with the activated form of an endogenous, untagged protein.

Animation G. Enzymatic substrate sensors report covalent peptide modifications by intramolecular FRET. The animation shows phosphorylation producing a change in the conformation of a peptide attached to two variants of GFP. This leads to intramolecular FRET.

Animation H. Permutation: Conformational changes affect the fluorescence of a GFP mutant fused to the target protein. In the animation, the protein that changes conformation is fused in frame with a GFP mutant and produces a change in the fluorescence of the GFP mutant in response to ligand binding.

Animation I. Bimolecular fluorescence complementation (BiFC) reports protein interactions. GFP can be divided into two nonfluorescent halves and each half attached to a protein domain. When the two domains interact, the GFP halves are united to produce fluorescence.

Citation: F. Gaits, K. Hahn, Shedding light on cell signaling: Interpretation of FRET biosensors. Sci. STKE 2003, pe3 (2003).

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