Biology Reference
In-Depth Information
Ehrhardt & Shaw, 2006; Pastuglia & Bouchez, 2007; Wasteneys & Ambrose, 2009
).
Although the dominant life phase of most plants lacks a discrete microtubule organizer,
gamma-tubulin and the associated proteins of the core ring complex are present in
plants (
Nakamura, Ehrhardt, &Hashimoto, 2010
). Live-cell imaging studies using fluo-
rescent transgenes in
A. thaliana
have revealed that gamma-tubulin localizes transiently
to the cell cortex and nucleates new polymers adjacent to the plasma membrane (
Murata
et al., 2005
). At the resolution of light microscopy, the gamma-tubulin nucleation com-
plexes localize primarily on existingmicrotubule lattices, and new polymers are formed
parallelorat40
angles to existing microtubules (
Murata et al., 2005
).
Microtubule organization emerges from the combination of the dynamic behav-
ior of microtubules, the action of various MAPs, and the geometry of cell (
Ehrhardt,
2008; Ehrhardt & Shaw, 2006; Pastuglia & Bouchez, 2007; Wasteneys & Ambrose,
2009
). Plant genomes encode MAPs similar to those found in other organisms such
as kinesins, katanins, MAP65's, EB1's, XMAP215, and CLASP (
Ambrose, Shoji,
Kotzer, Pighin, & Wasteneys, 2007; Burk, Liu, Zhong, Morrison, & Ye, 2001;
Chan, Calder, Doonan, & Lloyd, 2003; Hamada, 2007; Jiang & Sonobe, 1993;
Kirik et al., 2007; Reddy & Day, 2001; Whittington et al., 2001
). The function of
sever
al of these proteins is similar to the animal or yeast counterpart. For example,
the
A. thaliana
catalytic subunit of katanin severs microtubules from cortical nucle-
ation sites, releasing the polymers for hybrid treadmilling (
Nakamura et al., 2010
).
However, other plant MAPs appear to have acquired plant-specific functions. Mu-
tation of two redundant
A. thaliana
MAP65 genes leads to plant growth defects
but not dramatically disorganized microtubule arrays (
Lucas et al., 2011
). These data
suggest that these proteins have additional functions beyond array organization
(
Lucas et al., 2011
). Additionally, there are MAPs specific to the plant lineage, such
as the MAP70 family (
Korolev, Buschmann, Doonan, & Lloyd, 2007
). In plants,
there is much microtubule biology yet to be discovered and live-cell imaging of fluo-
rescent protein fusions will help researchers uncover this science.
This review focuses on techniques used to image microtubules and MAPs in liv-
ing cells of
A. thaliana
. The first step is to build transgenic plants that express a fluo-
rescent protein fused to a protein of interest in the appropriate genetic background.
After transgenic lines are generated, live-cell image data are collected with confocal
microscopy and subsequently analyzed using different software packages.
15.1
PROTOCOLS
15.1.1
Building transgenic A. thaliana
Transgenes can be introduced into the
A. thaliana
genome quickly and easily using
the bacterium
Agrobacterium tumefaciens
(
Clough & Bent, 1998
).
A. tumefaciens
is
a soil-dwelling plant pathogen that can stably transfer a segment of plasmid DNA
into the nuclear genome of several dicotyledonous plant species (
Lee & Gelvin,
2008; Pitzschke &Hirt, 2010
). There are multiple ways to transform
A. thaliana
with
