Understanding CRY2 interactions for optical control of intracellular signaling

Abstract

Arabidopsis cryptochrome 2 (CRY2) can simultaneously undergo light-dependent CRY2–CRY2 homo-oligomerization and CRY2–CIB1 hetero-dimerization, both of which have been widely used to optically control intracellular processes. Applications using CRY2–CIB1 interaction desire minimal CRY2 homo-oligomerization to avoid unintended complications, while those utilizing CRY2–CRY2 interaction prefer robust homo-oligomerization. However, selecting the type of CRY2 interaction has not been possible as the molecular mechanisms underlying CRY2 interactions are unknown. Here we report CRY2–CIB1 and CRY2–CRY2 interactions are governed by well-separated protein interfaces at the two termini of CRY2. N-terminal charges are critical for CRY2–CIB1 interaction. Moreover, two C-terminal charges impact CRY2 homo-oligomerization, with positive charges facilitating oligomerization and negative charges inhibiting it. By engineering C-terminal charges, we develop CRY2high and CRY2low with elevated or suppressed oligomerization respectively, which we use to tune the levels of Raf/MEK/ERK signaling. These results contribute to our understanding of the mechanisms underlying light-induced CRY2 interactions and enhance the controllability of CRY2-based optogenetic systems.

Optical activation of TrkB receptors

“…The result agrees with our previous report that the same design for TrkA receptors, Opto-iTrkA + CIBN-CAAX, can activate TrkA signaling better than other methods exploiting full-length receptors or membrane-bound intracellular domain of the receptors [24]. To further improve the activation efficiency in the future, CRY2 variants with enhanced homo-interacting abilities, such as CRY2high [41], CRY2clust [42] or CRY2oligo [43], can be incorporated into the Opto-iTrkB + CIBN-CAAX construct. We expect that the Opto-iTrkB + CIBN-CAAX strategy would be a powerful tool to interrogate TrkB signaling.…”

“…Cells were then illuminated with 200 μW/cm 2 intermittent blue light (5 sec on/5 sec off) for 24 hours using a custom-built LED array inside a CO2 incubator. Similar illumination conditions have been tested in previous reports and shows no obvious toxicity [24,41,45]. Additional sets of transfected cells were kept in the dark as control groups.…”

Molecular flux control encodes distinct cytoskeletal responses by specifying SRC signaling pathway usage

“…To this end, we modulated OS oligomers recruitment rates to adhesive sites by anchoring them on membrane through co-expression of CIBN-GFP-Caax. Indeed, CRY2 oligomerization and CRY2-CIBN heterodimerization coexist as they are supported by different regions of CRY2 22 . First, we determined if membraneassociated OS oligomers (OS + CIBN-GFP-Caax) could also relocalized to adhesive sites after sustained TIRF illumination ( Fig.…”

“…S2C, D). Finally, reducing clustering properties of CRY2 by using CRY2 low mutant (CRY2-1 to 488-EED,22 ) slightly reduced OS relocalization in adhesive sites without blocking it (Fig.S2C)showing that lower-size oligomers (probably dimers) can support this relocalization.…”

Clustering of CD3ζ is sufficient to initiate T cell receptor signaling

“…1a). Under blue light illumination, Cry2 self-associates into clusters in a reversible manner (Duan et al, 2017). We fused the construct to mCherry to visualize the protein under 594 nm excitation and named the final construct 'lightinduced clustering of CD3ζ' (LIC-CD3z, Fig.…”