Myosin-Mediated Vesicular Transport in the Extruded Cytoplasm of Characean Algae Cells

This chapter describes a stable and reproducible, in vitro, experimental system that facilitates the study of myosin and actin-based organelle movement, using video and electron microscopy of extruded cytoplasm of the giant cells of the characean algae. This assay determines that the characean algal cytoplasm displays at least two myosin-mediated organelle movements. One population of vesicles moves along actin bundles at an average speed of 11μm s^-1, while the other vesicle class is translocated at velocities up to 60 μm s^-1. On the basis of three-dimensional reconstruction of the cytoplasm obtained by fast-freeze electron microscopy, it is predicted that the myosin-dependent movements of the endoplasmic reticulum in these cells are responsible for the streaming of the whole cytoplasm. Chara corallina and Nitella flexilis are two of the most commonly used characean algae and are easy to maintain. Organelles moving along actin bundles can be visualized, using differential interference contrast (DIC) optics via conventional Koehler illumination, fiberoptic illumination, or critical illumination. The extruded cytoplasm contains areas where actin bundles and vesicular organelles are the only components present.