3-D RECONSTRUCTION OF RBL-2H3 ER AND PLASMA MEMBRANES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Drs. Wilson and Oliver are cell biologists with over 30 years experience in the analysis of signal transduction through the high affinity IgE receptor, Fc[epsilon]RI, of mast cells and in innovative electron microscopy to study the membrane topography of receptors and signaling proteins. Our work was the first to describe the changes in cell shape and receptor topography induced by Fc[epsilon]RI crosslinking, to describe the rapid clustering of IP3 receptors following elevations in intracellular calcium and, most recently, to establish that Fc[epsilon]RI signaling occurs in multiple distinct membrane microdomains. We have worked previously with biophysicists and mathematical modelers on aspects of Ca2+ mobilization in mast cells and on the modeling of IgE receptor redistribution during mast cell signaling. We now have two distinct modeling projects, involving applied mathematics and computing specialists from the UNM Dept. of Mathematics as well as computational scientists at Sandia National Laboratories. We propose to use the NCMIR's tomographic resources to:1) Determine the 3-dimensional volume of the endoplasmic reticulum in RBL-2H3 cells. We showed previously that Type 2 IP3 receptors form large clusters within the endoplasmic reticulum within minutes of sustained elevations in calcium induced by receptor activation or calcium ionophore (Wilson et al, 1998). For our current modeling project, that attempts to predict the effects of IP3 receptor clustering on the filling state of the ER calcium store, we need accurate measurements of the endoplasmic reticulum volume, shape and distribution. We are encouraged by the successful reconstruction of the ER in Purkinje cells (Martone et al, 1993). Because the two cell types are so different, our modeling project will need to be based upon actual TEM measurements in RBL cells. We will integrate the ER volume data with IP3 cluster number and distribution data obtained by confocal microscopy and ultra-cryo immunogold labeling.