This is a proposal for funds to purchase a freeze fracture instrument and a high pressure freezer. The freeze fracture machine is required for determination of: (1) typed and distribution of proteins in membranes; (2) patterns of vesicle fusion; (3) relationships between extracellular matrix components; and (4) the role of non-bilayer lipids in membrane function. The research proposed includes analysis of natural and artificial membranes, comparison of control and experimental samples, and investigation of plants and protists. (Minor users will also examine fungi, animals, and DNA molecules.) Freeze fracture, which involves cleaving and replicating cells and membranes, is the only technique that allows structural observation of intramembrane proteins and lipid phases. Therefore, it is absolutely required for this research. If freeze fracture micrographs are to be meaningfully interpreted, it is best for the sample to be uncryoprotected, but frozen so fast that vitrification occurs before the formation of large ice crystals that destroy ultrastructure. The high pressure freezer, which briefly applies pressure and cryogen together to lower the rate of freezing required to avoid ice crystal formation, is the only instrument that will allow good freezing to occur below the surface layer of an uncryoprotected sample. Therefore, it greatly reduces artifacts, enhances meaningful interpretation of replicas, and increases the efficiency of freeze fracture experimentation.
