Organization of Mammalian Cytoplasm

ABSTRACT Although the role of macromolecular interactions in cell function has attracted considerable attention, important questions about the organization of cells remain. To help clarify this situation, we used a simple protocol that measures macromolecule release after gentle permeabilization for the examination of the status of endogenous macromolecules. Treatment of Chinese hamster ovary cells with saponin under carefully controlled conditions allowed entry of molecules of at least 800 kDa; however, there were minimal effects on internal cellular architecture and protein synthesis remained at levels comparable to those seen with intact cells. Most importantly, total cellular protein and RNA were released from these cells extremely slowly. The release of actin-binding proteins and a variety of individual cytoplasmic proteins mirrored that of total protein, while marker proteins from subcellular compartments were not released. In contrast, glycolytic enzymes leaked rapidly, indicating that cells contain at least two distinct populations of cytoplasmic proteins. Addition of microfilament-disrupting agents led to rapid and extensive release of cytoplasmic macromolecules and a dramatic reduction in protein synthesis. These observations support the conclusion that mammalian cells behave as highly organized, macromolecular assemblies (dependent on the actin cytoskeleton) in which endogenous macromolecules normally are not free to diffuse over large distances.