An Automated Method for Defining Microtubule Length Distributions

This chapter presents two examples of automated data acquisition and evaluation and examines various statistical problems, with examples of actual experiment data and a detailed program listing for implementing this automated method. Length distribution measurements often provide the only available way to determine important parameters in the study of biological polymers. For example, because most polymerization mechanisms tend to be endwise, it is necessary to know the number of polymer ends in solution to study polymerization or depolymerization kinetics. This can be determined from knowledge of the total protein in polymer form (usually determined by turbidity) and the average polymer length. The initial rates determined by turbidity have agreed with the electron microscopic number concentration measurements to within a few percent. Accurate determinations of microtubule distribution shapes have enabled theoretically predicting the entire time course of the depolymerization reaction on the basis of these electron microscopic measurements. These results demonstrate that electron microscopy can be used to accurately determine microtubule lengths in solution. In addition, length distribution techniques may be used to determine the mode of action of other microtubule assembly effectors.