Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
2001-04-05
2004-01-27
Redding, David A. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S007100, C435S007800, C435S287100
Reexamination Certificate
active
06682927
ABSTRACT:
FIELD OF THE INVENTION
The present invention concerns method and apparatus for detecting binding interactions in cells. The methods and apparatus are particularly suitable for the high through-put screening of cell arrays and combinatorial libraries.
BACKGROUND OF THE INVENTION
A significant fraction of known cellular signaling proteins have been shown to translocate to or dissociate from the plasma membrane as part of their activation cycle. In particular, the recruitment of cytosolic proteins by activated receptors and plasma membrane signaling proteins is a general principle in receptor-mediated signal transduction (Pawson, T. (1995)
Nature
373, 573-580; Ullrich, A. & Schlessinger, J. (1990)
Cell
61, 203-212; Pfister, et al. (1985)
Science
228, 891-893; Hunter, T. (1987)
Cell
50, 823-829; Pawson, T. & Scott, J. D. (1997)
Science
278, 2075-2080). Translocation is often transient with active signaling components dissociating from the plasma membrane and acting on cytosolic and nuclear targets. Recruitment processes are exemplified by the binding of cytosolic SH2-domain containing proteins to tyrosine phosphorylated plasma membrane receptors (Koch, et al. (1991)
Science
252, 668-674; Kypta, et al. (1990)
Cell
62, 481-492) and by the binding of cytosolic signaling enzymes to GTP bound small G-proteins at the plasma membrane (Moodie, et al. (1993)
Science
260, 1658-1661; Stokoe, et al.
Science
264, 1463-1467; Leevers, et al. (1994)
Nature
369, 411-414). In addition to direct recruitment by protein-protein binding interactions, protein-lipid binding interactions are also important for translocation (Nishizuka, Y. (1992)
Science
258, 607-614; Rameh, L. E. & Cantley, L. C. (1999)
J. Biol. Chem.
274, 8347-8350). Lipid recruitment is exemplified by the translocation of PH-domain containing proteins in response to receptor-mediated production of plasma membrane phosphatidylinositol lipids (Ferguson, et al. (1995)
Cell
83, 1037-1046), C1-domain containing proteins in response to plasma membrane diacylglycerol production, and C2-domain containing proteins in response to calcium mediated binding interactions with negatively charged lipids in the plasma membrane (Nishizuka, Y. (1992)
Science
258, 607-614; Newton, A. C. (1995)
Curr. Biol.
5, 973-976).
Why does translocation to the plasma membrane play such a ubiquitous role in signal transduction? First, most of the cellular interactions with the extracellular environment are mediated by receptors located in the plasma membrane. Activated receptors often serve as a scaffold for signaling proteins that have to be recruited for a particular signaling function. Second, plasma membrane translocation concentrates signaling proteins at the membrane and enhances the frequency of intermolecular collisions. Translocation then serves as an intermediate signaling step that enhances the effective on-rate for target binding or the Michaelis constant for enzyme action (Haugh, J. M. & Lauffenberger, D. A. (1997)
Biophys. J.
72, 2014-2031).
Over the last few years, confocal imaging measurements were used to monitor the plasma membrane translocation of signaling proteins over time (Sakai, et al. (1997)
J. Cell Biol.
139, 1465-1476; Venkateswarlu, et al. (1998)
Curr. Biol.
8, 463-466; Barak, et al. (1997)
J. Biol. Chem.
272, 27497-27500; Oancea, et al. (1997)
J. Cell Biol.
140, 485-498; Stauffer, T. & Meyer, T. (1997)
J. Cell Biol.
139, 1447-1454; Stauffer, et al. (1998)
Curr. Biol.
8, 343-346; Kontos, et al. (1998)
Mol. Cell Biol.
18: 4131-4140; Oancea, E. & Meyer, T. (1998)
Cell
95, 307-318; Parent, et al. (1998)
Cell
95, 81-91; Meili, et al. (1999)
EMBO J.
18, 2092-2105; Watton, S. J. & Downward, J. (1999)
Curr. Biol.
9, 433-436). Although successful for many proteins, this approach was limited to cell types where the confocal resolution was sufficient to separate the plasma membrane from the cytosol and where the translocation involved a significant fraction of the cytosolic protein. Nevertheless, these imaging studies showed that single cell time-course measurements of translocation events can give important insights into the activation mechanism of enzymes (Oancea, E. & Meyer, T. (1998)
Cell
95, 307-318), into spatial gradients of second messengers (Parent, et al. (1998)
Cell
95, 81-91; Meili, et al. (1999)
EMBO J.
18, 2092-2105; Watton, S. J. & Downward, J. (1999)
Curr. Biol.
9, 433-436) and into the single cell kinetics of specific signaling steps (Stauffer, T. & Meyer, T. (1997)
J. Cell Biol.
139, 1447-1454; Oancea, E. & Meyer, T. (1998)
Cell
95, 307-318).
Biomolecular or combinatorial arrays have provided a means for the high throughput screening of chemical libraries. See, e.g., U.S. Pat. No. 5,143,854. A variety of specific techniques for carrying out the automated screening of such arrays have been developed, including the evanescent scanning of a pixel array. See U.S. Pat. No. 5,633,724.
A disadvantage of combinatorial arrays is that they provide an in vitro rather than an in vivo assay. In vitro binding assays can seldom provide an accurate measure of how binding will actually occur in vivo, particularly for intracellular binding events, because the complexity of the intracellular environment is difficult to replicate outside of the cell. Of course, the ultimate application of many screening assays is to develop in vivo applications for the compounds being screened. Accordingly, there is a continued need for new in vivo screening techniques that can be readily adapted to automated or high throughput screening.
SUMMARY OF THE INVENTION
A first aspect of the present invention is an apparatus for screening for translocation of a first protein of interest in vivo in a cell. The apparatus comprises:
(a) a total internal reflection member having a surface portion. If desired, the surface portion can be divided into separate and discrete segments.
(b) A cell contacted to the surface portion by the plasma membrane of the cell, the protein having a fluorescent group conjugated thereto. If desired, different cells can be contacted to different ones of the separate and discrete segments.
(c) A light source operatively associated with the total internal reflection member and positioned for directing a source light into the member to produce an evanescent field adjacent the surface portion, with the evanescent field extending into a first portion of the cell adjacent the plasma membrane, with the evanescent field being weaker in a second portion of the cell, the fluorescent group emitting light when in the first portion of the cell and emitting less light when in the second portion of the cell (i.e., less light as compared to the amount emitted when the same fluorescent group is in the first portion of the cell).
(d) A light detector operatively associated with the total internal reflection member and configured to detect emitted light from the cell
The emission of more or less light from the cell indicates the translocation of the first protein between the first and second portions of the cell.
The cell or cells may further contain a second protein of interest located in either the first portion of the cell or the second portion of the cell, whereby the emission of more or less light from the cell indicates the presence or absence of specific binding between the first and second proteins of interest. When the second protein is located in the first portion of the cell, the emission of more light indicates the specific binding of the proteins of interest, and the emission of less light indicates the lack of such binding. When the second protein is located in the second portion of the cell, the emission of less light indicates the specific binding of the proteins of interest, and the emission of more light indicates the lack of such binding. First and second proteins of interest may be members of a specific binding pair. Either or both of the first and second proteins of interest may be expressed by a nucleic acid carried by the cell; either of the first and second proteins of interest
Meyer Tobias
Teruel Mary N.
Duke University
Myers Bigel & Sibley Sajovec, PA
Redding David A.
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