Biology Reference
In-Depth Information
CHAPTER
Architecture
21
Susanne Bechstedt and Gary J. Brouhard
Department of Biology, McGill University, Montreal, Quebec, Canada
Fluorescence-Based
Assays for Microtubule
CHAPTER OUTLINE
Introduction ............................................................................................................ 344
21.1 Materials........................................................................................................347
21.2 Methods .........................................................................................................348
21.2.1 Tubulin and Microtubule Preparations .......................................... 348
21.2.2 Setup for the Single-Molecule Fluorescence Assay ........................ 348
21.2.3 Comparison of Controlled 13-pf with Controlled 14-pf
Microtubules............................................................................. 348
21.2.4 Comparison of Mixed Populations of Microtubules with
an Internal 14-pf Control ............................................................ 349
21.2.5 Purification of DCX-GFP for Labeling 13-pf Microtubules............... 351
21.3 Discussion......................................................................................................352
Acknowledgments ................................................................................................... 352
References ............................................................................................................. 352
Abstract
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. Obser-
vation of microtubule architecture has required the imaging of microtubules by elec-
tron 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
