This application is being submitted by a group of investigators who propose to purchase a transmission electron microscope (TEM) for their NIH.-funded research programs. All are using TEM to answer important questions in basic molecular, cellular, and developmental biology. We propose to replace a 25 year old TEM (Philips 301) that is no longer going to be supported by the manufacturer, and for which there are no longer many replacement parts available, with a modern """"""""workhorse"""""""" instrument that has digital imaging capacity. After considering many options, we have chosen a Philips/FEI 208S microscope with Advantage R Digital CCD camera system because of its versatility, low cost, and the outstanding technical service provided by Philips in this geographic region. The 4 major and 4 minor user laboratories account for 9 NIH - and l N.S.F.-funded research projects that cover a wide range of topics, all of which greatly benefit from analysis by TEM and immunoelectron microscopy. The topics covered by the major users of this instrument include membrane trafficking in the secretory and endocytic pathways of mammalian cells (Dr. Brown), the structure and function of the actin cytoskeleton in polarized epithelial cells and in the yeast Saccharomyces cerevesiae (Dr. Bretscher), the role of microtubules in chromosome segregration in S. cerevesiae (Dr. Huffaker), and the assembly and packaging of Rous sarcoma virus (Dr. Vogt). Other research topics that also benefit from TEM analysis, but on a less consistent basis and therefore forming a minor user group, include the regulation of metaphase-to-anaphase transition by mitotic checkpoints in Xenopus and yeast (Dr. Chen), the role of the nuclear lamina protein Ya that is essential for the transition from meiosis to mitosis in the fertilized Drosophila egg (Dr. Wolfner), establishment of polarity in the early embryo of C. elegans (Dr. Kemphues), and the role cholesterol in membrane structure (Dr. Feigenson). The members of this group will continue to utilize TEM in their research, and, in fact, we predict that in the """"""""post-genome sequencing"""""""" era, an increasing number of projects will require analyses by EM and immunoelectron microscopy.
