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. We use the NCMIR's and NBCR's tomographic resources to: (1) determine the 3-dimensional volume of the endoplasmic reticulum in RBL-2H3 cells and (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 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. 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. 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. We previously used immunogold labeling of membrane sheets to map distributions of receptors and associated signaling molecules in discrete microdomains of the plasma membrane. Previous work from the Oliver group has mapped distribution of receptors by scanning electron microscopy, using backscattered electron detection for gold particle imaging.