A detailed view of cytoarchitecture is essential to understanding kinetics and mechanisms of biochemical reactions in living cells, and for understanding transport and localization of intracellular components. Rheological, biochemical, and electron microscopic evidence suggests that cytoskeletal elements interact to form a three-dimensional cytoplasmic network. Measurements of solvent viscosity in living cells indicate that rotational diffusion of molecules in cytoplasm is essentially the same as in dilute solution, while their translational diffusion is significantly hindered, apparently by a combination of collisions with other solute molecules and sieving by the cytoskeletal network. Even very small solutes such as ions and metabolites may diffuse 3 to 4 times slower in cytoplasm than in dilute aqueous solution. The diffusion of particles >26 nm in diameter is further constrained by exclusion from some subcompartments. Many of the intracellular components involved in protein synthesis, respiration and particle transport are also absent from such compartments, raising the possibility that they represent functional as well as structural differentiations of the cytoplasm. Excluding compartments are filled with bundles and meshworks of actin microfilaments. An actin-binding protein, ABP280, that crosslinks F-actin into isotropic networks is also concentrated in excluding compartments. Since F-actin / ABP280 networks in vitro are very elastic and stable, the presence of these two cytoskeletal proteins in excluding compartments suggests they are specialized for a mechanical function. The objectives of this proposal are to test several models for the organization of the cytoskeletal network that hinders the diffusion of macromolecules, to test the hypothesis that exclusion of large particles from excluding compartments is due to molecular sieving by a gel network, to identify actin-binding proteins that may be components of the cytoarchitecture of excluding compartments, and to attempt to implicate F-actin and ABP280 more directly in the exclusion of particles by selectively disrupting their function. A multidisciplinary approach will be employed, combining microinjection of proteins and inert fluorescent tracer particles into living cells, fluorescence recovery after photobleaching to measure intracellular diffusion coefficients, digital fluorescence microscopy to study the dynamic localization of injected molecules and the response of the cells to them, fluorescent antibody localization of endogenous molecules in cells that have been fixed, gold antibody localization to obtain a high resolution picture of the three-dimensional organization of molecules in the cytoskeleton of excluding compartments, and molecular genetics techniques to disrupt the function of ABP280. Ultimately, information obtained from this research should lead to an understanding of the effects of cytoarchitecture on diffusion and localization of intracellular components, and stimulate testable hypotheses regarding the function and regulation of excluding compartments. %%% Much current thinking about cellular biochemistry and metabolism is extrapolated from assays performed in homogeneous, dilute solution. However, the interior of a cell is neither dilute, nor homogeneous. The intracellular concentration of certain enzymes is orders of magnitude higher than the concentrations commonly used to assay enzyme activity in vitro, and the total protein concentration may be as high as 200 to 300 mg/ml. Sophisticated experiments employing a variety of biophysical and other techniques have demonstrated the existence of "structure" (other than the better- understood membrane-bound compartmentation) within what has until recently been considered an amorphous cytoplasm. This project will further define the nature and function of this cytoplasmic structure. The work is significant not only for what it can help us understand about the function of the living cell, but also potentially as a model for a new generation of synthetic materials of functionally defined porosities.
