2.3 Cell Plating
on Coverslips
1. A 6-well plate for cell culture.
2. Glass coverslips: 25 mm in diameter, 0.13–0.17 mm thickness.
3. Fibronectin solution: 20 μg/mL fibronectin from bovine
plasma diluted in 100 mM NaHCO3 (pH 8.5).
4. A mounting holder for round 25 mm coverslips.
2.4 Stable Cell Line
Alternatively, to transient transfection, stable cell lines expressing
the two optogenetic plasmids can be obtained and studied over
months (see Note 3).
Use lentiviral infections to produce stable cell lines easily.
1. Viral particles containing the CIBN-GFP-CAAX and ITSNCRY2-mCherry fusion genes.
2. A FACS-sorter to select cells with appropriate expression levels
of the two partners of the dimerizer system (see Note 4) and to
limit cell-to-cell variability.
2.5 Microscopy
Setup for Optogenetics
Imaging
and Activation
The cellular response as well as the recruitment of ITSN-CRY2mCherry to the plasma membrane are best imaged with TIRF
microscopy, but can also be imaged with a spinning disk microscope, a fast scanning laser confocal microscope, or by epifluorescence. The TIRF setup allows fast imaging and accurate
quantification of fluorescence signals at the membrane in contact
with the coverslip without interference from the cytoplasmic signal.
1. A microscope with an atmosphere at 37
C and 5% CO 2 .
2. A high magnification objective (40–100Â).
3. GFP and mCherry fluorescence filter sets.
4. Bright-field imaging possibility, ideally DIC (Differential Interference Contrast) with a red long-pass filter in the light path
(cutoff wavelength above 550 nm) to avoid CRY2 activation by
blue wavelengths contained in white light.
5. A FRAP module using a 488 nm laser to shine blue light in
subcellular regions of the cells (see Note 5). Alternatively, any
system allowing to illuminate specifically a Region of Interest
within the field of view with blue light could also work (see
Note 6).
3 Methods
By shining blue light at cells expressing the optoGEF-Cdc42 system, it changes the conformation of CRY2. This increases the
affinity between CRY2 and CIBN, thus promoting the relocation
of ITSN-CRY2-mCherry at the plasma membrane (where GFPCIBN-CAAX is located). This allows ITSN to interact with the
Control of Cell Migration Using Optogenetics
417
on Coverslips
1. A 6-well plate for cell culture.
2. Glass coverslips: 25 mm in diameter, 0.13–0.17 mm thickness.
3. Fibronectin solution: 20 μg/mL fibronectin from bovine
plasma diluted in 100 mM NaHCO3 (pH 8.5).
4. A mounting holder for round 25 mm coverslips.
2.4 Stable Cell Line
Alternatively, to transient transfection, stable cell lines expressing
the two optogenetic plasmids can be obtained and studied over
months (see Note 3).
Use lentiviral infections to produce stable cell lines easily.
1. Viral particles containing the CIBN-GFP-CAAX and ITSNCRY2-mCherry fusion genes.
2. A FACS-sorter to select cells with appropriate expression levels
of the two partners of the dimerizer system (see Note 4) and to
limit cell-to-cell variability.
2.5 Microscopy
Setup for Optogenetics
Imaging
and Activation
The cellular response as well as the recruitment of ITSN-CRY2mCherry to the plasma membrane are best imaged with TIRF
microscopy, but can also be imaged with a spinning disk microscope, a fast scanning laser confocal microscope, or by epifluorescence. The TIRF setup allows fast imaging and accurate
quantification of fluorescence signals at the membrane in contact
with the coverslip without interference from the cytoplasmic signal.
1. A microscope with an atmosphere at 37
C and 5% CO 2 .
2. A high magnification objective (40–100Â).
3. GFP and mCherry fluorescence filter sets.
4. Bright-field imaging possibility, ideally DIC (Differential Interference Contrast) with a red long-pass filter in the light path
(cutoff wavelength above 550 nm) to avoid CRY2 activation by
blue wavelengths contained in white light.
5. A FRAP module using a 488 nm laser to shine blue light in
subcellular regions of the cells (see Note 5). Alternatively, any
system allowing to illuminate specifically a Region of Interest
within the field of view with blue light could also work (see
Note 6).
3 Methods
By shining blue light at cells expressing the optoGEF-Cdc42 system, it changes the conformation of CRY2. This increases the
affinity between CRY2 and CIBN, thus promoting the relocation
of ITSN-CRY2-mCherry at the plasma membrane (where GFPCIBN-CAAX is located). This allows ITSN to interact with the
Control of Cell Migration Using Optogenetics
417