In vitro assays to study the tracking of shortening microtubule ends and to measure associated forces

Abstract

Accurate segregation of mitotic chromosomes relies in part on a strong linkage between the kinetochores and the plus ends of spindle microtubules (MTs). These attachments are maintained even as the MTs shorten from their kinetochore-associated ends, and despite the large variability in the magnitude of load from the chromosomal "cargo." Analysis of the underlying mechanisms has recently been facilitated by the identification and purification of various kinetochore complexes. In this chapter we review some existing approaches to study the interaction of these protein complexes with the ends of shortening MTs in vitro. Specifically, we describe the application of a "segmented" MT technique, which allows quantitative characterization of the tracking of the shortening MT ends by fluorescent proteins and protein-coated beads, as well as controlled measurement of the associated forces. There is a marked similarity between these methods and the approaches that are used to study the motions and forces produced by ATP-dependent motor enzymes walking on coverslip-attached, stable MTs. However, optical resolution at the shortening ends of coverslip-tethered MTs is not as good and the thermal noise is high. Furthermore, there are significant differences in the mechanisms of motions of microbeads driven by motors and by MT depolymerization, as well as in the interpretation of the resulting forces. Clearly, the depolymerization-driven motions are difficult to study and the corresponding phenomenology and theories are more complex than in the motors field. We hope, however, that the relatively straightforward assays based on "segmented" MTs, which are described below, will become a routine methodology, thereby helping to advance the studies of the MT-depolymerization-dependent motility.