Chapter 20: Automated spatial mapping of microtubule catastrophe rates in fission yeast.

Microtubules (MTs) are cytoskeletal polymers whose spatial organization is dynamically regulated, depending on their biological function during different cell cycle stages. Growing MT ends are, for example, specifically targeted towards the cortex of motile or growing cells during interphase or towards chromosomal attachment sites during mitosis. An important parameter that cells use to control the average length of MTs, and thus the distance over which these targeting processes may operate, is the so-called catastrophe frequency f(cat): the rate at which MTs switch from a growing to a shrinking state. To understand how spatial targeting and the local control of f(cat) are related, quantitative in vivo measurements are needed that allow for the measurement of f(cat) in a spatially resolved way. Since catastrophes are intrinsically stochastic events, it is essential to acquire enough statistics to obtain the underlying rate constant f(cat). Here, we present automated image processing methodology, developed using GFP-tubulin expressing fission yeast cells, that makes it possible to measure f(cat) both spatially resolved and with high statistical accuracy. Although certain aspects of the analysis are specific to the system under investigation the basic concepts of the methodology are applicable to any kind of movies of fluorescently labeled MTs.