4. Use bright-field (with the red filter in the light path) and the
mCherry signal from ITSN-CRY2-mCherry to find transfected
cells with the desired shape and neighborhood crowding density (ideally no or few neighbors). Set up the TIRF so as to
image only the signal from the membrane. Cells with a fairly
high expression level of ITSN-CRY2-mCherry and maximal
expression level of CIBN-GFP-CAAX should be chosen for
the experiment to work (see Note 4), although it is not always
possible to test the latter as visualization of CIBN-GFP-CAAX
using blue light illumination will transiently activate Cdc42 in a
uniform fashion throughout the cell.
5. Draw a region of interest (ROI) where blue light will be shined
using the FRAP control software: target the cell edges, with a
ROI ranging from 5 to 50 μm
2 approximately. For the cell to
migrate in the direction dictated by the ROI, the ROI should
encompass a few micrometers within the cytoplasm and a bigger area outside the cell.
6. Set up the imaging and activation frequency. One activation
pulse every 10 s (100 ms pulses at approximately 10% power of
a 488 nm, 100 mW laser, see Note 7) is sufficient for strong
activation. In order to see the pre-activation and postactivation states, start these activation pulses from image number 20 on, and continue the movie after blue light illumination
are stopped (see Note 8).
7. When all parameters are selected, launch the experiment.
Recruitment of ITSN-CRY2-mCherry to the basal plasma
membrane should be visible through a local increase of red
fluorescence after a few seconds (see Note 9). If the microscope
does not have auto-focus possibilities, make sure there is no
drift of focus.
8. In the case of long experiments (>2 h), the cell starts to fully
invade the ROI. It might then be necessary to adapt the activation ROI so that it remains localized at the cell border.
4 Notes
1. Many signaling pathways can be specifically targeted (activated
or inactivated) using optogenetics. First, because many
GTPases are activated by specific GEFs, activation can usually
be achieved by recruiting one of these GEFs to the relevant
membranes. Similarly, optogenetics-driven actuation (activation or inhibition) can be achieved by locally depleting the
inhibitors or activators of the targeted pathway, respectively.
This can be done by recruiting these regulators to some cellular
compartments where they cannot activate their targets [15] or
Control of Cell Migration Using Optogenetics
419
mCherry signal from ITSN-CRY2-mCherry to find transfected
cells with the desired shape and neighborhood crowding density (ideally no or few neighbors). Set up the TIRF so as to
image only the signal from the membrane. Cells with a fairly
high expression level of ITSN-CRY2-mCherry and maximal
expression level of CIBN-GFP-CAAX should be chosen for
the experiment to work (see Note 4), although it is not always
possible to test the latter as visualization of CIBN-GFP-CAAX
using blue light illumination will transiently activate Cdc42 in a
uniform fashion throughout the cell.
5. Draw a region of interest (ROI) where blue light will be shined
using the FRAP control software: target the cell edges, with a
ROI ranging from 5 to 50 μm
2 approximately. For the cell to
migrate in the direction dictated by the ROI, the ROI should
encompass a few micrometers within the cytoplasm and a bigger area outside the cell.
6. Set up the imaging and activation frequency. One activation
pulse every 10 s (100 ms pulses at approximately 10% power of
a 488 nm, 100 mW laser, see Note 7) is sufficient for strong
activation. In order to see the pre-activation and postactivation states, start these activation pulses from image number 20 on, and continue the movie after blue light illumination
are stopped (see Note 8).
7. When all parameters are selected, launch the experiment.
Recruitment of ITSN-CRY2-mCherry to the basal plasma
membrane should be visible through a local increase of red
fluorescence after a few seconds (see Note 9). If the microscope
does not have auto-focus possibilities, make sure there is no
drift of focus.
8. In the case of long experiments (>2 h), the cell starts to fully
invade the ROI. It might then be necessary to adapt the activation ROI so that it remains localized at the cell border.
4 Notes
1. Many signaling pathways can be specifically targeted (activated
or inactivated) using optogenetics. First, because many
GTPases are activated by specific GEFs, activation can usually
be achieved by recruiting one of these GEFs to the relevant
membranes. Similarly, optogenetics-driven actuation (activation or inhibition) can be achieved by locally depleting the
inhibitors or activators of the targeted pathway, respectively.
This can be done by recruiting these regulators to some cellular
compartments where they cannot activate their targets [15] or
Control of Cell Migration Using Optogenetics
419