TY - JOUR
T1 - Light control of plasma membrane recruitment using the Phy-PIF system
AU - Toettcher, Jared E.
AU - Gong, Delquin
AU - Lim, Wendell A.
AU - Weiner, Orion D.
N1 - Funding Information:
We thank Anselm Levskaya for advice on PCB purification and light system manipulation. This work was partially supported by the National Institutes of Health grant GM084040, the Cancer Research Institute Postdoctoral Fellowship (J. E. T.), the American Cancer Society Fellowship (D. G.).
PY - 2011
Y1 - 2011
N2 - The ability to control the activity of intracellular signaling processes in live cells would be an extraordinarily powerful tool. Ideally, such an intracellular input would be (i) genetically encoded, (ii) able to be turned on and off in defined temporal or spatial patterns, (iii) fast to switch between on and off states, and (iv) orthogonal to other cellular processes. The light-gated interaction between fragments of two plant proteinstermed Phy and PIFsatisfies each of these constraints. In this system, Phy can be switched between two conformations using red and infrared light, while PIF only binds one of these states. This chapter describes known constraints for designing genetic constructs using Phy and PIF and provides protocols for expressing these constructs in mammalian cells, purifying the small molecule chromophore required for the system's light responsivity, and measuring light-gated binding by microscopy.
AB - The ability to control the activity of intracellular signaling processes in live cells would be an extraordinarily powerful tool. Ideally, such an intracellular input would be (i) genetically encoded, (ii) able to be turned on and off in defined temporal or spatial patterns, (iii) fast to switch between on and off states, and (iv) orthogonal to other cellular processes. The light-gated interaction between fragments of two plant proteinstermed Phy and PIFsatisfies each of these constraints. In this system, Phy can be switched between two conformations using red and infrared light, while PIF only binds one of these states. This chapter describes known constraints for designing genetic constructs using Phy and PIF and provides protocols for expressing these constructs in mammalian cells, purifying the small molecule chromophore required for the system's light responsivity, and measuring light-gated binding by microscopy.
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U2 - 10.1016/B978-0-12-385075-100017-2
DO - 10.1016/B978-0-12-385075-100017-2
M3 - Article
C2 - 21601096
AN - SCOPUS:79957527511
SN - 0076-6879
VL - 497
SP - 409
EP - 423
JO - Methods in enzymology
JF - Methods in enzymology
ER -