Abstract
Transmembrane receptors are the predominant conduit through which cells sense and transduce extracellular information into intracellular biochemical signals. Current methods to control and study receptor function, however, suffer from poor resolution in space and time and often employ receptor overexpression, which can introduce experimental artifacts. We report a genetically-encoded approach, termed Clustering Indirectly using Cryptochrome 2 (CLICR), for spatiotemporal control over endogenous transmembrane receptor activation, enabled through the optical regulation of target receptor clustering and downstream signaling using non-covalent interactions with engineered Arabidopsis Cryptochrome 2 (Cry2). CLICR offers a modular platform to enable photocontrol of the clustering of diverse transmembrane receptors including FGFR, PDGFR, and integrins in multiple cell types including neural stem cells. Furthermore, light-inducible manipulation of endogenous receptor tyrosine kinase (RTK) activity can modulate cell polarity and establish phototaxis in fibroblasts. The resulting spatiotemporal control over cellular signaling represents a powerful new optogenetic framework for investigating and controlling cell function and fate.
Highlights
Transmembrane receptors are the predominant conduit through which cells sense and transduce extracellular information into intracellular biochemical signals
As Cry[2] optogenetic clustering had been well characterized using cytoplasmic proteins[16], we first assessed the feasibility of Clustering Indirectly using Cryptochrome 2 (CLICR) by investigating whether we could optically cluster a Cryptochrome 2 (Cry2)-binding target in the cytoplasm and whether indirect clustering could induce signal activation (Fig. 1b, Supplementary Fig. 1)
We investigated whether a CLICR-induced avidity increase was sufficient to elicit translocation of a cytoplasmic Cry[2] fusion to the plasma membrane, which would be necessary for CLICR activation of membrane receptors (Fig. 1a)
Summary
Transmembrane receptors are the predominant conduit through which cells sense and transduce extracellular information into intracellular biochemical signals. We report a genetically encoded approach, termed Clustering Indirectly using Cryptochrome 2 (CLICR), for spatiotemporal control over endogenous transmembrane receptor activation, enabled through the optical regulation of target receptor clustering and downstream signalling using noncovalent interactions with engineered Arabidopsis Cryptochrome 2 (Cry[2]). Utilized approaches to study transmembrane receptor signalling[1,2] often employ overexpression of receptors and receptor fusions, which fundamentally alter how cells respond to extracellular signals and frequently induce hypersensitive or constitutive signal activation[3,4,5]. The emerging field of optogenetics has offered several methods to optically control and study numerous signalling phenomena with spatiotemporal precision[8,9,10,11,12,13,14,15,16,17], each currently relies on the ectopic expression of the signalling protein of interest. We re-designed and implemented Cry[2] clustering to optically target, cluster and regulate endogenous transmembrane proteins in a modular strategy called Clustering Indirectly using Cryptochrome 2, or CLICR
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