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the use of constantly improving light-inducible dimerization/
oligomerization systems (i.e., optogenetic tools) becomes more
and more popular. Using light as the sole activator allows unprecedented spatiotemporal resolution with minimal side effects. This
allows the intervention of signal transduction in a most sophisticated manner [2, 3].
One optogenetic tool that has been employed in the activation
of various kinases is the blue light-responsive cryptochrome 2/
cryptochrome-interacting basic-helix-loop-helix 1 (CRY2/CIB1)
system [4–6]. Cryptochromes are flavoprotein photoreceptors
found in bacteria, higher plants, and animals. They were first identified in Arabidopsis thaliana, where they regulate growth and
developmental processes [7]. Cryptochromes contain a C-terminal
cryptochrome extension and an N-terminal photolyase homology
region (PHR) that noncovalently binds flavin adenine dinucleotide
(FAD). Irradiation with blue light of wavelengths ranging from
390 nm to 480 nm leads to FAD reduction, which converts cryptochrome into its biological active conformation [8]. The activated
Arabidopsis thaliana CRY2 can interact with CIB1, which is
involved in the mechanism of floral initiation. Besides the interaction with CIB1, photoexcited CRY2 can also form homomers.
Light-activated CRY2 forms homo- and heteromers within seconds
and dissociates in the dark within minutes in a repeatable manner.
Optogenetic systems have been generated based on the lightinduced oligomerization of the PHR region of CRY2 (aa 1—489)
and the N-terminus of CIB1 (CIBN; aa 1—170). The characteristics
of the CRY2-based optogenetic are especially suited for the control
of signaling events in mammalian cells [3, 9] (Fig. 1). For instance,
blue light-induced clustering of CRY2PHR fused to the protein
kinase RAF as well as the recruitment of chimeric RAF to membrane-bound CIBN activates the mitogen-activated protein kinase
cascade (MAPK) signaling pathway [4, 5, 10]. To gain control of
the PI3K/AKT pathway, CRY2PHR either fused to the interSRC-homology 2 (iSH2) domain of the regulatory p85α subunit
of PI3K or to the protein kinase AKT were recruited to a membrane-anchored CIBN to activate signaling upon blue light irradiation [6, 11]. Due to its high relevance constant efforts to
further improve this system are undertaken (e.g., enhanced oligomerization) [12, 13].
In this chapter, we describe a detailed approach of how to use the
CRY2PHR/CIBN optogenetic tool to control AKT1 (optoAKT)
to activate the PI3K/AKT signaling pathway. A detailed description
of the experimental setup, as well as a step-by- step instruction of the
experimental procedure, will be given. By following the instructions
provided, the experienced scientist will be able to generate, either
transiently or stably, optoAKT- expressing cells. Upon irradiation
with blue light AKT1, fused to the PHR domain of CRY2, is
recruited toward a membrane-bound CIBN. The light-dependent
Wignand W.D. Mühlhäuser et al.
the use of constantly improving light-inducible dimerization/
oligomerization systems (i.e., optogenetic tools) becomes more
and more popular. Using light as the sole activator allows unprecedented spatiotemporal resolution with minimal side effects. This
allows the intervention of signal transduction in a most sophisticated manner [2, 3].
One optogenetic tool that has been employed in the activation
of various kinases is the blue light-responsive cryptochrome 2/
cryptochrome-interacting basic-helix-loop-helix 1 (CRY2/CIB1)
system [4–6]. Cryptochromes are flavoprotein photoreceptors
found in bacteria, higher plants, and animals. They were first identified in Arabidopsis thaliana, where they regulate growth and
developmental processes [7]. Cryptochromes contain a C-terminal
cryptochrome extension and an N-terminal photolyase homology
region (PHR) that noncovalently binds flavin adenine dinucleotide
(FAD). Irradiation with blue light of wavelengths ranging from
390 nm to 480 nm leads to FAD reduction, which converts cryptochrome into its biological active conformation [8]. The activated
Arabidopsis thaliana CRY2 can interact with CIB1, which is
involved in the mechanism of floral initiation. Besides the interaction with CIB1, photoexcited CRY2 can also form homomers.
Light-activated CRY2 forms homo- and heteromers within seconds
and dissociates in the dark within minutes in a repeatable manner.
Optogenetic systems have been generated based on the lightinduced oligomerization of the PHR region of CRY2 (aa 1—489)
and the N-terminus of CIB1 (CIBN; aa 1—170). The characteristics
of the CRY2-based optogenetic are especially suited for the control
of signaling events in mammalian cells [3, 9] (Fig. 1). For instance,
blue light-induced clustering of CRY2PHR fused to the protein
kinase RAF as well as the recruitment of chimeric RAF to membrane-bound CIBN activates the mitogen-activated protein kinase
cascade (MAPK) signaling pathway [4, 5, 10]. To gain control of
the PI3K/AKT pathway, CRY2PHR either fused to the interSRC-homology 2 (iSH2) domain of the regulatory p85α subunit
of PI3K or to the protein kinase AKT were recruited to a membrane-anchored CIBN to activate signaling upon blue light irradiation [6, 11]. Due to its high relevance constant efforts to
further improve this system are undertaken (e.g., enhanced oligomerization) [12, 13].
In this chapter, we describe a detailed approach of how to use the
CRY2PHR/CIBN optogenetic tool to control AKT1 (optoAKT)
to activate the PI3K/AKT signaling pathway. A detailed description
of the experimental setup, as well as a step-by- step instruction of the
experimental procedure, will be given. By following the instructions
provided, the experienced scientist will be able to generate, either
transiently or stably, optoAKT- expressing cells. Upon irradiation
with blue light AKT1, fused to the PHR domain of CRY2, is
recruited toward a membrane-bound CIBN. The light-dependent
Wignand W.D. Mühlhäuser et al.