Understanding how the dynamics and distributions of membrane molecules relate to the regulation of cellular function is the long- term goal of this project. Several instrumental techniques will be used to study the lateral and rotational motions of molecules involved in lymphocyte stimulation by antigens and mitogens. Fluorescence photobleaching recovery examines molecular lateral motions, while time-resolved phosphorescence anisotropy and polarized fluorescence depletion measure protein rotational relaxation. Both fluorescence energy transfer and scanning fluorescence correlation spectroscopy provide information on the distributions of specific molecules on cell surfaces. One research goal concerns the mechanisms by which B cells present antigens to T helper cells. Lateral and rotational motions of Ia on B cells capable of presenting antigenic peptides to antigen-specific T cells will be studied to identify possible changes in Ia aggregation or conformation caused by peptide binding. Polyclonal and monoclonal hapten-specific B cells will be used to present antigen to T cells. The relation between antigen structure and concentration and subsequent presentation efficiency will be examined to learn how antigen encounter with the B cell membrane initiates antigen processing and expression of antigenic peptides. Antigen-Ia-T cell receptor complexes will be examined using environmentally-sensitive fluorescent peptides and fluorescence energy transfer methods to probe how T cell encounter modifies antigen-Ia interactions. The second area of work deals with T-independent antigen and mitogen interaction with B cells. Lateral diffusion of sIg-antigen aggregates on cloned DNP-specific B cells as functions of antigen concentration and epitope density will be measured by fluorescence photobleaching recovery and compared with accompanying B cell differentiative responses. The size and composition of antigen-immunoglobulin aggregates formed under conditions leading to stimulation and tolerance of the same DNP- specific cell lines will be measured for comparison with recently- completed models. Flow cytometric techniques will be used together with scanning fluorescence correlation spectroscopy. The state of B cell lipids during high zone tolerance induced by T- independent antigens and during activation by lipopolysaccharide will be explored by fluorescence photobleaching recovery.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI021873-04
Application #
3132323
Study Section
Experimental Immunology Study Section (EI)
Project Start
1985-04-01
Project End
1991-03-31
Budget Start
1988-04-01
Budget End
1989-03-31
Support Year
4
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Colorado State University-Fort Collins
Department
Type
Schools of Arts and Sciences
DUNS #
112617480
City
Fort Collins
State
CO
Country
United States
Zip Code
80523
Meiklejohn, B I; Rahman, N A; Roess, D A et al. (1997) 5-iodonaphthyl-1-azide labeling of plasma membrane proteins adjacent to specific sites via energy transfer. Biochim Biophys Acta 1324:320-32
Woodard, S L; Aldo-Benson, M; Roess, D A et al. (1995) Flow cytometric analysis of T-independent antigen binding to dinitrophenyl-specific cells. J Immunol 155:163-71
Young, R M; Arnette, J K; Roess, D A et al. (1994) Quantitation of fluorescence energy transfer between cell surface proteins via fluorescence donor photobleaching kinetics. Biophys J 67:881-8
Londo, T R; Rahman, N A; Roess, D A et al. (1993) Fluorescence depletion measurements in various experimental geometries provide true emission and absorption anisotropies for the study of protein rotation. Biophys Chem 48:241-57
Kenny, N; Rahman, N A; Barisas, B G et al. (1993) Class I major histocompatibility complex antigens are not associated with the LH/CG receptor on ovine luteal cells. Biochim Biophys Acta 1220:94-100
Boullier, J A; Peacock, J S; Roess, D A et al. (1992) Protein and lipid lateral diffusion in normal and Rous sarcoma virus transformed chick embryo fibroblasts. Biochim Biophys Acta 1107:193-9
Roess, D A; Rahman, N A; Kenny, N et al. (1992) Molecular dynamics of luteinizing hormone receptors on rat luteal cells. Biochim Biophys Acta 1137:309-16
Rahman, N A; Pecht, I; Roess, D A et al. (1992) Rotational dynamics of type I Fc epsilon receptors on individually-selected rat mast cells studied by polarized fluorescence depletion. Biophys J 61:334-46
Chakrabarti, A; Matko, J; Rahman, N A et al. (1992) Self-association of class I major histocompatibility complex molecules in liposome and cell surface membranes. Biochemistry 31:7182-9
Bulva, C J; Printen, J; Melnykovych, G et al. (1991) Corticosteroid effects on lipid lateral diffusion in CEM-C1 and CEM-C7 acute lymphoblastic leukemia cells. Biochim Biophys Acta 1094:134-7

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