C H A P T E RE I G H T E E N
Synthetic Physiology: Strategies for
Adapting Tools from Nature for
Genetically Targeted Control of Fast
Brian Y. Chow,1,2Amy S. Chuong,1Nathan C. Klapoetke,1and
Edward S. Boyden
2. Molecular Design and Construction
3. Transduction of Microbial Opsins into Cells for
4. Physiological Assays
The life and operation of cells involve many physiological processes that take
place over fast timescales of milliseconds to minutes. Genetically encoded
technologies for driving or suppressing specific fast physiological processes
in intact cells, perhaps embedded within intact tissues in living organisms, are
critical for the ability to understand how these physiological processes contrib-
ute to emergent cellular and organismal functions and behaviors. Such “syn-
thetic physiology” tools are often incredibly complex molecular machines, in
part because they must operate at high speeds, without causing side effects.
We here explore how synthetic physiology molecules can be identified and
deployed in cells, and how the physiology of these molecules in cellular con-
texts can be assessed and optimized. For concreteness, we discuss these
Methods in Enzymology, Volume 497
ISSN 0076-6879, DOI: 10.1016/B978-0-12-385075-1.00018-4
#2011 Elsevier Inc.
All rights reserved.
Synthetic Neurobiology Group, The Media Laboratory and McGovern Institute, Departments of Biological
Engineering and Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge,
1Authors contributed equally
2Future location: Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania,
methods in the context of the “optogenetic” light-gated ion channels and
pumps that we have developed over the past few years as synthetic physiology
tools and widely disseminated for use in neuroscience for probing the role of
specific brain cell types in neural computations, behaviors, and pathologies. We
anticipate that some of the insights revealed here may be of general value for
the field of synthetic physiology, as they raise issues that will be of importance
for the development and use of high-performance, high-speed, side-effect free
physiological control tools in heterologous expression systems.
The life and operation of cells involve many physiological processes
that take place over fast timescales of milliseconds to minutes. These
physiological changes include variations in cell membrane potential and
cellular ionic composition; changes in protein conformation; posttransla-
tional modification, localization, and interaction; and other biochemical
and mechanical processes, all occurring at length scales ranging from nan-
ometers to meters. Technologies for driving or suppressing specific fast
physiological processes in intact cells, perhaps embedded within intact
tissues in living organisms, are critical for the ability to understand how
those physiological processes contribute to emergent cellular andorganismal
functions and behaviors. For example, the ability to drive a specific physio-
logical process can reveal precisely which functions that process is sufficient
to initiate or sustain, whereas the ability to suppress a specific physiological
process can reveal the set of functions for which the process is necessary.
Such precision physiological control technologies may, of course, also serve
therapeutic purposes if they offer the ability to remedy a pathway thrown
into disarray in a disease context, ideally while leaving other pathways
A diversity of molecular tools have been developed that allow the
precision control of physiological processes—including high-specificity
pharmacological compounds, caged chemicals that can be activated by
pulses of light, and tools whose physiological impact is unleashed by admin-
istration of heat or radiofrequency energy. This ongoing effort has led to a
number of physiological control tools that are partly or entirely genetically
encoded, and therefore easy to use in genetic model organisms in conjunc-
tion with commonly available transgenic strategies, for example, viruses for
delivery to specific mammalian cells embedded within intact organ systems.
One might call the set of capabilities opened up by these tools “synthetic
physiology,” because these tools enable a synthetic approach to studying
physiological pathways, with an emphasis on perturbation of specific path-
ways, to see what their influence is on other pathways. Many of the
Brian Y. Chow et al.
synthetic physiology tools in widespread use have come to be known as
“optogenetic,” because they enable specific physiological processes to be
controlled by light, and thus enable temporally and spatially precise control
of physiology with microscopes, lasers, and other common laboratory
optical equipment, often without the need for exogenous chemical delivery
(helpful for use in vivo). Such light-driven tools, or prototypes of tools, exist
for applications including driving of protein–protein interactions (Kennedy
et al., 2010; Levskaya et al., 2009; Yazawa et al., 2009), enzyme activity (Wu
et al., 2009), intracellular signaling (Schroder-Lang et al., 2007), and many
other fast changes (Moglich and Moffat, 2010). A widely used set of
optogenetic tools are the microbial rhodopsins, molecules that respond to
light by translocating ions from one side of the plasma membrane to the
other, thus enabling electrical activation or silencing of electrically excitable
cells such as neurons, in response to pulses of light (Boyden et al., 2005;
Chow et al., 2010; Gradinaru et al., 2008, 2010; Han and Boyden, 2007;
Zhang et al., 2007a). For example, channelrhodopsins, microbial opsins
from algae, admit cations into cells in response to light, depolarizing the
cells; halorhodopsins, opsins from archaea, pump in chloride in response to
light, resulting in cellular hyperpolarization; archaerhodopsins and bacter-
iorhodopsins, also isolated from archaea (and other kingdoms), pump out
protons, also resulting in cellular hyperpolarization. In the mammalian
nervous system, these molecules do not require any exogenous chemical
supplementation for their operation, and thus can be treated as fully geneti-
cally encoded. The hyperpolarization opsins are used to enable optical
silencing of genetically targeted neurons in order to see what neural dynam-
ics, behaviors, and pathologies they are necessary for, whereas the depolar-
izing opsins are used to drive neural activity in genetically targeted neurons,
to assess which downstream neural computations and behaviors causally
result. Both sets of tools are in widespread use for investigating the roles that
specific cells play within the nervous systems of species ranging from
Caenorhabditis elegans to nonhuman primate (see the following references
for some early papers in the field; Adamantidis et al., 2007; Alilain et al.,
2008; Aravanis et al., 2007; Arenkiel et al., 2007; Atasoy et al., 2008; Bi et al.,
2006; Douglass et al., 2008; Farah et al., 2007; Han et al., 2009a; Huber et al.,
2008; Ishizuka et al., 2006; Lagali et al., 2008; Li et al., 2005; Liewald et al.,
2008; Mahoney et al., 2008; Nagel et al., 2005; Petreanu et al., 2007; Schroll
et al., 2006; Toni et al., 2008; Wang et al., 2007; Zhang and Oertner, 2007;
Zhang et al., 2007b, 2008).
Although one of the goals of synthetic biology is to be able to regard
such tools as “black box parts” (Canton et al., 2008; Carr and Church, 2009;
Endy and Brent, 2001), whose internal workings can be hidden beneath an
abstraction layer, the genetically encoded tools in use for synthetic physiol-
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