This project determines the structure of neuronal and glial cytoplasm, particularly as it pertains to axoplasmic transport, and the organization of the cytoplasm.  Living cells or tissues are directly rapid-frozen and the structure of their cytoplasm is determined by one of two methods, freeze-etching or freeze-substitution.  Axons in turtle optic nerves have different cytoplasmic domains, each characterized by specific types of filaments and by their content of organelles.  Cultured myocytes, grown on grids, frozen, freeze-substituted, and examined directly at high voltages in an electronmicroscope have a cytoplasmic ground substance consisting of fine filaments instead of a microtubular meshwork, and distinct cytoplasmic domains characterized by different types of organelle movements.  Filaments are isolated from the axoplasm of the squid giant axon along which organelles continue to move for many hours, at 1-2 um per sec, provided ATP is present.  These organelles and filaments are below the limit of the light microscope so fast digital image resolution processing of differential interference contrast images is required to visualize them.  Filaments previously observed with the light microscope and then examined in the electronmicroscope turn out to be single microtubules; organelles move very close to these tubules.  Because organelles of all sizes, including mitochondria, move at the same rate, all the organelle movements of fast anterograde and retrograde axoplasmic transport may be powered by a single molecular motor with other cytoplasmic structures determining their final rate.  Because organelle movements are blocked by metabolic inhibitors even in the presence of ATP, we now believe that the molecular motor is powered by an electrochemical gradient across the organelle membrane.