Biology Reference
In-Depth Information
and onlymicrotubuleswhose entire length remainedwithin focus should bemeasured.
The length of the microtubule throughout the time lapse is typically measured 2-3
times and the average graphed over time to construct a “lifetime history plot.”
Using the lifetime history plots, microtubule polymerization and depolymeriza-
tion events are typically identified as four or more consecutive time points spanning
>
mwith a linear regression fit of
R
2
0.85. Attenuations, or pauses, are defined
as four or more time points during which length change did not exceed
0.5
m
m. Be-
cause the criteria for specific events generally require a sustained behavior, that is,
multiple consecutive time points, brief periods for some microtubules will remain un-
classified. Catastrophes are scored as transitions frompolymerization or paused states
into depolymerization, and rescues are scored as transitions from depolymerization
into paused or polymerization states. To calculate catastrophe frequency, the number
of catastrophe events is divided by the total observable lifetime spent in polymeriza-
tion and paused states for all analyzed microtubules. Similarly, to determine rescue
frequency, the number of rescues is divided by total time spent in depolymerization.
0.2
m
22.6
LOCALIZATION OF MICROTUBULE-ASSOCIATED
PROTEINS IN VIVO
Budding yeast utilizes a conserved set of microtubule-associated proteins to regulate
microtubule behavior during processes such as spindle positioning and spindle elon-
gation. Here, we outline the use of quantitative fluorescence microscopy to determine
whether specific mutations alter the interactions of microtubule-associated proteins
with microtubules. The technique involves tagging the endogenous copy of a micro-
tubule regulator with yellow fluorescent protein (YFP) and monitoring cyan fluores-
cent protein (CFP)-Tub1-labeled microtubules in the same cell.
22.6.1
Yeast strains for localization of microtubule-associated
proteins
Engineer cells to express CFP-Tub1. A useful plasmid for this purpose is pMG130,
which has the CFP variant Cerulean fused to
TUB1
(
Table 22.1
). Digest, integrate,
and screen as described for
GFP-TUB1
above.
Fuse the protein of interest to YFP to allow colocalization with CFP-labeled
microtubules. The simplest method utilizes fragment-mediated homologous recom-
bination. A fragment containing YFP and a selection marker is amplified by PCR
from a plasmid template. Many plasmids useful for this are commonly available
(
Janke et al., 2004
). The forward and reverse PCR primers are designed to contain
>
40 bases of homology just prior to and after the stop codon, respectively, of the tar-
geted gene. Potential transformants should be screened for YFP fluorescence and
isolated as described for
GFP-TUB1
. Successful fusion with the desired gene can
be verified by PCR using primers that anneal inside the gene of interest and YFP.
Note
: The functionality of fusion proteins is frequently increased by introducing a
soluble flexible linker of 6-8 amino acids between the two proteins.
