Biology Reference
In-Depth Information
rab5 S34N DNA construct, we were able to observe that, for some GPCRs (e.g.,
D1R, D2R, and NK-1R), internalization was instrumental to obtain a maximum
EGFR-GFP dimer density, whereas in other cases (AT1aR and beta2AR), results
indicated that internalization was not necessary for observing full transactivation
as measured by RTK dimerization.
1.2.2
Application to RTK transactivation in a native system: neurons
expressing dopamine receptors
One of the greatest advantages of SpIDAhas been demonstrated using endogenous sys-
tems (
Doyon et al., 2011; Sergeev, Godin, et al., 2012; Swift et al., 2011
). For exemple,
in one of these systems SpIDA was used to show that the brain-derived neurotrophic
factor (BDNF) receptor TrkB is transactivated by dopamine receptors subtypes
1 and 2 (D1R and D2R, respectively). This was acheived using primary culture of stria-
tal neurons (
Swift et al., 2011
) obtained from transgenic mice expressing fluorescent
reporter genes tdTomato, under the control of the D1R gene, and EGFP, under the con-
trol of the D2R gene (
Shuen, Chen, Gloss, & Calakos, 2008; Zhang, Burke, Calakos,
Beaulieu, & Vaucher, 2010
). Expression of these reporter genes allowed locating
two different populations of neurons expressing predominantly either D1R or D2R.
TrkB dimerization was measured by staining cell surface receptors with a pri-
mary antibody on nonpermeabilized neurons and detected using secondary anti-
bodies conjugated to an Alexa Fluor 647 moiety. The SpIDA method allowed for
quantification of TrkB oligomeric states in response to either BDNF (direct activa-
tion) or the nonselective dopamine receptor agonist apomorphine (transactivation) in
specific neurons (D1 in red or D2 in green) (
Fig. 1.2
).
This result demonstrates the usefulness of this image analysis method to quantify
RTK transactivation by GPCR in situ. Given the wide availability of antibodies
for many targets of interest and the quality of secondary fluorescent antibody
conjugates available commercially, SpIDA can be used to investigate a wide variety
of biological systems involving protein homooligomerization in native tissue with
endogenous expression.
1.3
PROCEDURE FOR SpIDA
Here, we describe the steps for detector calibration, image acquisition, and SpIDA
analysis using CLSM images acquired from fluorescently labeled cell or tissue sam-
ples. The overall process is straightforward for any user trained in biological fluo-
rescence imaging (
Fig. 1.3
).
1.3.1
Material and apparatus
SpIDA was validated using images obtained by CLSM, but, in theory, SpIDA can
also be used with total internal reflection fluorescence (TIRF) microscopy. To appro-
priately perform SpIDA, one needs to determine the suitable parameters to work in
