Assessment of Spindle Shape Control by Spindle Poleward Flux Measurements and FRAP Bulk Analysis

In plants, the segregation of genetic material is achieved by an acentrosomal, mitotic spindle. This macromolecular machinery consists of different microtubule subpopulations and interacting proteins. The majority of what we know about the assembly and shape control of the mitotic spindle arose from vertebrate model systems. The dynamic properties of the individual tubulin polymers are crucial for the accurate assembly of the spindle array and are modulated by microtubule-associated motor and non-motor proteins. The mitotic spindle relies on a phenomenon called poleward microtubule flux that is critical to establish spindle shape, chromosome alignment, and segregation. This flux is under control of the non-motor microtubule-associated proteins and force-generating motors. Despite the large number of (plant-specific) kinesin motor proteins expressed during mitosis, their mitotic roles remain largely elusive. Moreover, reports on mitotic spindle formation and shape control in higher plants are scarce. In this chapter, an overview of the basic principles and methods concerning live imaging of prometa- and metaphase spindles and the analysis of spindle microtubule flux using fluorescence recovery after photobleaching is provided.