intracellular gradients of Rho GTPase activity, thereby allowing to
guide cell migration and to recapitulate EMT [7]. However, this is
somehow technically difficult, especially if high spatial and fast
temporal resolutions are required or if experiments are done
in vivo. Recently, optogenetics emerged as a powerful tool to
control signaling activities with an unprecedented spatiotemporal
resolution [8–11]. By using light-sensitive protein domains derived
from plants, it allows researchers to control the conformation or
dimerization of synthetic polypeptides, thus making it possible to
govern quantitatively the activity of proteins of interest in time and
space with light [12, 13]. Rho GTPases are activated by Guanine
Exchange Factors (GEFs) that promote the exchange from GDP to
GTP. Optogenetic control of GEF localization at subcellular scale
thus enables one to modulate locally the activity of Rho GTPases
(Fig. 1) and to impose the direction and slope of intracellular
gradients of these polarity signals in a quantitative fashion. One
can then force cells to migrate and control the direction and speed
of locomotion [14]. In this chapter, as an example of optogenetic
manipulation of Rho GTPases, we explain how to manipulate
quantitatively Cdc42 activity by controlling its GEF Intersectin1
catalytic domain (ITSN) at the subcellular level in order to guide
cell migration (see Notes 1 and 2). Moreover, we detail some
critical parameters for successful implementation and optimization
of this technique.
2 Materials
2.1 Plasmids
and Transfection
1. Two plasmids that encode the fusion proteins ITSN-CRY2mCherry and CIBN-GFP-CAAX (optoGEF-Cdc42), with the
expression controlled by strong eukaryotic promoter such as
the CMV promoter. These plasmids can be shared upon
request.
2. Transfection reagent: X-treme gene 9, or equivalent. X-treme
gene 9 allows a strong expression of the transgenes in Hela
cells, but can be replaced by any other transfection system
adapted to the chosen cell type.
2.2 Cell Culture
1. HeLa cells.
Alternately the CRY2/CIBN optogenetic system has been
used by us and others on a wide range of cell types (MDCK
cells, NIH 3 T3 fibroblasts, RPE1, etc.) without encountering
specific issues.
2. Cell culture medium: DMEM (Dulbecco’s modified Eagle’s
medium) supplemented with 10% fetal calf serum.
416
Leo Valon and Simon de Beco
guide cell migration and to recapitulate EMT [7]. However, this is
somehow technically difficult, especially if high spatial and fast
temporal resolutions are required or if experiments are done
in vivo. Recently, optogenetics emerged as a powerful tool to
control signaling activities with an unprecedented spatiotemporal
resolution [8–11]. By using light-sensitive protein domains derived
from plants, it allows researchers to control the conformation or
dimerization of synthetic polypeptides, thus making it possible to
govern quantitatively the activity of proteins of interest in time and
space with light [12, 13]. Rho GTPases are activated by Guanine
Exchange Factors (GEFs) that promote the exchange from GDP to
GTP. Optogenetic control of GEF localization at subcellular scale
thus enables one to modulate locally the activity of Rho GTPases
(Fig. 1) and to impose the direction and slope of intracellular
gradients of these polarity signals in a quantitative fashion. One
can then force cells to migrate and control the direction and speed
of locomotion [14]. In this chapter, as an example of optogenetic
manipulation of Rho GTPases, we explain how to manipulate
quantitatively Cdc42 activity by controlling its GEF Intersectin1
catalytic domain (ITSN) at the subcellular level in order to guide
cell migration (see Notes 1 and 2). Moreover, we detail some
critical parameters for successful implementation and optimization
of this technique.
2 Materials
2.1 Plasmids
and Transfection
1. Two plasmids that encode the fusion proteins ITSN-CRY2mCherry and CIBN-GFP-CAAX (optoGEF-Cdc42), with the
expression controlled by strong eukaryotic promoter such as
the CMV promoter. These plasmids can be shared upon
request.
2. Transfection reagent: X-treme gene 9, or equivalent. X-treme
gene 9 allows a strong expression of the transgenes in Hela
cells, but can be replaced by any other transfection system
adapted to the chosen cell type.
2.2 Cell Culture
1. HeLa cells.
Alternately the CRY2/CIBN optogenetic system has been
used by us and others on a wide range of cell types (MDCK
cells, NIH 3 T3 fibroblasts, RPE1, etc.) without encountering
specific issues.
2. Cell culture medium: DMEM (Dulbecco’s modified Eagle’s
medium) supplemented with 10% fetal calf serum.
416
Leo Valon and Simon de Beco