Fluorescence-based assays for microtubule architecture.

In vitro fluorescence-based assays have enabled the direct observation of single microtubule-associated proteins (MAPs) alongside the measurement of microtubule growth and shrinkage. Fluorescence-based assays have not, however, been able to address questions of "microtubule architecture." Tubulin can form diverse polymer structures in vitro. Importantly, microtubules nucleated spontaneously have different numbers of protofilaments (pfs), ranging from 11-pf to 16-pf, as well as sheet-like structures, indicating flexibility in tubulin-tubulin bonds. This structural diversity influences microtubule dynamics and the binding of MAPs to microtubules. Observation of microtubule architecture has required the imaging of microtubules by electron microscopy (EM). Because EM requires chemical fixation or freezing, it has not been possible to observe, in real time, how microtubule dynamics might influence structure and vice versa; it also remains technically challenging to directly observe some MAPs, especially small ones, by EM. It is therefore imperative to develop fluorescence-based assays that enable the direct, real-time observation of microtubule architecture alongside growth, shrinkage, and MAP binding. In this chapter, we describe our efforts to control microtubule architecture for fluorescence-based assays. We also describe how microtubule structure can be probed with the help of GFP-tagged doublecortin, a MAP that binds preferentially to 13-pf microtubules.