Biology Reference
In-Depth Information
genetically encoded and targeted Ca
2
þ
reporters, especially those based on
apoaequorin. We also list examples of the organisms, tissues, and cells that
have been transfected with apoaequorin or an apoaequorin-BRET complex, as
well as of the organelles and subcellular domains that have been specifically
targeted with these bioluminescent Ca
2
þ
reporters. In addition, we summarize
the various techniques used to load the apoaequorin cofactor, coelenterazine,
and its analogs into cells, tissues, and intact organisms, and we describe recent
advances in the detection and imaging technologies that are currently being
used to measure and visualize the luminescence generated by the aequorin-
Ca
2
þ
reaction within these various cytoplasmic domains and subcellular
compartments.
I. Introduction
One of the most significant recent developments in the Ca
2
þ
signaling field has
been the general acceptance of the wide-spread heterogeneity of Ca
2
þ
activity
within individual cells; not only at rest, but also most importantly, during stimula-
tion (
Berridge, 2009; Rizzuto and Pozzan, 2006; Rutter et al., 2006; Whitaker,
2008
). This has led to the concept of dynamic subcellular ''Ca
2
þ
microdomains.''
As suggested by
Rizzuto and Pozzan (2006)
, this term (especially with regard to its
spatial dimensions) has several di
erent meanings depending on one's area of
interest. In this chapter, however, like Rizzuto and Pozzan, we use the term in a
general way to describe Ca
2
þ
dynamics that do not involve the entire cell cyto-
plasm, but that remain localized to a specific cytoplasmic domain, or occur within
a particular organelle or its periorganellar environment. Thus, one of the current
challenges researchers are facing in the field of Ca
2
þ
imaging is that of resolving
changes in [Ca
2
þ
] within, and between, various subcellular microdomains.
An e
V
ective strategy to address this challenge that is common to both fluorescence-
and luminescence-based imaging techniques is to exclusively visualize Ca
2
þ
dynamics in specific microdomains using genetically encoded and targeted Ca
2
þ
reporters (GET-CRs). These come in two general forms, fluorescent GET-CRs
and bioluminescent GET-CRs, respectively. At the other end of the size spectrum
is the exciting prospect of imaging Ca
2
þ
signals derived from GET-CRs within
freely moving, large, organisms, for example, adult mice (
Rogers
V
et al., 2007
).
erent set of technical challenges to researchers in the Ca
2
þ
This presents a di
V
imaging field.
Fluorescent GET-CRs include the camgaroos (
Baird et al., 1999; Griesbeck
et al.,2001
), G-CaMPs (
Nakai et al., 2001; Ohkura et al., 2005
), pericams (
Nagai
et al., 2001
), case-sensors (
Souslova et al., 2007
), grafted EF-hands (
Zou et al.,
2007
), and cameleon-types (
Miyawaki et al.,1997;Ishiiet al.,2006
;
Tsuruwaka
et al., 2007
; and reviewed by
Zorov et al., 2004; McCombs and Palmer, 2008
).
Bioluminescent GET-CRs include single protein entities such as aequorin
