Biology Reference
In-Depth Information
10.2
IMAGING GTP CAPS AND GTP ISLANDS USING
CENTROSOME-BASED MICROTUBULE ASSEMBLY OR
ENDOGENOUS MICROTUBULE ELONGATION IN
PERMEABILIZED CELLS
After microtubule depolymerization, centrosomes retain their capability to nucleate
new microtubules in the presence of free GTP-tubulin once cells are permeabilized
and cytosol proteins are extracted. This procedure was adapted from that used by
Brinkley et al. (1981)
. Here, permeabilized cells are used to nucleate oriented micro-
tubules and to control their regime of growth. A similar procedure can be undertaken
using permeabilized cells without prior microtubule depolymerization to elongate
microtubules in the pericellular region and observe their GTP-bound tubulin
domains.
10.2.1
Cell plating, culture, and treatment
10.2.1.1
Prior to assembly of centrosome-nucleated microtubules
HeLa or RPE-1 cells are plated on glass coverslips coated with gelatin and cultured
until they reach 50% confluence. Cellular microtubules are depolymerized in two
steps: first cells are incubated at 37
C for 1 h 30 min in 10
m
M nocodazole and then
put on ice for 2 h in the presence of nocodazole. The drug is then washed out by three
rinses in ice-cold culture medium and immediately extracted on ice using PEM sup-
plemented with 0.1% Triton X-100 (three extractions of 1 min). Cells are rinsed
twice on ice with PEM to remove Triton.
10.2.1.2
Prior to extracellular microtubule elongation
Cells plated on gelatin and cultured as described above are permeabilized in the per-
meabilization buffer and rinsed twice in PEM-G without Triton.
10.2.2
Microtubule growth
For both assays, microtubule growth is performed using purified porcine brain tubu-
lin (see
Section 3.2.1
) diluted at 0.25 g/L in PEM supplemented with 1 mMGTP. To
elongate extracellularly endogenous microtubules, the addition of 200-500
m
M
adenosine triphosphate (ATP) greatly enhances the number of polymerization-
competent microtubules (
Infante, Stein, Zhai, Borisy, & Gundersen, 2000
). For
centrosome-nucleated microtubules, extracted cells are kept on ice before polymer-
ization; tubulin assembly starts upon shifting the temperature to 37
C.
In both assays, assembly is performed for 5-30 min. At the end of the assembly
period, soluble tubulin is removed by gently washing coverslips with warm PEM-G
and optionally with 1
m
M Taxol prior to MB11 labeling, fixation, and colabeling if
required. GTP-tubulin labeling is performed as described previously.
