Diffusive and nondiffusive proteins in vivo

Proteins within the living cell potentially possess different spatial localizations and a wide range of diffusivities . At one extreme, proteins may be essentially nondiffusive-that is, highly localized structural components of the cytoplasm and/ or nucleus, with very long or infinite residence-times. An extensive, proteinaceous cytomatrix network consisting ofthe polymerized proteins of microtubules, microfilaments, and intermediate filaments, as well as a host of tubuleand filament-associated proteins that apparently serve as structural interconnectors and functional modulators ofthe cytomatrix, does pervade the cell (1) . Among the recognized cytomatrix proteins, intermediate filament proteins appear to be archetypical nondiffusive proteins . At the other extreme, some proteins may diffuse freely, with essentially zero residence-times, within the aqueous phase which percolates through the interstices of the cytomatrix . The existence of such an aqueous intracellular phase is demonstrated by the free diffusion oftracer macromolecules microinjected into cells (2) . Movements of freely diffusing proteins within the cell would be hindered only by random collisions with organelles and the structured proteins and water of the cytomatrix . Between these nondiffusive and diffusive extremes, proteins may be partially cytomatrix associated and partially diffusive, with intermediate residence-times . Actin and tubulin, for example, seem to exist in both diffusive and nondiffusive forms . Largely on the basis of the extensive in vitro biochemistry of these two proteins, it is thought that the dynamic exchange of proteins between diffusive (monomer) and nondiffusive (polymer) states can be fundamental to their roles in cellular function (3-5) . It is not known how proteins are distributed among these classes in vivo . Notions about the intracellular states of proteins are usually derived from experiments that disrupt (homogenize, fix, or permeabilize) cellular structure in aqueous media . Some proteins, normally diffusive in vivo, may precipitate onto structured elements during such treatments . Conversely, to assume that proteins that are soluble after such treatments (so-called "cytosolic" proteins) are actually diffusive in the living cell is patently unwise (6). It has not been possible to distinguish diffusive proteins populating the aqueous phase in vivo from nondiffusive intracellular proteins or to measure the diffusive/nondiffusive partitioning ofindividual proteins within intact cells .