title: Correlating in Vitro and in Vivo Activities of Light-Inducible Dimers: A Cellular Optogenetics Guide.
abstract: Light-inducible dimers are powerful tools for cellular optogenetics, as they can be used to control the localization and activity of proteins with high spatial and temporal resolution. Despite the generality of the approach, application of light-inducible dimers is not always straightforward, as it is frequently necessary to test alternative dimer systems and fusion strategies before the desired biological activity is achieved. This process is further hindered by an incomplete understanding of the biophysical/biochemical mechanisms by which available dimers behave and how this correlates to in vivo function. To better inform the engineering process, we examined the biophysical and biochemical properties of three blue-light-inducible dimer variants (cryptochrome2 (CRY2)/CIB1, iLID/SspB, and LOVpep/ePDZb) and correlated these characteristics to in vivo colocalization and functional assays. We find that the switches vary dramatically in their dark and lit state binding affinities and that these affinities correlate with activity changes in a variety of in vivo assays, including transcription control, intracellular localization studies, and control of GTPase signaling. Additionally, for CRY2, we observe that light-induced changes in homo-oligomerization can have significant effects on activity that are sensitive to alternative fusion strategies.
authors.affiliation: Department of Biochemistry & Biophysics, ‡Department of Cell Biology & Physiology, §UNC Lineberger Comprehensive Cancer Center, ∥Howard Hughes Medical Institute, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
authors.forename: Ryan A
authors.initials: RA
authors.lastname: Hallett
authors.affiliation: Department of Biochemistry & Biophysics, ‡Department of Cell Biology & Physiology, §UNC Lineberger Comprehensive Cancer Center, ∥Howard Hughes Medical Institute, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
authors.forename: Seth P
authors.initials: SP
authors.lastname: Zimmerman
authors.affiliation: Department of Biochemistry & Biophysics, ‡Department of Cell Biology & Physiology, §UNC Lineberger Comprehensive Cancer Center, ∥Howard Hughes Medical Institute, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
authors.forename: Hayretin
authors.initials: H
authors.lastname: Yumerefendi
authors.affiliation: Department of Biochemistry & Biophysics, ‡Department of Cell Biology & Physiology, §UNC Lineberger Comprehensive Cancer Center, ∥Howard Hughes Medical Institute, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
authors.forename: James E
authors.initials: JE
authors.lastname: Bear
authors.affiliation: Department of Biochemistry & Biophysics, ‡Department of Cell Biology & Physiology, §UNC Lineberger Comprehensive Cancer Center, ∥Howard Hughes Medical Institute, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
authors.forename: Brian
authors.initials: B
authors.lastname: Kuhlman
mesh_terms: D000818:Animals; D029681:Arabidopsis Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D056931:Cryptochromes / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D007700:Kinetics; D008027:Light; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D062308:Optogenetics / Q000379:methods*; D011554:Pseudopodia / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism / Q000528:radiation effects; D013347:Subcellular Fractions / Q000378:metabolism; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques
chemical_list: D029681:Arabidopsis Proteins; C103618:CRY2 protein, Arabidopsis; D056931:Cryptochromes
keywords: TULIP; cell signaling; cryptochrome2 (CRY2); dimerization; iLID; optogenetics