System and method for identifying proteins involved in force-initiated signal transduction

• US 6,982,150 B2
• Filed: 08/10/2004
• Issued: 01/03/2006
• Est. Priority Date: 02/11/2002
• Status: Expired due to Fees

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: Sheetz, Michael P., Sawada, Yasuhiro
• Primary Examiner(s): REDDING, DAVID A
• Application Number:
  US10/914,924
• Publication Number:
  US 20050084843A1
• Time in Patent Office: 511 Days
• Field of Search: 435/7.2, 435/7.21, 435/7.23, 435/7.32, 435/287.1, 435/288.3
• US Class Current: 435/7.2
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5011 for testing antineoplastic activity
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5076 involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• CPC Class Codes: G01N 33/567 utilising isolate of tissue or organ as binding agent
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins

Abstract

The present invention relates to a cytoskeletal system composed of purified components which allow for the identification of key proteins involved in mechanotransduction. The system comprises a cytoskeleton network deficient in force-dependent proteins, exogenous proteins in contact with the cytoskeleton network, a means for applying a force to the cytoskeleton network, and a means for identifying any of the exogenous proteins that bind to the cytoskeleton network when a force is applied to the cytoskeleton network, wherein proteins identified are involved in mechnotransduction. The invention further relates to a method for identifying proteins involved in mechanotransduction using the cytoskeletal system of the present invention. The identification of key players involved in mechanotransduction allows for the identification of agents capable of inhibiting or enhancing mechanotransduction.

Claims

1. A system for the identification of proteins involved in mechanotransduction comprising:

a cytoskeleton network deficient in force-dependent proteins,

exogenous proteins in contact with the cytoskeleton network,

means for applying a force to the cytoskeleton network; and

means for identifying any of the exogenous proteins that bind to the cytoskeleton network when a force is applied to the cytoskeleton network,

wherein exogenous proteins identified as being bound to the cytoskeleton network with the force applied thereto are identified as proteins involved in mechanotransduction.

2. The system of claim 1, wherein the cytoskeleton network is attached to a deformable substrate having a surface area, and the force is applied to the cytoskeleton network by deforming the substrate so as to apply the force to the cytoskeleton network.

3. The system of claim 2, wherein the deformable substrate comprises an elastomeric material.

4. The system of claim 2, wherein the cytoskeleton network is deformed by the force applied to thereto.

5. The system of claim 1, wherein the cytoskeleton network is prepared from cultured cells.

6. The system of claim 5, wherein the cells are treated with a component to obtain the cytoskeleton network.

7. The system of claim 6, wherein the component is selected from the group consisting of alcohols, enzymes and detergents.

8. The system of claim 7, wherein the detergent is a non-ionic detergent.

9. The system of claim 8, wherein the detergent is saponin.

10. The system of claim 5, wherein the cells are selected from the group consisting of primary cells and established cell lines.

11. The system of claim 10, wherein the established cell line is selected from the group consisting of L-929 cells, HEK 293 cells, tumor cells, muscle cells, hepatocytes, cardiac smooth muscle cells, hair cells, neuronal cells, osteoblasts, osteocytes, chondrocytes, keratinocytes and endothelial cells.

12. The system of claim 11, wherein the cell line is L-929 cells.

13. The system of claim 2, wherein the deformable substrate is the bottom of a tissue culture dish.

14. The system of claim 2, wherein the deformable substrate is deformed by the application of physical force thereto.

15. The system of claim 14, further comprising a stretch frame on which the substrate is mounted, wherein the substrate is deformed by expanding the stretch frame.

16. The system of claim 2, wherein the substrate is deformed by stretching the substrate to expand the surface area thereof.

17. The system of claim 16, wherein the surface area is expanded from about 5% to about 50% of the surface area of the substrate prior to stretching.

18. The system of claim 17, wherein the surface area is expanded by about 10% of the surface area of the substrate prior to stretching.

19. The system of claim 2, wherein the substrate is deformed by contraction so as to reduce the surface area of the substrate.

20. The system of claim 19, wherein the surface area is reduced from about 5% to about 35% of the surface area of the substrate prior to contraction thereof.

21. The system of claim 20, wherein the surface area is reduced by about 10% of the original surface area of the substrate prior to contraction thereof.

22. The system of claim 1, wherein the exogenous proteins are prepared from cells.

23. The system of claim 22, wherein the cells are selected from the group consisting of L929 cells, HEK 293 cells, tumor cells, muscle cells, hepatocytes, cardiac smooth muscle cells, hair cells, neuronal cells, osteoblasts, osteocytes, chondrocytes, keratinocytes and endothelial cells.

24. The system of claim 1, wherein the exogenous proteins are radiolabeled.

25. The system of claim 1, wherein the exogenous proteins are biotinylated.

26. A method of identifying proteins involved in mechanotransduction comprising:

providing a cytoskeleton network deficient in force-dependent proteins;

contacting the cytoskeleton network with exogenous proteins,

applying a force to the cytoskeleton network, and

identifying the exogenous proteins that bind to the cytoskeleton network when the force is applied thereto, wherein the exogenous proteins that bind to the cytoskeleton network are identified as proteins involved in mechanotransduction.

27. The method of claim 26, wherein the cytoskeleton network is prepared by

growing cells in culture on a deformable substrate having a surface area,

treating the cells with a component to obtain a cytoskeleton network deficient in force-dependent proteins, and

deforming the substrate to apply a force to the cytoskeleton network.

28. The method of claim 27, wherein the deformable substrate comprises an elastomeric material.

29. The method of claim 27, wherein the deformable substrate deforms when a force is applied thereto.

30. The method of claim 27, wherein the cytoskeleton network remains attached to the deformable substrate.

31. The method of claim 27, wherein the deformable substrate is a part of a tissue culture dish.

32. The method of claim 27, wherein the cells are mammalian cells.

33. The method of claim 32, wherein the cells are selected from the group consisting of primary cells and established cell lines.

34. The method of claim 33, wherein the established cell lines are selected from the group consisting of tumor cells, muscle cells, hepatocytes, cardiac smooth muscle cells, hair cells, neuronal cells, osteoblasts, osteocytes, chondrocytes, keratinocytes and endothelial cells.

35. The method of claim 27, wherein the component is selected from the group consisting of alcohols, enzymes and detergents.

36. The method of claim 27, wherein the detergent is a non-ionic detergent.

37. The method of claim 36, wherein the detergent is saponin.

38. The method of claim 27, wherein the substrate is deformed by mechanically expanding a stretch frame that is attached to the substrate.

39. The method of claim 27, further comprising stretching the substrate to expand the surface area of the substrate.

40. The method of claim 39, wherein the surface area is expanded from about 5% to about 50% of the surface area of the substrate prior to the stretching thereof.

41. The method of claim 40, wherein the surface area is expanded about 10% of the surface area of the substrate prior to the stretching thereof.

42. The method of claim 27, further comprising contracting the substrate to reduce the surface area thereof.

43. The method of claim 42, wherein the surface area of the substrate is reduced from about 5% to about 35% of the surface area of the substrate prior to the contraction thereof.

44. The method of claim 43, wherein the surface area is reduced about 10% of the surface area of the substrate prior to the contraction thereof.

45. The method of claim 26, wherein the exogenous proteins are prepared from cells.

46. The method of claim 45, wherein the cells are selected from the group consisting of L929 cells, HEK 293 cells, tumor cells, muscle cells, hepatocytes, cardiac smooth muscle cells, hair cells, neuronal cells, osteoblasts, osteocytes, chondrocytes, keratinocytes and endothelial cells.

47. The method of claim 26, further comprising radiolabeling the exogenous proteins.

48. The method of claim 26, further comprising biotinylating the exogenous proteins.

49. A method of identifying an agent that inhibits or enhances mechanotransduction comprising:

a) providing a cytoskeleton network deficient in force-dependent proteins;

b) contacting the cytoskeleton network with exogenous proteins in the absence of the agent;

c) applying a force to the cytoskeleton network;

d) measuring an extent of binding of each of the exogenous proteins to the cytoskeleton network having the force applied thereto in the absence of the agent; and

e) contacting the cytoskeleton network with the exogenous proteins in the presence of the agent;

f) measuring the extent of binding of each of the exogenous proteins to the cytoskeleton network having the force applied thereto in the presence of the agent; and

g) determining from the measurements in steps d) and f) whether the presence of the agent increases or decreases the extent to which each of the exogenous proteins bind to the cytoskeleton network so as to determine whether the agent inhibits or enhances mechanotransduction.

50. The method of claim 49, wherein the cytoskeleton network is prepared by

growing cells in culture on a deformable substrate,

treating the cells with a component to obtain a cytoskeleton network deficient in force-dependent proteins, and

deforming the substrate so as to apply a force to the cytoskeleton network.

51. The method of claim 50, wherein the substrate deforms in shape by applying a force thereto.

52. The method of claim 50, wherein the cytoskeleton network remains attached to the substrate.

53. A method for identifying an agent capable of inhibiting or enhancing mechanotransduction comprising:

a) providing a cytoskeleton network deficient in force-dependent proteins;

b) contacting the cytoskeleton network with an identified force-dependent protein in the absence of an agent;

c) applying a force to the cytoskeleton network;

d) measuring an extent of binding of the identified force-dependent protein to the cytoskeleton network having the force applied thereto in the absence of the agent;

e) contacting the cytoskeleton network with the identified force-dependent protein in the presence of the agent;

f) measuring an extent of binding the identified force-dependent protein to the cytoskeleton network having the force applied thereto in the presence of the agent; and

g) determining from the measurements in steps d) and f) whether the presence of the agent increases or decreases the extent the identified force-dependent protein binds to the cytoskeleton network having the force applied thereto so as to determine whether the agent inhibits or enhances mechanotransduction.