This work seeks to engineer, optimize, and validate new tools and strategies to control protein activity with light, which can be delivered with spatial and temporal resolution. In previous work, we pioneered use of the Arabidopsis thaliana blue light photoreceptor cryptochrome 2 (CRY2) as an optogenetic module. We developed a light-induced dimerization platform based on interaction with a partner protein, CIB1, which can be used to control activity and localization of target proteins. In recent work, we developed a new photoregulated module, CRY2olig, that induces protein oligomerization with light. The broad aims of this project are to further develop the CRY2/CIB and CRY2olig technologies allowing control of protein dimerization and oligomerization with light. Specifically, we aim to optimize CRY2/CIB dimerizers with improved and extended properties for cell biology applications (Aim 1).
In Aim 2, we will apply these dimerizers to control activity of a set of important and versatile regulatory enzymes, allowing manipulation of DNA recombination, protein cleavage, and protein labeling with light. Importantly, we will carry out further optimization and validation of a promising light-activated Cre recombinase, activity of which we will test in vivo in rodent brain.
In Aim 3, we will develop and validate methods to transiently and locally disrupt protein activity using CRY2 and CRY2olig. Such tools will enable researchers to quickly and precisely disrupt function, avoiding compensatory effects that can occur with genetic ablation or knockdown studies.
The goals of this research are to develop novel tools for to regulate protein activity using light. These tools, allowing precise spatial, temporal, and dose dependent control of biological processes, will open up vast new experimental avenues, allowing better understanding of pathways important in health and disease.
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|Cook, Sarah G; Bourke, Ashley M; O'Leary, Heather et al. (2018) Analysis of the CaMKII? and ? splice-variant distribution among brain regions reveals isoform-specific differences in holoenzyme formation. Sci Rep 8:5448|
|Pathak, Gopal P; Spiltoir, Jessica I; Höglund, Camilla et al. (2017) Bidirectional approaches for optogenetic regulation of gene expression in mammalian cells using Arabidopsis cryptochrome 2. Nucleic Acids Res 45:e167|
|Liu, Qi; Tucker, Chandra L (2017) Engineering genetically-encoded tools for optogenetic control of protein activity. Curr Opin Chem Biol 40:17-23|
|Taslimi, Amir; Zoltowski, Brian; Miranda, Jose G et al. (2016) Optimized second-generation CRY2-CIB dimerizers and photoactivatable Cre recombinase. Nat Chem Biol 12:425-30|
|Spiltoir, Jessica I; Strickland, Devin; Glotzer, Michael et al. (2016) Optical Control of Peroxisomal Trafficking. ACS Synth Biol 5:554-60|
|Schindler, Suzanne E; McCall, Jordan G; Yan, Ping et al. (2015) Photo-activatable Cre recombinase regulates gene expression in vivo. Sci Rep 5:13627|
|Hanson, M Gartz; Fregoso, Veronica L; Vrana, Justin D et al. (2014) Peripheral nervous system defects in a mouse model for peroxisomal biogenesis disorders. Dev Biol 395:84-95|
|Pathak, Gopal P; Strickland, Devin; Vrana, Justin D et al. (2014) Benchmarking of optical dimerizer systems. ACS Synth Biol 3:832-8|
|Taslimi, Amir; Vrana, Justin D; Chen, Daniel et al. (2014) An optimized optogenetic clustering tool for probing protein interaction and function. Nat Commun 5:4925|
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