Going Solo: Measuring the Motions of Microtubules with an In Vitro Assay for TIRF Microscopy

Microtubule (MT) plus-end-tracking proteins (+TIPs) specifically associate with the plus ends of growing MTs, thereby determining, in many different ways, the dynamic behavior of the MTs. Over the past years, a variety of fluorescently tagged +TIPs have been purified. Their reconstitution together with other purified components involved in MT plus-end-tracking, and analysis by total internal reflection fluorescence microscopy, has helped to elucidate some of the crucial mechanisms underlying the motion of MTs. For example, +TIP dwell time and association rate, and key MT dynamic instability parameters can be measured in a controlled cell-free environment. In this chapter, we have aimed to describe in an accessible and practical manner how we carry out these assays in our lab. We cover basic steps such as the preparation of glass and sample chambers through to the details of the in vitro +TIP assay. When appropriate, we mention common problems providing practical help to overcome potential issues.