Macromolecular Organization and Function of the Aqueous Cytoplasm

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The proposed research is the continuation of a long range program aimed at understanding the structure, properties and metabolic activities of the aqueous compartments of animal cells: nucleoplasm, interiors of cytoplasmic membrane-bound organelles, and the cytosol, or aqueous cytoplasm. Although these compartments make up about 75% of the mass and volume of most cells, and are the sites of much metabolic activity, very little is known about the details of their properties and organization. Current work focuses on the aqueous cytoplasm, and employs chiefly cultured mouse fibroblasts (L cells) which have been permeabilized with dextran sulfate (DSP cells). They retain most macromolecules that are released into the medium by harsher treatments (detergent extraction or homogenization): those macromolecules commonly thought to be freely dissolved ("soluble") in the aqueous cytoplasm. Retention of most of these macromolecules in DSP cells occurs in spite of lesions ("pores") in their plasma membranes that are large enough to allow them to escape. This research is designed to test the hypothesis that most "soluble" macromolecules defined by extraction and classical cell fractionation are not actually free in intact cells but are instead organized, and associated loosely with the cytomatrix. The hypothesis will be tested in DSP cells by an examination of the enzymes of glycolysis (long believed to be "soluble") using a variety of metabolic probes and high voltage electron microscopy. The efflux of these enzymes (and proteins in general) from DSP cells exposed to treatments designed to perturb postulated cytomatrix-enzyme associations will be studied by electrophoretic and enzyme activity assays. Studies on DS permeabilization of other cell types will also be initiated. Although little is known about the organization of the cytoplasm of living cells at the macromolecular level, it has long been overlooked as a subject of rigorous study because "cytoplasm" has generally been regarded as a simple solution of proteins and small molecules. Several lines of recent evidence from cell biology and biochemistry suggest that this is an oversimplified and misleading view of the cell. The proposed research will provide much needed data on the state of organization of proteins and small molecules in cells and could lead to a fundamentally different and more realistic concept of cellular structure and function.