in ways that sometimes make optogenetic experiments poorly
favorable. If this is the case, other systems like iLID or TULIP
could solve the problem.
4. Except for the photoactivatable Rac system, all the other systems are light-gated dimerizers at chemical equilibrium
between the bound and free forms of the dimer. While the
dissociation constant is unaffected by light, the association
constant of the dimers is modified under light exposure.
Thus, the dissociation constant at equilibrium, K D , varies
from high values in the dark to low value under light. For
optogenetic activation to be efficient but only triggered when
light is shone, cellular concentrations of the optogenetic components have to be higher than the K D of the lit state and lower
than the K D of the dark state. Since it is usually difficult to
control the level of expression in cellular systems, it is important to choose the optogenetic dimer with the largest difference between the K D s in the dark and in the lit state for the
chosen cell line. The values of some of these constants have
been measured, but not all [23]. In the case of the CRY2/
CIBN system, this requirement translates into fairly high
expression levels of CIBN and CRY2 for the system to work.
However, both components should not be equally expressed: it
is important that the concentration of CIBN-GFP-CAAX is in
excess, so that it is not easily saturated with ITSN-CRY2mCherry and that large amounts of the latter can be recruited
to the plasma membrane. It is also important that ITSNCRY2-mCherry is not expressed too strongly otherwise it can
spontaneously form clusters in the cytoplasm. Even though the
K D in the illuminated state of the CRY2/CIBN couple has not
been reported, it is sometimes difficult in some cell lines to
reach expression levels that are high enough for its activation by
illumination, suggesting this K D is quite high. If this is the case,
it can be worth testing a system with a lower K D , like the iLID/
SSPB nano couple.
5. All the optogenetic systems are not activated by the same
wavelengths. Therefore, they are not all compatible with the
same fluorophores. Mainly, the photoactivatable Rac, CRY2/
CIBN, LOV/TULIP and iLID systems are activated by a wide
range of blue light (400–530 nm approximately), while the
Phytochrome B is activated by red light (centered around
650 nm). The wavelength of any other readout or additional
fluorophore has thus to be compatible with this excitation
spectrum as well as with potential fluorophores already
attached to the system itself (ex: CIBN-GFP, CRY2-mCherry).
Taking this into consideration, it limits the number of
fluorophores that can be used without activating the dimerization. In the case of the use of the CRY2/CIBN system all
Control of Cell Migration Using Optogenetics
421
favorable. If this is the case, other systems like iLID or TULIP
could solve the problem.
4. Except for the photoactivatable Rac system, all the other systems are light-gated dimerizers at chemical equilibrium
between the bound and free forms of the dimer. While the
dissociation constant is unaffected by light, the association
constant of the dimers is modified under light exposure.
Thus, the dissociation constant at equilibrium, K D , varies
from high values in the dark to low value under light. For
optogenetic activation to be efficient but only triggered when
light is shone, cellular concentrations of the optogenetic components have to be higher than the K D of the lit state and lower
than the K D of the dark state. Since it is usually difficult to
control the level of expression in cellular systems, it is important to choose the optogenetic dimer with the largest difference between the K D s in the dark and in the lit state for the
chosen cell line. The values of some of these constants have
been measured, but not all [23]. In the case of the CRY2/
CIBN system, this requirement translates into fairly high
expression levels of CIBN and CRY2 for the system to work.
However, both components should not be equally expressed: it
is important that the concentration of CIBN-GFP-CAAX is in
excess, so that it is not easily saturated with ITSN-CRY2mCherry and that large amounts of the latter can be recruited
to the plasma membrane. It is also important that ITSNCRY2-mCherry is not expressed too strongly otherwise it can
spontaneously form clusters in the cytoplasm. Even though the
K D in the illuminated state of the CRY2/CIBN couple has not
been reported, it is sometimes difficult in some cell lines to
reach expression levels that are high enough for its activation by
illumination, suggesting this K D is quite high. If this is the case,
it can be worth testing a system with a lower K D , like the iLID/
SSPB nano couple.
5. All the optogenetic systems are not activated by the same
wavelengths. Therefore, they are not all compatible with the
same fluorophores. Mainly, the photoactivatable Rac, CRY2/
CIBN, LOV/TULIP and iLID systems are activated by a wide
range of blue light (400–530 nm approximately), while the
Phytochrome B is activated by red light (centered around
650 nm). The wavelength of any other readout or additional
fluorophore has thus to be compatible with this excitation
spectrum as well as with potential fluorophores already
attached to the system itself (ex: CIBN-GFP, CRY2-mCherry).
Taking this into consideration, it limits the number of
fluorophores that can be used without activating the dimerization. In the case of the use of the CRY2/CIBN system all
Control of Cell Migration Using Optogenetics
421