Biology Reference
In-Depth Information
oil immersion objective. The end of an axoneme that possesses more, faster growing,
and longer microtubules than the opposite end is designated as the plus-end. The
real-time, 10-min videos are analyzed using Real Time Measurement (RTM II) soft-
ware, and the data are collected using IgorPro (MediaCybernetics, Bethesda, MD,
USA). Microtubules are considered to be growing if they increase in length by
>
0.3
m
m at a rate of
0.3
m
m/min. Shortening events are identified by a
1-
m
mlength
>
change at a rate of
2
m
m/min. We calculate the catastrophe frequency by dividing the
total number of catastrophes (transitions to shortening) by the time the microtubules are
growing and in the attenuated state. The rescue (transition from shortening to growing)
frequency is calculated as the total number of rescue events divided by the total time
shortening. Dynamicity is calculated as the sum of the total growth length and the total
shortening length divided by the total time.
Table 12.1
shows the effects of Gs
a
QL and
Gs
a
-derived peptide (270-284) on microtubule dynamics.
12.1.4
Cellular consequences of Gs
a
interaction with
tubulin/microtubules
Several forms of evidence obtained from
in vitro
binding assays clearly show
that tubulin directly interacts with Gs
a
. In cells, this interaction and function of
tubulin-Gs
a
complex also occur (
Yu et al., 2009
). In PC12 cells, activated Gs
a
inter-
actswith tubulin and increasesmicrotubule dynamic instability. In addition, the ability
of Gs
a
to induce dynamic instability results in neurite-like process outgrowth in PC12
cells. A flowchart illustrating the cellular functional assays is shown in
Fig. 12.5
.
12.1.4.1
Binding studies in cells
In order to show that Gs
a
and tubulin are complexed in cells, HA-tagged Gs
a
is
expressed in PC12 cells. The Gs
a
is then activated by either adding cholera toxin
(which inactivates GTPase of Gs
a
) or mutational activation (Q227L). The Gs
a
-
tubulin complexes are determined by coimmunoprecipitation using anti-HA
antibody to pull down HA-tagged Gs
a
and anti-tubulin antibody to detect the tubulin
presence in the complex. Immunofluorescence techniques also reveal colocalization
of Gs
a
and tubulin in PC12 cells after cholera toxin activation (
Yu et al., 2009
).
12.1.4.1.1
Immunoprecipitation
PC12 cells infected with denoviruses coding for GFP, Ad/GFP, Ad/Gs
a
, or Ad/
Gs
a
Q227L
are cultured for 40 h and then washed twice in PBS. Cells are lysed in
500
m
l of lysis buffer on ice for 30 min. The lysate is collected and cleared by
centrifuging at 12,000
g
for 20 min at 4
C. After adjusting protein concentration
to equal amounts for each sample, the supernatant (450
m
l) is transferred to 1.5-ml
microcentrifuge tubes and incubated with agarose beads coated with antimouse IgG
for 1 h at 4
C with continuous gentle inversion. The agarose beads are pulled down
by centrifuging at room temperature and discarded. The lysate is then incubated with
5
m
l of monoclonal antibody against HA for 20 h at 4
C, and then the antibody/ly-
sate mixture is incubated with agarose beads coated with antimouse IgG for 2 h at
