Michael Aaron Edidin, Ph.D. - US grants

Lateral mobility and organization of cell surface molecules are important in normal cell functions, for example, response of receptors to their ligands and presentation of antigens to T cells. Even the general principles of lateral organization of membranes are not well understood. Our work concentrates on the mobility and associations of class I MHC antigens. These properties of the antigens appear to be affected by the organization of membranes into domains, and by the extent of glycosylation of membrane proteins. We will characterize lateral diffusion of the antigens in glycosylation-defective mutant cells and will use both fluorescence photobleaching and digital video microscopy to characterize the spatial organization of a variety of class I antigens in fibroblasts and lymphocytes. We will use flow cytometric measurements of quenching of Terbium-labeled antibodies to determine local diffusion coefficients and to select variants in lateral diffusion for function studies. The function of class I antigens in antigen presentation will be investigated by measuring energy transfer between class I antigens labeled with fluorescent beta-2m and fluorescent viral antigens. Differences in the mobility of class I antigens in different lymphocyte subsets will also be investigated.

A flow cytometer/cell sorter is requested for the use of a group of eleven investigators in two departments, Biology and Biophysics, on The Johns Hopkins Homewood campus. Ongoing projects which would benefit from the use of a sorter cover four main areas: 1) cell surfaces and receptors, 2) gene expression, transfection and somatic cell genetics, 3) chromosome structure and biochemistry and 4) carcinogenesis. The sorter's proposed uses range from the daily preparation of single hepatocytes for analysis of cell adhesion, to development of separation methods for non-parenchymal liver cells (for the study of asialoglycoprotein receptors), to the cloning of hybridomas or of mutant or transfected cells (for biochemical and biophysical studies of lipid metabolism or studies of membrane function). Analytical use of the instrument will range over detection of antigen expression in mouse cells transfected with human genes, receptor binding assays, and energy transfer measurements of distances between molecules on the cell surface. The machine will be supported by user fees. It is expected to be largely used by the group, but as techniques for separation are fully developed, time will be available for use by others on the campus or from nearby institutions.

The experiments all aim to understand the relationship between major histocompatibility haplotype and the binding of ligands to specific cell surface receptors. Two different systems of ligand receptor binding will be studied for effects of H-2 and HLA on binding. Peptide hormone binding to fibroblasts, fat cells and liver membranes will be studied as a function of H-2 type in order to extend our previous observations on glucagon and insulin binding. The new experiments will concentrate on glucagon, insulin and epidermal growth factor (EGF) binding and thus the results will be extended to new cell types and to a hormone not previously studied by us, EGF. In addition, we will examine the effect of monoclonal anti-H-2 antibodies on peptide hormone binding. Some fluorescent-labelled antibodies and hormones will be used to determine the proximity of peptide hormone receptors and MHC antigens on cell membranes. We except that a monoclonal anti-HLA and EGF will be the most suitable combination for initial experiments, since we have shown a functional interaction between these two cell surface components. Cell-cell adhesion is the second system of receptor-ligand interactions to be studied as a function of H-2 type. We will use a new assay for the determination of adhesion rates of intact fibroblasts to one another. We will also use monoclonal anti-H-2 antibodies to perturb this adhesion system and another in which single cells adhere to a monolayer. All of these experiments should help to better define the function of class I MHC antigens in cells other than those of the immune system.

Our goal is to show the extent to which MHC antigens influence ligand binding by peptide hormone receptors and in turn are modified as a consequence of ligand binding to specific receptors. We have shown as association between HLA (genotype and phenotype) and the affinity of insulin receptors on B lymphoblasts. We will now further analyze the genetic associations between HLA and insulin receptor affinity and extend the analysis to the physical relationships between the antigens and receptor molecules. The genetic analysis will proceed through examination of mutant lymphoblasts, deleted for expression of some HLA antigens, which have been transfected with genes for the missing antigens. We will also attempt to rescue expression of HLA antigens in Daudi cells, to examine the effects of such rescue on insulin binding by Daudi. The physical analysis will take two approaches: 1. an attempt, based on precedents in the literature, to co-precipitate insulin receptors, detected by their endogenous tyrosine kinase activity, with class I MHC antigens, using specific anti-receptor and anti-class I monoclonal antibodies and 2. determination of the proximity of class I MHC antigens and insulin receptors in intact cells, using resonance energy transfer between endogenously (Beta-2-m) labeled class I antigens, and insulin receptors which we have shown can be specifically labeled with biotinyl insulin and avidin phycobilliproteins. Energy transfer will be measured in the flow cytometer in terms of donor quenching sensitized emission of acceptor. We plan to extend our work on the metabolic effects of epidermal growth factor binding on the display and phosphorylation state of wild-type and mutant class I MHC antigens, using flow cytometry to quantitate the display and standard immunochemical and biochemical approaches to determine the phosphorylation state of the antigens associated with changes in the display.

Tracking of single marker-particles is a powerful method for studying the dynamics of cell surfaces. However, only a few types of particles are available for tracking and applications of these particles are limited. We propose to develop and characterize sensitive, specific fluorescent marker immunoliposomes useful for particle tracking and other optical techniques. We will explore phospholipid compositions and fluorescent labels that give the brightest possible liposomes and study ways of conjugating antibodies to these liposomes to yield paucivalent particles. The behavior of the immunoliposomes on the cell surface will be characterized in terms of patching, capping and lateral diffusion. Development of the labels should allow application of particle tracking and laser tweezers techniques to a wide range of cell types, included rounded and thick cells whose images are not easily used in present single particle techniques.

Clustering and immobilization of membrane proteins are important in creating and maintaining the polarized epithelial cell phenotype. We have found that newly-synthesized molecules of a glycosylphosphatidylinositol(gpi)-anchored protein, gD-1-DAF, are immobile and clustered when they first reach the apical surface of polarized MDCK cells but are neither immobile nor clustered upon reaching the surface of mutant, non-sorting MDCK. We will investigate the way in which clustering of gpi-anchored proteins functions in the targeting of these proteins to the apical surface of polarized epithelial cells. Clustering and immobilization of gpi-anchored proteins will be probed in plasma membranes, transport vesicles, Golgi-derived vesicles and liposomes and will be compared between normal and sorting-defective cells. Three different fluorescence physical techniques will be used for these probes, FPR, to measure lateral diffusion, RET, to measure molecular proximity and PFD to measure rotational diffusion. Biochemical and immunochemical techniques will complement the fluorescence techniques. The same approach will be used for some transmembrane-anchored apical membrane proteins, allowing comparison of requirements for sorting and targeting of two structurally different classes of membrane proteins.

Epithelial cells, which comprise 50% of all cells in the body [and account for 90% of all human cancers] have as a defining function the creation and maintenance of physiological compartments of the body. Epithelial cells not only physically separate different environments; they regulate selective exchange between them. This requires that the cells' opposing surfaces be functionally differentiated; hence, epithelial cells are functionally polarized. This polarization is accomplished through cooperative formation and dynamic maintenance of junctional complexes between adjacent cells and by the selective or differential delivery and retention of membrane proteins and lipids to one pole or the other each cell. The projects in this program study selective delivery of components to apical and basolateral membrane domains. A variety of perspectives are taken and different techniques are used, but at the same time there is a common focus in all four projects on the Golgi complex, considering its lateral organization and heterogeneity, traffic, composition, and integrity and maintenance of its spatial orientation. Edidin (Project 1) focuses on the lateral organization of Golgi membrane and plasma membrane proteins, particularly GPI-proteins, and lipids, required for the polarized traffic of these molecules. Hubbard (Project 3) focuses on intracellular routes and mechanisms for the polarized delivery and retention of membrane proteins, with emphasis on the endosomal/transcytotic system. Machamer (Project 4) focuses on the mechanisms of the organization and composition of the Golgi complex that important for sorting lipids and proteins destined for the plasma membrane, while maintaining its own integrity. Schroer (Project 6) focuses on the cytoskeletal machinery that maintains the polarized intracellular architecture of epithelial cells and on the routes traveled by vesicles between cytoplasmic membranes and between cytoplasm and surface.

It is proposed to purchase a scanning laser confocal microscope with two-photon capabilities to measure the 3D conformation of living cells, tissues, macromolecular assemblies, and novel materials. With this new microscope, one can (i) slice clean, thin optical sections out of thick fluorescent specimens and light scattering materials, (ii) view specimen planes parallel to the line of sight, (iii) penetrate deep into light-scattering tissues, living cells, complex fluids, and complex materials, and (iv) gain 3-D views at very high resolution. This new microscope will compliment the experimental techniques currently used by Hopkins faculty, active in research projects in biomedical engineering, chemical engineering, materials science and biology. This proposal details four projects where the new SLCM will have a dramatic impact. These projects include: (1) the development of a high resolution particle-tracking instrument coupled to the new confocal microscope to probe the in vitro and in vivo mechanical properties of biopolymers; (2) studying the lateral organization of membrane proteins in epithelial cells emphasizing the packaging of glycosylphos-phatidylinositol-anchored proteins, GPI-proteins, for targeted delivery to the apical surface of morphologically polarized cells; (3) studying the molecular mechanisms by which chromatin insulators affect enhancer-promoter interactions; in particular, studying the gypsy insulator, a sequence located in the 5' transcribed untranslated region of the gypsy retrotransposon of Drosophila; (4) monitoring the process of transfecting cells and micro-organelles translocations with high spatial and time resolution. One of the primary educational objectives for acquiring a confocal microscope is to train Hopkins scientists and engineers to use one of the most versatile and promising tools of investigation in the life and materials sciences. Of course, such a powerful instrument cannot be used properly and to its full potential with out proper training. Therefore, two of the Co- PIS will be teaching a new course entitled "Practical Confocal Microscopy" to be offered during the January intersession to advanced graduate students in science and engineering who are interested in learning how to use the confocal microscope in their graduate research.

DESCRIPTION (Adapted from abstract): The cell surface mediates the flow of information and metabolites between a cell and its environment. The model of cell surface membranes is evolving from one that emphasizes mobility and autonomy of membrane constituent molecules, to another that emphasizes the lateral concentration of membrane proteins and lipids into patches that are often interpreted as showing that these molecules are confined in membrane domains. While a variety of experiments report the existence of membrane domains, the mechanisms of patch formation, and by implication, the mechanisms of domain creation are largely unknown. Specific interactions between molecules are involved in the formation of very small patches of proteins or lipid but the mechanisms that create large membrane domains, 100s of nm in diameter, are unknown. Without understanding these mechanisms one cannot understand the importance or relevance of membrane domains for cell surface membrane function in normal and abnormal cells. The PI has developed a model, a numerical simulation model based on experimental data, for large-scale domain formation in cell plasma membranes. The model assumes no specific interactions between membrane proteins and lipids. Rather, it depends upon the lateral diffusion coefficients of membrane proteins and lipids, upon the occurrence and stability of barriers to lateral mobility, and upon vesicle traffic to and from the surface. The PI finds that both vesicle traffic, and dynamic barriers to lateral mobility are required to create and maintain lateral heterogeneities in membranes consistent with domains 100s of nm in diameter. In the absence of either barriers to lateral mobility or vesicle traffic, lateral diffusion randomizes the distribution of membrane molecules. The model implies that the apparent concentration of proteins and lipids in membrane domains may be a nonspecific consequence of membrane physics and cell metabolism. The PI proposes to test his model using cells in which the barriers to lateral mobility are defective, sph/sph, alpha-spectrin-deficient, erythroleukemia cells, and cells in which vesicle traffic from and to the surface is inhibited.

This award supports the development of a new kind of fluorescent microscope. This microscope, dubbed the Holoskop, when developed, will allow the viewing of microscopic specimens in 3 dimensions (3D) in real-time.

In the last decade, advances in cell science have made it apparent that there is rapid intracellular movement of small molecules and proteins. However there is currently no microscope available to view these events in real time. Green fluorescent proteins obtained from jellyfish and other aquatic organisms (GFP) have been isolated and sequenced. Thus GFP can be expressed in cells as part of the expression of proteins of biological interest by linking the DNA from GFP with the DNA of the protein of interest. Such expression enables one to follow the movement of target proteins of biological importance by following the fluorescence of the GFP tag.

These fluorescent protein construct probes, and other organic fluorescent probes, combined with advances in optical sectioning utilizing another type of microscopy called confocal microscopy, have revolutionized studies in 3D localization and dynamic trafficking of proteins, ions, and messenger molecules. In practice, the 3D imaging of probes is derived from stacks of images taken through the z-plane in cells or tissues. Thus fluorescent molecules identifying cells, structures or processes, which move rapidly in the x-y or x-y-z plane during the capture of the image stack, cannot at present be adequately resolved or studied. Knowledge of these fast processes and their interactions is of paramount importance to understanding the myriad of events in the normal and abnormal functioning of cells and tissues. The objective of this project is to develop a new and demonstrably faster microscope for 3D imaging. The Holoskop will utilize scanning holographic principles for accelerated 3D imaging without the need for collection of image stacks.

In addition to developing an important new tool for biology, this project will have considerable impact upon the educational and outreach activities inherent in the universities involved in this project. The Holoskop once developed, will aid scores of projects in a wide variety of biological science investigations since there is currently no method that can obtain the kind of information expected from this instrument.

DESCRIPTION (provided by applicant): Ligand-receptor interactions impart specificity to biological processes. Therefore, the development of specific agonists and antagonists, so called targeted therapy, offers the potential of selectively inhibiting or enhancing biological processes. However, since specific receptors are rarely expressed solely on the target cell of interest, much of the specificity of targeted therapy can be lost. The correct receptor may be specifically targeted but not necessarily solely on the cells of interest. For example, anti-CD3 antibodies are potent T cell agonists which activate the T Cell Receptor (TCR) signaling cascade. However, attempts to enhance specific T cell responses in vivo are marked by dramatic sequelae secondary to general and non-specific T cell activation. There are too many targets for anti-CD3. Hence, the potent ability of anti-CD3 to activate T cells has not yet been leveraged to enhance anti-pathogen or anti-tumor responses in vivo. Therefore a strategy to enhance the selectivity of targeted therapy to restrict it to the cells of interest is desirable. Recently, ourgroup has found that anti-CD3, when constrained to the surface of a nanoparticle, a quantum dot, selectively activates previously antigen-stimulated T cells, without activating na¿ve cells. The nanoboost to specific T cells may reflect the spatial matching of QD/CD3 to the clustered TCR of antigen-stimulated T cells, or may involve other mechanisms that selectively target activated over na¿ve T cells. In this proposal we aim to i) test mechanisms of the enhanced T cell responses to anti-CD3 on quantum dots ii) engineer other, novel, nanoparticles for nanoboost and iii) test the ability of anti-CD3 constrained on optimized nanoparticles to selectively boost protective vaccine responses to influenza virus in vivo.