Board Member: 2004 - 2007
Cell membranes are complex two-dimensional arrays of mobile, interacting molecules. My laboratory uses cell biology, biophysics, especially fluorescence methods, biochemistry and immunology to study membrane dynamics and organization in cells ranging from lymphocytes to epithelial cells.
All of our work on membranes arises from interests in transplantation immunology, especially in the cell biology of class I MHC molecules. We aim to understand: 1) the intracellular traffic of class I MHC molecules during and after peptide loading, 2) the relationship between plasma membrane biophysics and antigen presentation by class I molecules and 3) the way in which viral proteins interfere with both these processes.
Our methods of analysis concentrate on two microscope-based physical techniques, fluorescence recovery after photobleaching, FRAP, to measure lateral diffusion, and fluorescence resonance energy transfer, FRET, to measure molecular proximity and clustering. Work with these techniques as well as with other advanced microscopies, including deconvolution microscopy and total internal reflection microscopy, is done in a strong Departmental imaging facility with ample hardware and technical support.
Using genetically fluorescent class I MHC molecules (tagged with GFP and its derivatives) we have dissected the organization of these molecules and their specialized chaperones in the endoplasmic reticulum, ER. Thus far we know that nascent class I molecules remain in the ER after peptide loading and that they interact with a number of factors some of which may carriers for export from the ER. We also have some evidence that the ER membrane is organized into domains with specialized function. Experiments are planned to study ER domain organization in detail and to resolve the nature of class I-associated molecules regulating their ER export. The same reagents and methods can be used to understand the ways in which viral proteins that suppress expression interact with class I molecules in the ER.
We also investigate the way in which antigen presentation to T cells and NK cells is affected changes in organization of class I MHC molecules at the cells surface. We are concentrating on techniques that vary the mobility of the class I molecules and on methods that change the extent to which they are clustered. It appears that cholesterol depletion of plasma membranes has large effects on antigen presentation; these effects can be mimicked by drugs that affect the membrane skeleton. In parallel we are also studying mutant class I MHC molecules with altered lateral diffusion and almost have enough data to connect the two sets of phenomena into a surprising model about the relationship between lateral diffusion and antigen presentation.
It is thought that membrane proteins and lipids are not randomly distributed in the fluid lipid bilayer. Rather, both proteins and lipids may associate to form domains. Most of the evidence for these domains is indirect. Many domains are smaller than the resolution of the light microscope. We have built a "super-resolution" light microscope, a near-field scanning optical microscope, or NSOM, which images cells labeled with fluorescent antibodies, but with a resolution of better than 50nm. Using this microscope we plan to image the patchiness of surfaces of living cells, and to follow changes in this patchiness when cells present antigen.
A particular type of lipid domain, a 'lipid raft', has been suggested as important for trafficking of some membrane lipids and proteins, and for the assembly of signaling complexes after surface receptors bind their ligands. We are probing for these rafts using fluorescence resonance energy transfer. Our fluorescent probes may be either labeled antibodies, or variants of GFP.