Biology Reference
In-Depth Information
Note I
: Preparations of benomyl plates can vary. It is essential to include a
nonmutated strain and, if possible, strains with known sensitivity or resistance as
controls.
Note II
: To prepare benomyl plates, autoclave the YPD and agar with a stir bar,
cool to
65
C, and then add 10 mg/ml benomyl in DMSO dropwise while
stirring.
22.5
DIRECT ANALYSIS OF MICROTUBULE DYNAMICS IN VIVO
Live-cell imaging of cells expressing GFP-tagged
-tubulin allows individual astral
microtubules to be tracked at high temporal and spatial resolution. This approach
determines how microtubule behavior and parameters of dynamic instability are al-
tered by specific tubulin mutations.
a
22.5.1
Yeast strains for
microtubule dynamics analysis
Yeast strains are typically engineered to express an exogenous copy of
in vivo
-tubulin
fused to GFP under the
TUB1
promoter (
pTUB1-GFP-TUB1
) because GFP-tubulin
alone does not support cell viability. Two commonly used integrating plasmids,
pAFS125 and pMG3, are described in
Table 22.1
. To integrate
GFP-TUB1
, trans-
form cells with
a
g linearized plasmid and plate on appropriate SC-Ura or SC-Leu
media. Identify positive transformants by visually inspecting for GFP expression.
Generate clonal strains of the positive transformants by two rounds of single-colony
isolation on selection media.
1
m
Note
: If strains destined for
in vivo
microtubule analysis are not auxotrophic for
uracil and leucine, they may be made so by disrupting the
URA3
or
LEU2
gene
using “marker swap” cassettes (
Cross, 1997
).
22.5.2
Maintaining comparable
expression levels
Linearized plasmids can potentially integrate as tandem repeats, introducing multi-
ple copies of
pTUB1-GFP-TUB1
. Anecdotal evidence also suggests that increased
GFP-Tub1 is correlated with increased microtubule stability. Thus, it is advised to
ensure that strains used for the
in vivo
analysis of microtubule dynamics have com-
parable levels of GFP-Tub1.
A common and practical method of selecting strains with similar
GFP-TUB1
expression is to compare
GFP-TUB1
10 transformants after streaking for single colonies. Pre-
pare the strains for microscopy as described in the following section. With identical
excitation intensity and exposure times, compare the relative fluorescence signal
from individual astral microtubules among the strains. Typically,
50% will have
microtubules with similar fluorescence intensity, while a minority display noticeably
increased intensity. Select strains representative of the group with the lower basal
level of GFP-Tub1 fluorescence, which should be comparable to other strains
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