Visualization of Protein Interactions in Living Primary Lymphocytes

Tom Kerppola, Ph.D.

University of Michigan Medical Center

Funded in June, 2005: $300000 for 4 years
LAY SUMMARY . ABSTRACT . HYPOTHESIS . SELECTED PUBLICATIONS .

LAY SUMMARY

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Using Imaging to See How Immune Cell Actions Are Affected by Therapeutic Agents

The researchers will use new cellular imaging methods to study how therapeutic agents that either stimulate or suppress immune cells are affected by proteins that regulate immune system responses.

Proteins, which are expressed by genes to carry out specific functions such as immunity, are influenced by one another and also can modify the properties of other groups of proteins.  Both the interactions and modifications of proteins control immune system functioning.  For instance, protein interactions and modifications are essential for enabling immune cells to attack invaders, while not also inadvertently attacking the body’s own cells and creating “autoimmune” diseases.  The investigators have developed new imaging methods for directly visualizing protein interactions and modifications in a living immune lymphocyte, which ordinarily learns to recognize an invader and attack it.  They will use biomolecular fluorescence complementation analysis to image the location within the lymphocyte where a particular interaction or modification occurs.  If successful, this method could be used to identify normal and abnormal protein interactions in immune cells. 

Significance:  This new cellular imaging technique could provide an important tool for identifying normal and abnormal protein interactions that occur within immune cells.  Such identification eventually could lead to methods for strengthening interactions needed to fight disease, and for blocking those that otherwise could produce autoimmune diseases. 

 

ABSTRACT

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Visualization of Protein Interactions in Living Primary Lymphocytes

NFAT family proteins and other transcription factors function in both T cell activation and peripheral tolerance. We hypothesize that changes in transcription factor interactions and ubiquitination control the balance between T cell activation and anergy in response to an immune challenge. We propose to visualize interactions and ubiquitination of NFAT and Fos/Jun family proteins in immortalized and primary T cells. We will use bimolecular fluorescence complementation (BiFC) analysis to image protein interactions in living cells. This assay enables direct visualization of protein complexes in the normal cellular environment. We will use ubiquitin-mediated fluorescence complementation (UbFC) analysis to image proteins modified by ubiquitin-family peptides in living cells. This assay enables selective visualization of peptide conjugates without interference by an excess of unmodified proteins.

The results of these experiments will elucidate how the same transcription factors mediate both T cells activation and anergy. These studies will also identify molecular targets and provide experimental approaches for the development of therapeutic agents for immune disorders that are caused by altered protein interactions or modifications in T cells.

HYPOTHESIS

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Hypothesis:
Direct imaging of protein interactions and modifications in living primary lymphocytes will provide unique insight into the mechanisms that modulate T cell activation.

Goals:
The research will focus on the visualization of transcription factor interactions and ubiquitin family peptide modifications in cultured cell lines and primary lymphocytes.  The primary objective of the research is to identify the subcellular localization of protein complexes and ubiqutin family peptide conjugates in living cells.

Methods:
The studies will take advantage of the bimolecular fluorescence complementation (BiFC) and ubiquitin mediated complementation (UbFC) assays developed in the laboratory.  These methods enable direct visualization of protein interactions and modifications in living cells.

SELECTED PUBLICATIONS

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Kerppola T.K.  Bimolecular fluorescence complementation: visualization of molecular interactions in living cells.   Methods Cell Biol. 2008;85:431-70.

Kerppola T.K.  Visualization of molecular interactions by fluorescence complementation.  Nat Rev Mol Cell Biol. 2006 Jun;7(6):449-56.

Hu C.D., Grinberg A.V., and Kerppola T.K.  Visualization of protein interactions in living cells using bimolecular fluorescence complementation (BiFC) analysis.  Curr Protoc Cell Biol. 2006 Jan;Chapter 21:Unit 21.3.

Kerppola T.K.  Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.  Nat Protoc. 2006;1(3):1278-86.

Fang D. and Kerppola T.K.  Ubiquitin-mediated fluorescence complementation reveals that Jun ubiquitinated by Itch/AIP4 is localized to endo-lysosomes.  Proc Natl Acad Sci U S A. 2004 Oct 12;101(41):14782-7.  [Abstract] [Full text[PDF] 

Rajaram N. and Kerppola T.K.  Synergistic transcription activation by Maf and Sox, and their subnuclear localization are disrupted by a mutation in Maf that causes cataract. Mol Cell Biol. 2004 Jul;24(13):5694-709.  [Abstract] [Full text] [PDF]

Grinberg A.V., Hu C.D., and Kerppola T.K.  Visualization of Myc/Max/Mad family dimers and the competition for dimerization in living cells, Mol Cell Biol. 2004 May;24(10):4294-308.  [Abstract] [Full text] [PDF] 

Hu C.D. and Kerppola T.K.  Simultaneous visualization of interactions between multiple proteins in living cells using multicolor fluorescence complementation analysis. Nat Biotechnol. 2003 May;21(5):539-45. [Abstract[Full text] [PDF] 

Hu C.D., Chinenov Y., and Kerppola T.K.  Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation.  Mol Cell. 2002 Apr;9(4):789-98. [Abstract] [Full text]