A rapid tracking method for the quantitative analysis of organelle streaming velocity.

A key to understanding cytoskeletal mechanisms of eukaryotic cells is found in their internal motility. In many plant cell types, these motile events, termed "cytoplasmic streaming", are very impressive with rapid movement of organelles over long distances. Like many other features of cytoplasmic streaming, organelle velocity is determined by acto-myosin-related mechanisms. Therefore, the quantification of streaming velocity aids the characterization of important factors contributing to cytoskeleton function. Usually, the movement velocity varies greatly between particles and exhibits rapid changes. This complexity makes measurements very cumbersome and requires large cell numbers and a lot of imaging data for statistical evaluation. Focusing on a triplet of rapidly moving organelles in a single cell proved to be an efficient method for determining organelle displacement in a direct, standardized manner. This approach requires only a few cells and allows the evaluation of potential factors involved in cytoplasmic streaming with a relatively low temporal and technical effort. This chapter evaluates two examples that show the high sensitivity of the method in the detection of differences in organelle streaming velocities. These include the retardation of streaming upon myosin inhibition and a similar, but much less expected, response following the overexpression of actin-binding proteins.