STRUCTURAL AND DYNAMIC STUDIES OF MEMBRANE DOMAINS IN MAST CELLS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This is a project to apply ACERT's advanced ESR methods to gain detailed structural and dynamical information about the membrane domains involved in IgE-FeRI mediated activation of mast cells. It is based on the past collaboration that clearly demonstrates our capacity to obtain high resolution ESR spectra of a variety of spin labeled lipids within detergent resistant membranes (related to lipid rafts) (Ge et al, Biophys J., 77: 925, 1999) and plasma membrane vesicles (Ge et al, Biophys. J. 85: 1278, 2003) and to demonstrate distinctive phase-like properties as revealed from rotational rate constants, order parameters, and other physical characterization. Our past measurements on RBL mast cells (and three other cell types) showed for the first time distinctive regions of ordered and disordered lipids in living cells (Swamy et al, Biophys. J. 90: 4452-4465, 2006). In this collaboration we are investigating the effect of antigen-mediated receptor activation on the plasma membrane dynamic structure by spin label ESR. We will use the greater sensitivity of our 2D-ELDOR methods in comparative studies on plasma membrane vesicles and live cells and assess lipid exchange rates between the different phase-like regions. Our capacity to examine the plasma membranes of whole cells in conjunction with simpler subcellular membrane preparations and defined model membranes enables further characterization of membrane domains and refinement of our concepts of lipid rafts that appear to facilitate IgE-FceRI signaling.