Reticulomyxa is a large, multinucleated, freshwater protozoan with striking intracellular transport and cytoskeletal dynamics. Saltatory movement of individual organelles at a rate of up to 25um per second and cytoplasmic streaming are observed within the extensive reticulapodial network of fine strands and the naked cell body. In addition, the strands of the peripheral network are capable of rapid extension, retraction, splaying and fusion. The research proposed is aimed at elucidating the molecular mechanisms of the movement of organelles and reticulopodial strands. Preliminary studies, including a characterization of the structural organization of the network, the development of a stable and reproducible permeabilized cell model, and the preparation of monoclonal antibodies, have laid the groundwork for turning this into a useful model system. From these studies, a number of specific projects have emerged. To complete the characterization of the structural prerequisites for motility, the polarity of microtubules and actin filaments will be determined, and their three-dimensional organization will be analyzed in the frozen-hydrated state. The preparation and characterization of monoclonal antibodies will be continued. An assay for microtubule sliding in lysed reticulopodial strands will be developed. The effect of monoclonal antibodies on reactivated organelle movement and microtubule splaying will be assessed, and the antigens likely to be involved in organelle movements and network dynamics will be localized by immunoelectron microscopy. These studies are significant because they promise to improve our understanding of organelle and microtubule dynamics in the cell. They will also provide a new approach for analyzing the molecular basis of particle transport in the living cells.
