B George Barisas - US grants

Several instrumental techniques will be used to study lateral and rotational motions of membrane molecules involved in lymphocyte stimulation by antigens and in luteal cell activation by gonadotropins. Fluorescence photobleaching recovery (FPR) and image-intensified video microscopy (IIVM) examine molecular lateral motions while polarized fluorescence depletion (PFD) measures protein rotational relaxation. B lymphocytes decorated by a new technique with artificial antigen receptors exhibit polyclonal differentiative responses to specific antigens. The lateral and rotational mobilities on these cells of bound antigens, antigen receptors, integral membrane proteins and lipid probes will be measured. Results will be compared with data on natural antigen-specific cells to better understand the role of surface immunoglobulin in B cell activation. Membrane dynamic requirements for B cell presentation of T-dependent antigen to T helper cells will be investigated. The role of B cell Ia and accessory cell proteolytic processing of antigen should be clarified by these studies. Mathematical models for antigen interaction with cell surface receptors will be extended and their predictions tested against experimental data. Binding of various gonadotropins such as luteinizing hormone (LH) and human chorionic gonadotropin to LH receptors on luteal cells will be examined by FPR and PFD. This will show how mobilities of hormone-receptor complexes are related to the duration of progesterone responses. The initial distribution, hormone-induced redistribution, and sites of internalization of LH receptors will be sought by electron microscopy and by IIVM to assess the connection between receptor redistribution and luteal cell activation. LH receptor mobility will be artificially restricted by crosslinking to test whether changes in receptor dynamics actively affect luteal cell steroidogenic responses to gonadotropins. Instrumental facilities for FPR, PFD, and IIVM will be updated and expanded.

photochemistry; fluorescence;

lasers; biomedical equipment resource;

The long-range goal of this project is to explore the physicochemical organization of the B lymphocyte plasma membrane and to learn how changes in this organization may reflect and/or mediate primary events in cellular activation. The first part of this project concern the organization and distribution of lipids on membrane domains. Sizes, lipid composition, and fluidities of lipid domains will be examined using fluorescence photobleaching recovery and scanning fluorescence correlation spectroscopy. Studies of large unilamellar vesicles prepared from B cell membrane lipid will help assess whether cellular lipid organization is dependent on the presence of cell surface protein. Membrane lipid alteration via specific lipid enrichment, via cholesterol depletion through glucocorticoid treatment, and through temperature perturbation will be examined. The second area of work concerns the distribution and interaction of membrane proteins. Scanning fluorescence correlation spectroscopy experiments will explore whether proteins are localized into one or more types of protein-rich regions and whether specific lipid classes preferentially associate with particular proteins. Specific associations occurring between known B cell membrane proteins and other known or as-yet unidentified species will be assessed using fluorescence energy transfer and new immunochemically-targeted photoproximity labeling methods. The aggregation state of specific membrane proteins will be evaluated using time-resolved phosphorescence anisotropy. Finally, immediate changes in B lymphocyte membrane structure resulting from activation by three mechanisms, by anti-IgM treatment, by thymus-independent antigen-like ligands interacting with cells artificially decorated with specific receptors, and by lipopolysaccharide will be examined.

Class II molecules of the Major Histocompatibility Complex (MHC) are central to antigen presentation in T cell-dependent immune responses. The lateral and rotational dynamics and membrane distribution of Class II molecules have been implicated in signal transduction and antigen presentation. The cytoplasmic domains of Class II molecule alpha- and beta-chains are critical for normal functioning of these molecules and are major determinants of their membrane motions. In collaboration with Dartmouth Medical School, we will examine the effects of mutations within Class II molecules on their membrane dynamics and biological function. B cells expressing mutant Class II molecules with truncated and substituted cytoplasmic domains, as well as Class II molecules with mutations in the contact residues of the Class II (alpha- beta2) dimer, will be examined using biophysical methods to determine if specific Class II molecular structural features and domains have roles in modulating molecular dynamics and intermolecular associations. These phenomena have been implicated in regulating Class II functions including signal transduction in the B cell and antigen presentation to T cells. Specific aims include: l) Time-resolved phosphorescence anisotropy, polarized fluorescence depletion and fluorescence photobleaching recovery studies on cytoplasmic mutants of Class II to determine which amino acid residues regulate Class II molecular rotational and lateral motions. An additional aspect of this aim is to examine Class II molecules with mutations affecting signalling. The effect of mutations on molecular motions will be correlated with signalling defects and correlated with nearest neighbor analysis of protein-protein interactions which contribute and control Class II's membrane motions and physiology; 2) Lateral and rotational diffusion studies of wild type and mutant Class II molecules in the presence or absence of immunogenic peptides. These studies will examine the ability of peptides to regulate Class II aggregation in the presence or absence of cytoplasmic domain structures of Class II molecules; and 3) Examination of the effect of mutations in the contact amino acids between alpha-beta molecules in the Class II (alpha-beta)2 dimer. Class II molecular aggregation appears required for efficient signal transduction and antigen presentation. Studies in this aim will examine whether changes in dimer-forming ability, evaluated as rotational and energy transfer changes measurements, correlate with the efficacy of antigen presentation.