This application is a continuation of ongoing efforts in Dr. Verkman's laboratory to characterize the dynamics of solid and fluid phases in the intracellular and intraorganelle media. This program was last reviewed by the BBCA study section in June 1992. In this new application Dr. Verkman proposes to: 1)Measure solute diffusion in crowded aqueous-phase compartments in living cells. This effort will be focused on obtaining data about the motion of metabolites and enzyme-sized solutes using fluorescence photobleaching recovery, total internal reflection (TIR)-microfluorimetry, and time resolved fluorescence anisotropy. These experiments will be done in rat liver mitochondria, which possess, a 50 - 60 percent solids v/v, and in erythrocytes, whose solid concentration can be varied from 30 - 80 percent by changing the osmolarity of the medium. Labeling of the mitochondrial matrix will be accomplished by cell transfection with cDNAs encoding the green fluorescent protein (GFP), in fusion with a mitochondrial targeting sequence and endogenous enzymes, so as to be able to vary the size of the fusion product. These experiments will determine the effect on diffusion by the presence of solids. 2) To quantify and model the sieving properties of cytoplasm, nucleoplasm and the aqueous phase of trans-Golgi apparatus. These experiments will test the hypothesis that aqueous cellular compartments have a fluid phase that is low in viscosity but that is able to sieve large solutes, in a manner that is dependent on the size and the density of the fixed and mobile obstacles present in these compartments. These studies are motivated by preliminary results obtained in Dr. Verkman's laboratory, indicating that the rotational diffusion of dissolved solutes is little or not affected in these compartments, while the translational diffusion is greatly reduced. Several strategies are proposed to introduce the fluorescent reporters, such as GFP fusion constructs (to label cytoplasm and nucleoplasm) and fluorophor delivery by liposome encapsulation (to label the trans-Golgi space). These experiments will utilize methods such as FRAP, 3-d single particle tracking, and ratio imaging of a viscosity sensitive fluoorophore. The results will be analyzed using mathematical models of molecular sieving. 3) To quantify the submicroscopic intermolecular distances to measure specific membrane-skeletal distances and skeletal motions in living cells. The objective is to characterize the bilayer-to-skeletal protein distance in intact cells. Specifically, erythrocytes which have a dense spectrin matrix will be studied, as well as epithelial cells, which have an acting cytoskeleton. To carry out these studies, multiple angle total internal reflection will be used to characterize distances between 10 - 300nm, a range that falls between the higher limit of FRET and the lower limit of optical microscopy methods. These studies will address the hypothesis that the membrane skeleton of these cells is a mobile, dynamic structure.
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