We report an optogenetic method based on Arabidopsis thaliana cryptochrome 2 for rapid and reversible protein oligomerization in response to blue light. We demonstrated its utility by photoactivating the β-catenin pathway, achieving a transcriptional response higher than that obtained with the natural ligand Wnt3a. We also demonstrated the modularity of this approach by photoactivating RhoA with high spatiotemporal resolution, thereby suggesting a previously unknown mode of activation for this Rho GTPase.
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We thank S. Kumar for technical discussions and use of equipment and reagents, and J. McKay and S. Rammensee for experimental advice and assistance. We received the CRY2PHR-mCherry construct as a gift from C. Tucker (Duke University), constitutively active variants of Rac1, RhoA and Cdc42 as gifts from G.S. Martin (UC Berkeley), the full-length LRP6 construct as a gift from X. He (Harvard University) and CA-β-catenin as a gift from A. Asthagiri (Northeastern University). We would also like to thank M. Niewiadomska-Bugaj (Western Michigan University) for statistical analysis, A. Keung (UC Berkeley) for retroviral constructs encoding the Rho GTPases and A. Fritz (UC Berkeley) for retroviral constructs encoding CA-β-catenin and CA-GSK-3β. This work was supported by the US Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering, under Award nos. DE-SC0001216 and DE-SC0001874.
The authors declare no competing financial interests.
Supplementary Figures 1–9 (PDF 2374 kb)
Details of constructs used in this study. (XLSX 21 kb)
HEK 293T cells transiently transfected with CRY2-mCh were exposed to blue light pulses every 2 s. mCherry fluorescence was visualized before, during and after activation. Time is given in minutes:seconds. (AVI 7452 kb)
HEK 293T cells transiently transfected with CRY2-mCh were illuminated with blue light to induce cluster formation. A reconstructed z-stack sequence allows a 3D view at cluster morphology in a single cell. (AVI 9292 kb)
HEK 293T cells transiently transfected with CRY2-LRP6c were exposed to blue light pulses every 10 s. mCherry fluorescence was visualized during light illumination and its subsequent withdrawal. Time is given in minutes:seconds. (AVI 4918 kb)
HEK 293T cells transiently transfected with CRY2-RAC1 were exposed to continuous blue light pulses. mCherry fluorescence begins in the cytoplasm but rapidly translocates to the membrane upon activation. Blue-light state is indicated in the top left corner, and time is given in minutes:seconds. (AVI 2935 kb)
HEK 293T cells transiently transfected with CRY2-RHOA were exposed to blue light pulses every 5 s. mCherry fluorescence begins in the cytoplasm, translocates to the membrane upon activation and returns to the cytoplasm upon withdrawal of blue light. Blue-light state is indicated in the top left corner, and time is given in minutes:seconds. (AVI 12805 kb)
HEK 293T cells were either transfected with CRY2-RHOA (left panel) or cotransfected with mCh-RHOA and CRY2-GFP (middle and right, respectively) to determine whether fluorescence redistribution of Cry2-RhoA upon light induction was due to nonspecific effects of Cry2 photoactivation. Cry2-RhoA redistributes to vesicles and membrane, whereas mCh-RhoA localization does not change, despite Cry2-GFP cluster formation, supporting the hypothesis that induced RhoA clustering mediates protein redistribution within the cell. Samples are illuminated starting with frame 1. Time is in minutes:seconds. (AVI 30724 kb)
NIH 3T3 cells stably expressing CRY2-RHOA were focally illuminated (white circle), and cell contraction was observed. The leftmost cell, expressing higher amount of protein, exhibits visible, bright clusters upon activating illumination. Time is given in minutes:seconds. (AVI 51206 kb)
A representative 3T3 fibroblast expressing high levels of CRY2-RHOA shows distinct cluster formation upon light activation that appears to align to fibrillar structures. As activation continues, these clusters appear to retract inwards toward the cell center. Time is given in minutes:seconds. (AVI 17640 kb)
A 3T3 fibroblast expressing the CRY2-RHOA construct is excited sequentially in three regions. mCherry fluorescence allows visualization of cell morphology during cell excitation and relaxation. (AVI 7731 kb)
Comparing contractility of fibroblasts expressing CRY2-RHOA upon blue light exposure in the presence and absence of pathway inhibitors
3T3 fibroblasts were exposed to whole-field blue light illumination every 30 s, and the percentage of visibly contractile cells was observed in the presence and absence of pathway inhibitors Y-27632 and ML-7, which inhibit Rho-associated protein kinase and myosin light chain kinase, respectively. The video shows representative results, with time given in minutes:seconds. Quantitative results are depicted in Figure 3d of the main text. (AVI 22152 kb)
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Bugaj, L., Choksi, A., Mesuda, C. et al. Optogenetic protein clustering and signaling activation in mammalian cells. Nat Methods 10, 249–252 (2013). https://doi.org/10.1038/nmeth.2360
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