Biology Reference
In-Depth Information
involved in MT plus-end-tracking, and analysis by total internal reflection fluores-
cence microscopy, has helped to elucidate some of the crucial mechanisms underly-
ing 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 envi-
ronment. 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
þ
þ
TIP assay. When appropriate, we mention common problems providing practical
help to overcome potential issues.
INTRODUCTION
The fluorescence microscopy-based
in vitro
assay for measuring microtubule (MT)
behavior is essentially the reconstitution of (fluorescent) key components required
for MT growth. Besides tubulin, which is virtually always purified from brain
(
Miller & Wilson, 2010
) or purchased (Cytoskeleton Inc.), it also relies upon puri-
fied protein components such as EB proteins, which have to be “homemade” and
purified (
Honnappa, John, Kostrewa, Winkler, & Steinmetz, 2005; Komarova
et al., 2009
). Furthermore, it also requires the appropriate buffer conditions and es-
sential cofactors for MT growth, such as GTP. The first application of the reconsti-
tuted
in vitro
assay, as described here, to study the dynamic instability of MTs in the
presence of MT plus-end-tracking proteins (
þ
TIPs) was successfully carried out by
Bieling et al. in 2007 on the yeast
TIPs Mal3, Tea2, and Tip1 (
Bieling et al.,
2007
); the proteins were observed to interact on dynamically growingMTs. By label-
ing the proteins with the fluorescent markers GFP and mCherry, it was possible to fol-
low their binding interactions utilizing two-color total internal reflection fluorescence
(TIRF)microscopy. It is the use of this
in vitro
assay, as opposed to studying
þ
TIPs via
whole-cell imaging, that has allowed the dissection of the complex interactions of
þ
þ
TIPs with MTs over the past few years (
Brouhard et al., 2008; Dixit et al., 2009;
Honnappa et al., 2009; Maurer, Bieling, Cope, Hoenger, & Surrey, 2011; Zimniak,
Stengl,Mechtler, &Westermann, 2009
). Parameters that can be easilymeasured using
fluorescent
TIPs specifically recognize the
ends of growingMTs), the dwell time (or residency time) of the
þ
TIPs as markers are MT growth rate (
þ
TIP, and even, when
using single-molecule conditions, the association rate (
K
on
)(
Bieling et al., 2008;
Komarova et al., 2009; Montenegro Gouveia et al., 2010
). A major advantage of
the fluorescence microscopy-based MT
in vitro
system over that of traditional bio-
chemical assays is that it is possible to visualize and measure the interaction of the
proteins on specific sites of the MT, while classical assays measure ensemble MT be-
havior. By altering biochemical conditions, one can address specific questions such as
the effect of ionic strength on
þ
TIP binding (
Buey et al., 2011
) and investigate the
interplay of additional protein factors at the plus end (
Montenegro Gouveia et al.,
2010
). The MT
in vitro
assay is fairly simple to carry out. One complexity lies in
the reconstitution of
þ
the individual components which sometimes requires
