Method to detect interactions between cellular components in intact living cells, and to extract quantitative information relating to those interactions by fluorescence redistribution

A method for detecting if a compound induces interaction between two intracellular proteins comprising the steps of:

• (a) providing a cell that contains three heterologous conjugates,
• a first heterologous conjugate comprising an anchor protein that specifically binds to an internal structure within the cell conjugated to an interactor protein of type A
• a second heterologous conjugate comprising an interactor protein of type B conjugated to the first protein of interest
• a third heterologous conjugate comprising a second protein of interest conjugated to a detectable group,
• (b) inducing interaction of protein of type A with protein of type B through application of a dimerizer molecule;
• (c) detecting the intracellular distribution of the detectable group
• (d) repeating step (c) with and without the compound;
• an intracellular distribution of said detectable group mimicking the intracellular distribution of the anchor-protein;
• a change in intracellular distribution of the detectable group with the compound to being indicative that the compound induces interaction between the first and the second protein of interest.

A method for screening for compounds modulating the interaction between two intracellular proteins comprising the steps of:

• (a) providing a cell that contains three heterologous conjugates,
• a first heterologous conjugate comprising an anchor protein that specifically binds to an internal structure within the cell conjugated to an interactor protein of type A
• a second heterologous conjugate comprising an interactor protein of type B conjugated to the first protein of interest
• a third heterologous conjugate comprising a second protein of interest conjugated to a detectable group,
• (b) detecting the non-dimerized intracellular distribution of the detectable group;
• (c) inducing interaction of protein of type A with protein of type B through application of a dimerizer molecule;
• (d) detecting the dimerized intracellular distribution of the detectable group;
• (e) applying the test compound
• (f) detecting the intracellular distribution of the detectable group in the test-stage;
• the non-dimerized distribution forming the first, and the dimerized distribution forming the second end of the detection scale on which the intracellular distribution in the test-stage is measured such that if the intracellular distribution in the test-stage mimics the dimerized distribution, the compound has no effect on the interaction between the two proteins of interest, and if the intracellular distribution in the test-stage mimics the non-dimerized distribution the compound disrupts the interaction between the two proteins of interest.

A method for screening for compounds modulating the interaction between two intracellular proteins comprising the steps of:

• (a) providing at least three homogenous groups of homogenous cells that contains three heterologous conjugates,
• a first heterologous conjugate comprising an anchor protein that specifically binds to an internal structure within the cell conjugated to an interactor protein of type A
• a second heterologous conjugate comprising an interactor protein of type B conjugated to the first protein of interest
• a third heterologous conjugate comprising a second protein of interest conjugated to a detectable group;
• (b) applying the test compound to the first group of cells;
• (c) inducing interaction of protein of type A with protein of type B through application of a dimerizer molecule to all three groups of cells;
• (d) applying a competitive reference compound to the second group of cells;
• (e) detecting the intracellular distribution of the detectable group in the test-stage in the first one;
• (f) detecting the non-dimerized intracellular distribution of the detectable group in the second group;
• (g) detecting the dimerized intracellular distribution of the detectable group in the third group;
• the non-dimerized distribution forming the first, and the dimerized distribution forming the second end of the detection scale on which the intracellular distribution in the test-stage is measured such that if the intracellular distribution in the test-stage mimics the dimerized distribution, the compound has no effect on the interaction between the two proteins of interest, and if the intracellular distribution in the test-stage mimics the non-dimerized distribution the compound disrupts the interaction between the two proteins of interest.

A method for identifying novel interaction partners for a bait protein comprising the steps of:

• (a) providing a cell line where each cell contains two heterologous conjugates,
• the first heterologous conjugate comprising an anchor protein that can specifically bind to an internal structure within the cell conjugated to an interactor protein of class A
• the second heterologous conjugate comprising an interactor protein of class B conjugated to the bait protein
• (b) introducing into said cell line a cDNA library coding for prey proteins conjugated to a detectable group
• (c) detecting the intracellular distribution of the detectable group
• (d) inducing interaction of protein of type A with protein of type B through application of a dimerizer molecule
• (e) detecting the intracellular distribution of the detectable group
• the intracellular distribution of said detectable group mimicking the intracellular distribution of the anchor-protein only in the presence of said dimerizer molecule being indicative of binding between the bait and prey proteins
• (f) isolating prey conjugates that show indication of binding to the bait component.