Research Interests

Characterization of the detergent insolubility of the T cell Receptor complex and its interaction with detergent-resistant membranes

Background

A cell gains information from its environment by binding molecules called ligands. These bind to specific cell surface receptors which then transmit these signals to the interior of the cell. My research involves the receptor for foreign antigens on the surface of T cells. T cells are an important part of the immune system and aid in fighting disease. The T cell receptor (TCR) complex can be studied using easily grown T cell lines and antibodies that bind to the receptor, mimicking antigen binding. The TCR is a large multisubunit complex. Aggregating the TCR complexes on the cell surface is necessary to activate the cells. My experiments with students over the past few of years have shown that aggregation induces the receptors to associate detergent-resitant membranes (DRMs). These areas of the cell membrane, which are relatively resistant to solubilization by mild detergents, have been charachterized by scientists in several cell types and have been hypothesized to act as "rafts" to allow important molecules to come together during cellular signaling. DRMs are enriched in glycolipids, cholesterol, glycosylphosphatidyl inositol-linked proteins, and tyrosine kinases. These later molecules are especially interesting as they are essential to signaling by the TCR.

Possible research projects for students involve characterizing this association and addressing its importance in activation of the T cell response. We have had some success separating, purifying, and analyzing the aggregated TCR complexes and membrane domains using density gradient centrifugation. These DRMs are found in the low in density region of the gradient due to the high lipid to protein ratio. The TCR containing complexes are very large and can also be separated by gel filtration chromatography.

• Characterization of the different fractions of containing TCRs, trying to identify other components which copurify with the aggregated receptors and DRMs. We will focus on tyrosine kinases, and those proteins known to be associated with DRMs to see whether these domains are similar to those characterized in other cell types. Antibodies to the various proteins will be used to identify them after separation by gel electrophoresis. A another aspect of this project can be to compare the TCR complexes separated by centrifugation and those separated by chromatography.

• Most experiments thus far have involved using radioactively labeled antibodies to label the TCR. One project would be to develop nonradioactive labels that can be used to quantitate the amount of receptor. This project would use with fluorescently labeled antibodies, quantitating the amount of bound antibody using spectrofluorimetry. Preliminary results show this to be promising.

• Involvement of the cytoskeleton in the aggregated TCR complexes. A subset of the aggregated receptors are found in the pellet when separated by centrifugation, indicating a very high density which could be due to association with cytoskeletal elements. Microscopy experiments on other laboratories have shown that actin microfilaments can associate with DRMs. This project will involve fluorescent microscopy to test whether the TCR associates with microfilaments and/or known components of membrane domains after aggregation. Some cell biology background would be useful for this project.

Selected Publications

• Prinetti, A., V. Chigorno., S. Prioni, N. Loberto, N. Marano, G. Tettamanti, and S. Sonnino (2001) "Changes in the lipid turnover, composition and organization, as sphingolipid-enriched membrane domains, in rat cerebellar granule cells developing in vitro." J. Biol. Chemistry 276, 21136-21145.
  
  

• Prinetti, A., N. Marano, S. Prioni, V. Chigorno, L. Mauri, R. Casellato, G. Tettamanti, and S. Sonnino, (2000) Association of Src-family protein tyrosine kinases with sphingolipids in rat cerebellar granule cells differentiated in culture. Glycoconjugate Journal 17, 223-32.

• Marano, N., M. Crawford, and B. Govindan (1997) "Characterization of the detergent insolubility of the T cell receptor", Molecular Immunology 34, 967-976.

• Marano, N., M. Liotta, J. Slattery, D. Holowka and B. Baird (1993). "FceRI and the T cell receptor for antigen activate similar signaling pathways in T cell-RBL cell hybrids," Cellular Signalling 5, 155-167.

• Marano, N., D. Holowka and B. Baird (1989) "Bivalent binding of an anti-CD3 antibody to Jurkat cells induces association of the T cell receptor complex with the cytoskeleton," J. Immunol. 143, 931.

• Marano, N., B. Dietzschold, J. J. Earley, G. Schatteman, S. Thompson, P. Grob, A.H. Ross, M. Bothwell, B.F. Atkinson, and H. Koprowski (1987) "Purification and amino terminal sequencing of the human melanoma nerve growth factor receptor," J. Neurochemistry 48, 225.

• Ross, A.H., P. Grob, M. Bothwell, D.E. Elder C.S. Ernst, N. Marano, B.F.D. Ghrist, C.C. Slemp, M. Herlyn, B. Atkinson and H. Koprowski (1984) "Characterization of nerve growth factor receptor in neural crest tumors using monoclonal antibodies," Proc. Natl. Acad. Sci. 81, 6681.

Recent Student Research Projects

• "Insolubility of Lipid Rafts by Aggregation of T-cell Receptors in Human Jurkat Cells". Jane Mutoru, Summer 2003, Summer 2004, 2005-6 Honors Project (Senior Year Experience). Jane also worked on a project related to sphingosine and calcium influx, and presented a poster at the SLU Festival of Science. Jane was a Heuer Fellow and presented a poster at the 2006 ACS National Meeting in Atlanta. See note (5) below.
  
  
• Analysis of T Cell Lipids. Joel Blanchard, 2003-4 Senior Project (biology major).
  
  
• T Cell Lipids, Junior research project, Chris Hungerford, Spring 2003. (biology major)
  
  
• T Cell Lipid Analysis, Jacqueline Wanja Nyoro, Summer 2002 (6) and 2003 (7).
  
  
• T cell lipid analysis. Alexa Siddon, Hamilton College, Summer 2002 (6)
  
  
• Microscopy of T-Cell GM1 related to lipid rafts. Maureen Zagursky, Summer 2002. See notes (5,7) below.
  
  
• Analysis of T Cell Lipids in Activated vs. Non-activated Cells. Timothy Errington, 2001-02 Senior Research Project (5).
  
  
• "Functional Effects of Exogenous Lipids in T Cells." Stacey Reed, Senior Research, 2000-2001.
  
  
• "T Cells and Detergent Resistant Membranes", Kristy Luong (Merck Scholar), '98-'99
  

• "Investigating the Interaction between T Cell Receptors and Detergent Resistant Membrane Domains - Utilizing Fluorescence Microscopy", David E. Clark (Merck Scholar), '98-'99

• "Association of T cell receptors with detergent-resistant membrane domains", Lawrence Kring, '97-'98, summer research and biology honors project (Tom Budd, biology advisor) ^(1,3,4,6 )

• "Characterization of Detergent Insoluble TCR Complex and Associated Proteins using a Tyrosine Kinase Assay and Sucrose Gradient Ultracentrifugation", Aye Moe Thu Ma, Summer '96, Spring '97 Summer Research project and Honors project. ^{(1, 2, 4)}

• "Protein Tyrosine Kinase Activity in Human Leukemia T Cells: the Effects of Cellular Growth Conditions on Basic and Stimulated Tyrosine Kinase Activity" Josh Ford, '95 - '96, Biology Honors project, (Tom Budd, biology advisor) ⁽⁴⁾

• "Separation of Soluble and Insoluble T Cell Receptor Complexes Using Column Chromatography", Chris Phelps, '95 - 96 ⁽⁵⁾

• "Tyrosine Kinase Activity Stimulated by the T Cell " and "Characterization of the T Cell Receptor: Analysis of Soluble and Insoluble T Cell Antigen receptors and Associated Proteins." Ndegwa Njuguna, Summer '95, Fall '96^{, Spring '97 (see notes 1,2,3}) .

• "Purification of T cell Receptor Complexes", Heather Cromie, Spring '95. Purifying antibodies to the T cell receptor on mouse cells, using these antibodies to test whether the T cell receptor associates with the cytoskeleton on cells which lack the CD4 protein.
• NOTES:

  1. Pew faculty/student summer research program.
  2. Results included in my presentations to the Meeting of the Northern Immunogical Mountain Society (a regional Immunology meeting) (1994, 1995, 1996). Heather Cromie and Aye Moe Thu Ma also each attended one of these meetings.
  3. Student gave a presentation at the Cornell Summer Undergraduate Research Symposium.
  4. Student was selected for and gave a talk at the SLU Festival of Science.
  5. Student was coauther in a poster presentation at the 1996 FASEB Summer Research Conference on Receptors and Signal Transduction.
  6. Student presented a talk at an ACS undergraduate research conference at Skidmore College and coauthor at of a presentation I gave a the 1998 FASEB Summer Research Conference on Molecular Biophysics of Cellular Membranes. Merck Scholar.

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