METHODS FOR THE DETECTION OF MOLECULAR INTERACTIONS WITHIN CELLS

• US 20090131270A1
• Filed: 08/02/2005
• Published: 05/21/2009
• Est. Priority Date: 08/02/2004
• Status: Abandoned Application

• Current Assignee: Cellumen, Inc.
• Original Assignee: Cellumen, Inc.
• Inventors: Giuliano, Kenneth A., Taylor, D. Lansing
• Application Number:
  US11/573,121
• Publication Number:
  US 20090131270A1
• Time in Patent Office: Days
• US Class Current: 506/10
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures

Abstract

The invention provides a method of detecting the effect of an agent of interest on the interaction between two or more polypeptides introduced into a cell. The invention also provides a method for quantifying the interaction between at least two molecules of interest, which are introduced into a cell. A method for quantifying the effects of an agent of interest or the interaction between two molecules of interest on cellular constituents or functions in the same cells is also provided. The above inventive methods can be automatically quantified by a device, such as a HCS device, and utilized in the construction of a database.

Claims

1. A method for detecting the effect of an agent of interest on the interaction between two or more polypeptides, the method comprising:

a. introducing two or more polypeptides (e.g., a first polypeptide and a second polypeptide), into a cell under conditions wherein said polypeptides interact with each other, endogenous proteins, or a combination thereof, and wherein at least said first polypeptide is a biosensor comprising an interaction domain that interacts with one or more polypeptides, endogenous proteins, or a combination thereof and further comprising a reporter domain, and quantifying the interaction between one or more of said polypeptides, one or more of said endogenous proteins, or combinations thereof,

b. contacting said cell with an agent of interest and quantifying the interaction between said polypeptides, endogenous proteins, or a combination thereof, and

c. comparing the result of step (a) to that of step (b).

2. The method of claim 1, wherein said second polypeptide also is a biosensor comprising an interaction domain that interacts with polypeptides, endogenous proteins, or a combination thereof and further comprising a reporter domain.

3. The method of claim 1, wherein a reporter domain comprises a luminescent or fluorescent moiety.

4. The method of claim 2, wherein a reporter domain comprises a luminescent or fluorescent moiety.

5. The method of claim 3 where the luminescent or fluorescent moiety is GFP.

6. The method of claim 3, wherein the interaction between said polypeptides is evaluated by assessing a fluorescence of luminescence signal change.

7. The method of claim 4, wherein the interaction between said polypeptides is evaluated by assessing a fluorescence or luminescence signal change.

12. The method of claim 1, wherein said agent is a chemical, physical stimulus, environmental stimulus, electrical stimulus, or radiation.

18. The method of claim 6, wherein the interaction is quantified using fluorescence resonance energy transfer, fluorescence anisotropy, rotational difference, fluorescence lifetime change, fluorescence solvent sensitivity, and fluorescence quenching.

19. The method of claim 1, where the interaction is automatically quantified by a device.

20. The method of claim 19, wherein the device provides an array of locations which contain multiple cells, scans multiple cells in each of the locations containing cells to obtain luminescent or fluorescent signals from at least one luminescent or fluorescent reporter polypeptide within the cells;

measuring a luminescence or fluorescence intensity of the luminescent or fluorescent signals from at least one luminescent or fluorescent reporter polypeptide within a specific location in the cells; and

automatically calculating changes induced by the polypeptide of interest in one or more of the following;

a. a ratio of luminescent or fluorescent signal intensity from at least one luminescent or fluorescent reporter polypeptide in a specific location in the cells to luminescent or fluorescent signal intensity from at least one luminescent or fluorescent reporter polypeptide in a different specific location in the cells; and

b. a difference between luminescent or fluorescent signal intensity from the at least one luminescent or fluorescent reporter polypeptide in a specific location in the cells and luminescent or fluorescent signal intensity from at least one luminescent or fluorescent reporter polypeptide in a different specific location in the cells wherein the changes induced by the agent of interest indicate an effect of the agent of interest on the localization of the polypeptide from a first location in the cells to a second specific location in the cells.

26. The method of claim 1, wherein one or more of said polypeptides comprises a localization domain which causes said polypeptide to be expressed with a specific localization pattern in the cell, and the agent of interest disrupts said localization pattern.

27. The method of claim 26, where one or more of said polypeptides contains more than one localization domain.

28. The method of claim 26, where said localization domain is selected from the group consisting of a NES and NLS.

29. The method of claim 26, wherein the localization domain comprises or consists essentially of an amino acid sequence selected from the group consisting of

30. The method of claim 1, wherein at least one of said polypeptides comprises a protein, protein fragment, or protein interaction domain.

31. The method of claim 1, wherein at least one of said polypeptides comprises a protein and is expressed within in the cell under the transcriptional control of an inducible promoter.

32. The method of claim 1, wherein at least one of said polypeptides is produced outside of the cell and then introduced into the cell.

33. The method of claim 1, wherein at least one of said polypeptides is exogenous to said cell.

34. The method of claim 1, wherein at least one of said polypeptides is endogenous to said cell.

35. The method of claim 1, wherein at least two of said polypeptides are p53 and HDM2.

36. The method of claim 1, wherein at least one of said polypeptides has the sequence SEQ ID NO:

7.

37. The method of claim 1, wherein at least one of said polypeptides has the sequence SEQ ID NO:

8.

38. The method of claim 1, wherein at least one of said polypeptides has the sequence SEQ ID NO:

9.

39. The method of claim 1, wherein at least one of said polypeptides has the sequence SEQ ID NO:

10.

40. The method of claim 1, wherein at least one of said polypeptides has the sequence SEQ ID NO:

11.

41. The method of claim 1, wherein at least one of said polypeptides has the sequence SEQ ID NO:

12.

42. A method of High Content Screening (HCS) comprising the method of claim 1, wherein the measurements are taken using HCS techniques.

43. The method of claim 1, wherein said method is repeated using different molecules and/or agents of interest, whereby the results of said repeated assays are compiled into a database of quantified comparisons.

44. A database created using the method of claim 43.

45. The database of claim 44, comprising information on the interactions of molecules of interest with other molecules or agents and relating said interactions to additional cellular constituents and functions in the cells.

46. The database of claim 44, which is constructed by screening a library for molecules or agents that have an effect on the interaction between two or more molecules of interest.

47. The database of claim 44, which is constructed by screening a library for molecules or agents that disrupt the interaction between two or more cellular molecules.

8. A method for detecting the effect of an agent of interest on the interaction between two or more polypeptides, the method comprising:

a. introducing two or more polypeptides (e.g., a first polypeptide and a second polypeptide), into a cell under conditions wherein said polypeptides interact with each other, endogenous proteins, or a combination thereof, and wherein at least said first polypeptide is a biosensor comprising a reporter comprising a luminescent or fluorescent moiety, and quantifying the interaction between one or more of said polypeptides, one or more of said endogenous proteins, or combinations thereof by assessing a reversible fluorescence or luminescence signal change,

b. contacting said cell with an agent of interest and quantifying the interaction between one or more of said polypeptides, one or more of said endogenous proteins, or combinations thereof by assessing a fluorescence or luminescence signal change, and

c. comparing the result of step (a) to that of step (b).

9. The method of claim 8, wherein said second polypeptide also is a biosensor comprising a reporter comprising a luminescent or fluorescent moiety.

10. The method of claim 9 where the luminescent or fluorescent moiety is GFP.

11. The method of claim 8, wherein said first and/or said second polypeptide further comprises an interaction domain that interacts with one or more of said polypeptides, one or more of said endogenous proteins, or combinations thereof.

13. The method of claim 8, wherein said agent is a chemical, physical stimulus, environmental stimulus, electrical stimulus, or radiation.

14. A method for quantifying the interaction between at least two molecules of interest, the method comprising:

a. introducing each molecule of interest individually into a cell, wherein at least one of said molecules of interest comprises a reporter comprising a luminescent or fluorescent moiety, and quantifying the level of luminescence or fluorescence,

b. introducing said molecules of interest into a cell concurrently and quantifying the level of luminescence or fluorescence, and

c. comparing the result of step (a) to that of step (b).

15. The method of claim 14, where one of the molecules of interest is SH2.

16. The method of claim 14, wherein a molecule of interest is a polypeptide.

17. The method of claim 16, wherein the polypeptide further comprises an interaction domain that interacts with one or more of said polypeptides, one or more of said endogenous proteins, or combinations thereof.

21. A method for detecting the effect of an agent of interest on cellular constituents or functions in the same cells, the method comprising:

a. introducing two or more molecules that interact into a cell and quantifying the cellular constituent or function of interest,

b. contacting said cell with an agent of interest and quantifying the cellular constituent or function of interest, and

c. comparing the result of step (a) to that of step (b).

22. The method of claim 21, wherein a molecule is a polypeptide.

25. The method of claim 21, wherein the cellular constituents or functions of interest are selected from the group consisting of apoptosis;

necrosis;

cell cycle regulation;

nuclear morphology;

cellular DNA content;

histone H3 phosphorylation levels;

other kinase or phosphatase activities;

transcription factor activation;

tumor suppressor activation or induction;

organellar functions including mitochondrial potential, peroxisome number and size, or endosomal pH;

organization of the actin, microtubule, or intermediate filament cytoskeleton;

receptor internalization or translocation;

cell motility;

protease activation;

the heat shock response;

exocytosis;

endocytosis;

cellular hypertrophy or other shape changes; and

gene expression including coding and non-coding RNAs as well as proteins.

23. A method for quantifying the effect of the interaction between two molecules of interest on cellular constituents or functions in the same cells, the method comprising:

a. introducing each molecule of interest individually into a cell and quantifying the cellular constituent or function of interest,

b. introducing said molecules of interest into said cell concurrently and quantifying the cellular constituent or function of interest, and

c. comparing the result of step (a) to that of step (b).

24. The method of claim 23, wherein a molecule is a polypeptide.

48. A biosensor comprising a polypeptide comprising a sequence of amino acids comprising SEQ ID NO:

8, 10, or 12.

49. A nucleic acid encoding the biosensor of claim 48.

50. The nucleic acid of claim 49, which comprises a DNA sequence comprising SEQ ID NO:

7, 9, or 11.