II. Overview of Optogenetic Systems
and Their Applications
As mentioned above, optogenetics provides a
great way to probe complex biological phenomena. Specifically, the implementation of lightresponsive proteins has the potential to allow
deciphering the dynamics of cellular processes
with great detail as a result of its striking spatiotemporal resolution (Fig. 12.1) and minimal
invasiveness. In that context, different optogenetic systems have been reported to control
gene expression, DNA recombination, subcellular protein localization, protein activity, cell
morphology, cell signaling, and protein degradation (Zhang and Cui 2015; Shcherbakova
et al. 2015).
A. The Early Days of Optogenetics
A clear example of a modular system in the
early days of optogenetics can be tracked
down to 2005 when the light-sensitive microbial protein Channelrhodopsin-2 (ChR2) was
used to modulate mammalian neuronal activity
(Boyden et al. 2005). ChR2 is a cation channel
from the green algae Chlamydomonas reinhardtii, containing seven a-helix transmembrane domains and passing to an open state
by isomerization with the retinal chromophore
in response to blue light (~460 nm) (Nagel et al.
2003). Using a lentiviral transfection system,
ChR2 was expressed in neurons achieving the
control of synaptic transmission with an outstanding temporal resolution in the order of
milliseconds (Boyden et al. 2005). After this
successful optogenetic application, more lightsensitive ion channels (type-I opsins) were discovered and tested to excite/inhibit the generation of action potentials. For instance, archaeal
halorhodopsin has been utilized to repress neuronal firing by yellow light-induced transport of
chloride anions (Han and Boyden 2007). The
use of photoactivated ion channels in the neuroscience field led to great knowledge advances
in neuronal connectivity, being also employed
in the study of cell signaling processes in other
relevant mammalian tissues. In this sense,
ChR2 has been used to control Ca
2+ fluxes in
muscular cells, where this ion is one of the most
important secondary messengers in signal
transduction processes (Carafoli and Krebs
2016; Sebille et al. 2017; Mao et al. 2019). Similarly, the light-induced opening of ChR2 permitted to study the maturation and contraction
capacity of muscle cell lines, overcoming the
problems of traditional electric stimulation
(Bruegmann et al. 2010; Beiert et al. 2014). On
the other hand, type II opsins have been used to
manipulate cell signaling at the plasma membrane level, since they are G-protein-coupled
receptors (Mu ¨hlha ¨user et al. 2017). Depending
on the nature of the G protein, the receptor is
associated with different signaling pathways,
being able to activate or inhibit signal propagation. In that context, blue light has been used to
increase the concentration of inositol triphosphate (IP3), by activation of phospholipase C
(PLC) in HeLa cells expressing melanopsin
(Melyan et al. 2005).
However, the beginnings of optogenetics as
the direct means of control of any biological
process by light dates back to 2002. In that
year, a plant photoreceptor was inadvertently
used to implement a light-inducible expression
system in the budding yeast Saccharomyces
cerevisiae. Indeed, such experiments were not
intended to create an optogenetic device, but
instead to explore the interaction of two particular plant components. In Arabidopsis thaliana, phytochrome B (PhyB) senses red light
(~ 660 nm) through its chromophore phytochromobilin, passing to the active state and
binding the Protein Interacting Factor (PIF3
or PIF6) (Fig. 12.2a), which in turn activates
the expression of multiple genes (Fairchild
and Quail 1998; Quail 2010). Importantly, phytochrome B returns to its basal inactive state by
far-red illumination (~740 nm), configuring a
system with on and off states, and generating
the first description of a light-controlled gene
expression system (Shimizu-Sato et al. 2002).
In this seminal work, the light-inducible
system was based on the architecture of a
yeast two-hybrid assay, where GAL4 DNAbinding domain (DBD) and GAL4 transactivation domain (AD) were fused to PhyB and PIF3,
respectively (Shimizu-Sato et al. 2002). Thus, a
12 Exploiting Fungal Photobiology as a Source of Novel Bio-blocks for Optogenetic Systems
299
and Their Applications
As mentioned above, optogenetics provides a
great way to probe complex biological phenomena. Specifically, the implementation of lightresponsive proteins has the potential to allow
deciphering the dynamics of cellular processes
with great detail as a result of its striking spatiotemporal resolution (Fig. 12.1) and minimal
invasiveness. In that context, different optogenetic systems have been reported to control
gene expression, DNA recombination, subcellular protein localization, protein activity, cell
morphology, cell signaling, and protein degradation (Zhang and Cui 2015; Shcherbakova
et al. 2015).
A. The Early Days of Optogenetics
A clear example of a modular system in the
early days of optogenetics can be tracked
down to 2005 when the light-sensitive microbial protein Channelrhodopsin-2 (ChR2) was
used to modulate mammalian neuronal activity
(Boyden et al. 2005). ChR2 is a cation channel
from the green algae Chlamydomonas reinhardtii, containing seven a-helix transmembrane domains and passing to an open state
by isomerization with the retinal chromophore
in response to blue light (~460 nm) (Nagel et al.
2003). Using a lentiviral transfection system,
ChR2 was expressed in neurons achieving the
control of synaptic transmission with an outstanding temporal resolution in the order of
milliseconds (Boyden et al. 2005). After this
successful optogenetic application, more lightsensitive ion channels (type-I opsins) were discovered and tested to excite/inhibit the generation of action potentials. For instance, archaeal
halorhodopsin has been utilized to repress neuronal firing by yellow light-induced transport of
chloride anions (Han and Boyden 2007). The
use of photoactivated ion channels in the neuroscience field led to great knowledge advances
in neuronal connectivity, being also employed
in the study of cell signaling processes in other
relevant mammalian tissues. In this sense,
ChR2 has been used to control Ca
2+ fluxes in
muscular cells, where this ion is one of the most
important secondary messengers in signal
transduction processes (Carafoli and Krebs
2016; Sebille et al. 2017; Mao et al. 2019). Similarly, the light-induced opening of ChR2 permitted to study the maturation and contraction
capacity of muscle cell lines, overcoming the
problems of traditional electric stimulation
(Bruegmann et al. 2010; Beiert et al. 2014). On
the other hand, type II opsins have been used to
manipulate cell signaling at the plasma membrane level, since they are G-protein-coupled
receptors (Mu ¨hlha ¨user et al. 2017). Depending
on the nature of the G protein, the receptor is
associated with different signaling pathways,
being able to activate or inhibit signal propagation. In that context, blue light has been used to
increase the concentration of inositol triphosphate (IP3), by activation of phospholipase C
(PLC) in HeLa cells expressing melanopsin
(Melyan et al. 2005).
However, the beginnings of optogenetics as
the direct means of control of any biological
process by light dates back to 2002. In that
year, a plant photoreceptor was inadvertently
used to implement a light-inducible expression
system in the budding yeast Saccharomyces
cerevisiae. Indeed, such experiments were not
intended to create an optogenetic device, but
instead to explore the interaction of two particular plant components. In Arabidopsis thaliana, phytochrome B (PhyB) senses red light
(~ 660 nm) through its chromophore phytochromobilin, passing to the active state and
binding the Protein Interacting Factor (PIF3
or PIF6) (Fig. 12.2a), which in turn activates
the expression of multiple genes (Fairchild
and Quail 1998; Quail 2010). Importantly, phytochrome B returns to its basal inactive state by
far-red illumination (~740 nm), configuring a
system with on and off states, and generating
the first description of a light-controlled gene
expression system (Shimizu-Sato et al. 2002).
In this seminal work, the light-inducible
system was based on the architecture of a
yeast two-hybrid assay, where GAL4 DNAbinding domain (DBD) and GAL4 transactivation domain (AD) were fused to PhyB and PIF3,
respectively (Shimizu-Sato et al. 2002). Thus, a
12 Exploiting Fungal Photobiology as a Source of Novel Bio-blocks for Optogenetic Systems
299