Light control of plasma membrane recruitment using the Phy-PIF system.

TitleLight control of plasma membrane recruitment using the Phy-PIF system.
Publication TypeJournal Article
Year of Publication2011
AuthorsToettcher, JE, Gong, D, Lim, WA, Weiner, OD
JournalMethods Enzymol
Volume497
Pagination409-23
Date Published2011
ISSN1557-7988
KeywordsAnimals, Arabidopsis, Arabidopsis Proteins, Cell Line, Cell Membrane, Light, Microscopy, Confocal, Models, Molecular, Peptide Fragments, Phytochrome B, Plant Proteins, Protein Binding, Protein Conformation
Abstract

<p>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 proteins--termed Phy and PIF--satisfies 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.</p>

DOI10.1016/B978-0-12-385075-1.00017-2
Alternate JournalMeth. Enzymol.
PubMed ID21601096
PubMed Central IDPMC3121701
Grant ListGM084040 / GM / NIGMS NIH HHS / United States
R01 GM084040 / GM / NIGMS NIH HHS / United States
R01 GM084040-04 / GM / NIGMS NIH HHS / United States