Biological membranes are heterogeneous structures composed of membrane domains with discrete physical properties. Glycolipid-enriched membrane (GEM) domains, or lipid rafts, are one type of membrane domain that is crucial for efficient T cell signaling. Studies of GEM domains in T cells are therefore important for understanding the mechanisms underlying development of the T cell repertoire through T cell signaling, such as positive and negative selection, T cell anergy, and clonal expansion. Studies of GEM domains may also provide important insight into immunological diseases related to these events, such as allergy, autoimmunity, and graft rejection. Our research interests are determining the physical and biological properties of GEM domains in T cell signaling, and our recent studies have used fluorescence imaging to measure GEM domains in situ. Our experiments showed GEM domains are constitutively assembled into micron-size membrane patches through association with the actin cytoskeleton. Furthermore, we found that the GEM enriched patches are mobile and diffuse to the site of T cell signaling. Based on our findings, we propose the model that immune synapses form by an actin-dependent assembly of GEM domains at the site of T cell activation, and that GEM domains serve as a vehicle for targeting signaling molecules to the immune synapse and the T cell receptor (TcR). We will address this model with the following aims: 1) Measure the exchange of GEM-associated Lck with remaining cellular pools of protein using fluorescence photo bleaching techniques and determine the role of protein trafficking in T cell activation. For example, GEM-associated Lck is inactive relative to the Lck in the remaining fractions of the cells.
This aim will therefore show if active Lck from the non-GEM pool of the cell is targeted to GEM domains and immune synapses for T cell signaling; 2) Measure the dynamics of GEM domains during T cell signaling.
This aim will determine when domain clustering occurs relative to the onset of signals from the TcR. We will also determine if domain clustering coincides with mixing of GEM microdomains; 3) Identify the signals that initiate domain clustering by measuring the effects of over expression of dominant negative constructs of CD2 and Fyn; 4) Determine the effect of TcR antagonists on GEM domains and GEM-associated signaling proteins.
This aim will show if the altered T cell signaling that occurs with altered peptide ligands is the outcome of inefficient clustering of GEM domains and inhibition of GEM-associated Lck. This research will provide better understanding of an important class of membrane domains, and how their properties relate to human health and disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM070001-05
Application #
7253345
Study Section
Experimental Immunology Study Section (EI)
Program Officer
Marino, Pamela
Project Start
2003-07-01
Project End
2010-06-30
Budget Start
2007-07-01
Budget End
2010-06-30
Support Year
5
Fiscal Year
2007
Total Cost
$255,535
Indirect Cost
Name
Oklahoma Medical Research Foundation
Department
Type
DUNS #
077333797
City
Oklahoma City
State
OK
Country
United States
Zip Code
73104
Byrum, Jennifer N; Van Komen, Jeffrey S; Rodgers, William (2013) CD28 sensitizes TCR Ca²? signaling during Ag-independent polarization of plasma membrane rafts. J Immunol 191:3073-81
Chichili, Gurunadh R; Westmuckett, Andrew D; Rodgers, William (2010) T cell signal regulation by the actin cytoskeleton. J Biol Chem 285:14737-46
Chichili, Gurunadh R; Rodgers, William (2009) Cytoskeleton-membrane interactions in membrane raft structure. Cell Mol Life Sci 66:2319-28
Johnson, Corey M; Chichili, Gurunadh R; Rodgers, William (2008) Compartmentalization of phosphatidylinositol 4,5-bisphosphate signaling evidenced using targeted phosphatases. J Biol Chem 283:29920-8
Koenig, A; Russell, J Q; Rodgers, W A et al. (2008) Spatial differences in active caspase-8 defines its role in T-cell activation versus cell death. Cell Death Differ 15:1701-11
Van Komen, Jeffrey S; Mishra, Sudha; Byrum, Jennifer et al. (2007) Early and dynamic polarization of T cell membrane rafts and constituents prior to TCR stop signals. J Immunol 179:6845-55
Chichili, Gurunadh R; Rodgers, William (2007) Clustering of membrane raft proteins by the actin cytoskeleton. J Biol Chem 282:36682-91
Byrum, Jennifer; Rodgers, William (2005) Visualization of transfer of a fluorescently-labeled membrane raft protein to T cells using lentivirus. Gene Ther Mol Biol 9B:135-142
Rodgers, William; Farris, Darise; Mishra, Sudha (2005) Merging complexes: properties of membrane raft assembly during lymphocyte signaling. Trends Immunol 26:97-103
Rodgers, William; Smith, Kenneth (2005) Properties of glycolipid-enriched membrane rafts in antigen presentation. Crit Rev Immunol 25:19-30

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