3 D RECONSTRUCTION OF RBL 2H3 ER & 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 more than 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 raising and sustaining elevations in calcium induced by receptor activation or calcium ionophore. For our current modeling project, which 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. 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. 2) Reconstruct a 3-dimensional view of a "typical" resting and activated RBL-2H3 cell, reflecting the dramatic changes in surface topography (and potentially volume). We have recently used immunogold labeling of membrane sheets to map distributions of receptors and associated signaling molecules in discrete microdomains of the plasma membrane. Previously, the Oliver group has mapped receptor distribution by scanning electron microscopy, using backscattered electron detection for gold particle imaging. We aim to develop a geometric model of the shape of the RBL-2H3 rat mast cell and superimpose upon it the topographical distribution of selected transmembrane proteins. The model will contrast the distribution of signaling molecules on cells at rest and following signal initiation by crosslinking the Type I IgE receptor, FceRI. The model will be a statistical reconstruction generated through the integration of scanning and transmission electron microscopy images of cells labeled from either side of the membrane with immunogold particles specific for FceRI, membrane adaptors, signaling proteins and endocytic machinery. We envision that preliminary data for both of these aims can be accomplished using the same set of serial thick sections and tilt series. Samples have already been prepared (where the ER is stained selectively using the osmium ferrocyanide method of Forbes et al., 1977) and are embedded in Epon. Preliminary work began in October 2001 with the acquisition of a tilt series and subsequent reconstruction. This work proved promising, with additional work needed on section staining. In December 2001, Alex Smith visited the NCMIR, collected several tilt series and ran through the reconstruction techniques. Currently, we are converting the data into a form appropriate for the modeling software. Work on this project will continue with a visit by Stephen Jett in Spring 2004. He will collect more data and learn tomography reconstruction using