cell lines. Without much implementation, it is also possible to
do multiple cells activation at the same time (without recording
different ROIs for each position). One has to place the ROI in a
central area of the field of view, and then to select the different
XY positions based on the position of the fixed ROI.
7. When starting using the CRY2-CIBN optogenetic system, one
needs to know what is the amount of light required to activate
the dimerization. In the case of the CRY2/CIBN dimer, a
good reference value is to use similar light power and exposure
times as the ones required to do GFP imaging without photobleaching. This should be sufficient to generate CRY2 recruitment to the membrane. Then, it is also possible to control
quantitatively the level of activation. According to theory and
experiments [13], two main parameters are important: the
frequency of the activation pulses, and/or the light amount
per pulse. With this approach, it is possible to create different
levels of recruitment which can be quantified a posteriori. This
approach remains correct as long as there is no saturation of the
system, i.e., CIBN is in large excess at the membrane in comparison to CRY2 proteins, and that there is a large cytoplasmic
CRY2 pool available for activation, in order to avoid the plateauing of the recruitment.
Finally, it is also possible to create subcellular and/or multicellular gradients of activation from gradients of light, using a
DMD for example [14]. This can be achieved in a straightforward manner as a linear gradient of light will create a linear
recruitment of ITSN-CRY2-mCherry [13], at the condition
that the system is not saturated.
8. When illumination is stopped after optogenetic activation is
achieved, the optogenetic systems will revert to their initial
state with timescales that depend on their biochemical properties. For example, the CRY2/CIBN couple, with its half-life
time of 2 min, will be fully dissociated within 10 min. Hence,
membrane-recruited ITSN will diffuse back to the cytoplasm
and stop activating Cdc42. In our experiments with HeLa cells
and NIH-3T3 cells, stopping blue illumination resulted in a
quick loss of protrusions and a reshuffling of cell polarity—even
after hour-long optogenetic activations. However, that might
be dependent on cell types and experimental conditions.
9. It is sometimes necessary to quantify the recruitment of ITSNCRY2-mCherry to the plasma membrane. Measure the
mCherry fluorescence TIRF signal over time in the recruitment
area. The mCherry signal is proportional to the concentration
of ITSN proteins at the plasma membrane (Fig. 2). If fluorescence compensation is necessary, quantify the photobleaching
Control of Cell Migration Using Optogenetics
423
do multiple cells activation at the same time (without recording
different ROIs for each position). One has to place the ROI in a
central area of the field of view, and then to select the different
XY positions based on the position of the fixed ROI.
7. When starting using the CRY2-CIBN optogenetic system, one
needs to know what is the amount of light required to activate
the dimerization. In the case of the CRY2/CIBN dimer, a
good reference value is to use similar light power and exposure
times as the ones required to do GFP imaging without photobleaching. This should be sufficient to generate CRY2 recruitment to the membrane. Then, it is also possible to control
quantitatively the level of activation. According to theory and
experiments [13], two main parameters are important: the
frequency of the activation pulses, and/or the light amount
per pulse. With this approach, it is possible to create different
levels of recruitment which can be quantified a posteriori. This
approach remains correct as long as there is no saturation of the
system, i.e., CIBN is in large excess at the membrane in comparison to CRY2 proteins, and that there is a large cytoplasmic
CRY2 pool available for activation, in order to avoid the plateauing of the recruitment.
Finally, it is also possible to create subcellular and/or multicellular gradients of activation from gradients of light, using a
DMD for example [14]. This can be achieved in a straightforward manner as a linear gradient of light will create a linear
recruitment of ITSN-CRY2-mCherry [13], at the condition
that the system is not saturated.
8. When illumination is stopped after optogenetic activation is
achieved, the optogenetic systems will revert to their initial
state with timescales that depend on their biochemical properties. For example, the CRY2/CIBN couple, with its half-life
time of 2 min, will be fully dissociated within 10 min. Hence,
membrane-recruited ITSN will diffuse back to the cytoplasm
and stop activating Cdc42. In our experiments with HeLa cells
and NIH-3T3 cells, stopping blue illumination resulted in a
quick loss of protrusions and a reshuffling of cell polarity—even
after hour-long optogenetic activations. However, that might
be dependent on cell types and experimental conditions.
9. It is sometimes necessary to quantify the recruitment of ITSNCRY2-mCherry to the plasma membrane. Measure the
mCherry fluorescence TIRF signal over time in the recruitment
area. The mCherry signal is proportional to the concentration
of ITSN proteins at the plasma membrane (Fig. 2). If fluorescence compensation is necessary, quantify the photobleaching
Control of Cell Migration Using Optogenetics
423