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the plasma membrane, monomer and protomer densities are quantified and the
total receptor density can be extrapolated. Receptor internalization can also be
observed as a decrease in fluorescently labeled protein density at the plasma
membrane for fixed samples. For live-cell studies, photobleaching will also
contribute to a decrease in measured density as a function of time.
Therefore, SpIDA allows for the simultaneous quantification of oligomerization
and internalization. In addition, as shown in Fig. 1.1 , data obtained with SpIDA
can readily be used for pharmacological analysis with the Hill-Langmuir binding
isotherm model.
1.4 DISCUSSION
Overall, the image analysis described in this chapter allows for the quantification of
RTK membrane density, oligomeric state, and internalization. Of general impor-
tance, SpIDA is versatile and is not restricted to cell surface proteins. In reality,
as this is a postacquisition image analysis method, it may be applied to any optical
section obtained by CLSM from fixed or live preparations.
Furthermore, it is compatible with the analysis of both fluorescent-tagged
proteins and native proteins in primary cultured cells or tissues. However, some
limitations exist as with any image quantification method. In its present form,
SpIDA can only resolve monochromatic images, which limits its applicability for
complex heteromerization schemes. On the other hand, SpIDA can be applied simul-
taneously to images acquired at different wavelengths (i.e., different fluorophores)
from the same preparation and is, therefore, compatible with multiplexing. In theory,
higher-order arrangements can be studied with SpIDA (tetramers or pentamers), but
the limitation here is the complexity of possible configurations because of either non-
detected proteins (for antibody labeling) or nonfluorescent proteins that can be pre-
sent. Finally, it is important to keep in mind that in contrast to RET, SpIDA is not
providing information about direct protein-protein interactions but only about olig-
omerization within a common complex, which can be dependent upon other inter-
mediates such as adaptor proteins. While this may be a limitation for those
seeking to identify direct protein-protein interactions, it offers the advantage of en-
abling detection of regular, yet indirect, associations between proteins with complex
macromolecular arrangements.
A major advantage of SpIDA over RET techniques is that it allows quantification
of mixtures of oligomeric arrangements (e.g., provide numbers of both monomer and
dimer densities within a mixed distribution). In summary, SpIDA is a low-cost image
analysis method enabling quantitative assessment of oligomerization that is compat-
ible with most CLSM systems commonly used in biomedical research laboratories.
Its compatibility with CLSM and conventional immunocytochemical methods opens
the door to studies of oligomerization in native tissue, which, to date, were largely
restricted to expression systems.
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