Spatiotemporal control of cell signalling using a light-switchable protein interaction

Abstract

Genetically encodable optical reporters, such as green fluorescent protein, have revolutionized the observation and measurement of cellular states. However, the inverse challenge of using light to control precisely cellular behaviour has only recently begun to be addressed; semi-synthetic chromophore-tethered receptors1 and naturally occurring channel rhodopsins have been used to perturb directly neuronal networks2,3. The difficulty of engineering light-sensitive proteins remains a significant impediment to the optical control of most cell-biological processes. Here we demonstrate the use of a new genetically encoded light-control system based on an optimized, reversible protein–protein interaction from the phytochrome signalling network of Arabidopsis thaliana. Because protein–protein interactions are one of the most general currencies of cellular information, this system can, in principle, be generically used to control diverse functions. Here we show that this system can be used to translocate target proteins precisely and reversibly to the membrane with micrometre spatial resolution and at the second timescale. We show that light-gated translocation of the upstream activators of Rho-family GTPases, which control the actin cytoskeleton, can be used to precisely reshape and direct the cell morphology of mammalian cells. The light-gated protein–protein interaction that has been optimized here should be useful for the design of diverse light-programmable reagents, potentially enabling a new generation of perturbative, quantitative experiments in cell biology.

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Figure 1: The phytochrome–PIF interaction can be used to reversibly translocate proteins to the plasma membrane in a light-controlled fashion.
Figure 2: Confocal microscopy demonstrating the second-scale kinetics and photostability of the Phy–PIF photoswitchable membrane recruitment system.
Figure 3: Recruitment to the plasma membrane can be controlled spatially by simultaneously irradiating cells with patterned red and infrared light.
Figure 4: Rho-family G-protein signalling can be controlled by the light-activated translocation system.

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Acknowledgements

We thank B. El-Sady, J. Tepperman, K. Thorn, G. Kapp and members of the Voigt, Weiner and Lim laboratories for assistance and discussion. We thank Molecular Devices and Photonics Instruments for the loan and customization of a Mosaic spatial light modulator. Data for this study were acquired at the Nikon Imaging Center at UCSF. This work was supported by a NSFGR fellowship (A.L.); NIH R01 GM084040 and Searles Scholar Award.(O.D.W.); Packard Fellowship, the Howard Hughes Medical Institute, and NIH grants GM55040, GM62583 and EY016546 (NIH Roadmap Nanomedicine Development Centers) (W.A.L.); Pew Fellowship, the Office of Naval Research, Packard Fellowship, NIH EY016546, NIH AI067699, NSF BES-0547637, UC-Discovery and the SynBERC NSF ERC (C.A.V.).

Author Contributions Concept was conceived by A.L., W.A.L. and C.A.V.; experiments were executed by A.L.; spatiotemporal microscopy methods were developed by A.L. and O.D.W.; all authors were involved in interpretation of results and preparation of the manuscript.

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Correspondence to Wendell A. Lim.

Additional information

Plasmids will be available from Addgene (http://www.addgene.org).

Supplementary information

Supplementary Information

This file contains Supplementary Methods, Supplementary Table S1, Supplementary Data, Supplementary Movie Legends, Supplementary References and Supplementary Figures S1-S5 with Legends. (PDF 826 kb)

Supplementary Movie 1

This movie shows YFP membrane recruitment - see file s1 for full Legend. (MOV 246 kb)

Supplementary Movie 2

This movie shows YFP membrane dissociation - see file s1 for full Legend. (MOV 149 kb)

Supplementary Movie 3

This movie shows oscillating YFP translocation - see file s1 for full Legend. (MOV 1986 kb)

Supplementary Movie 4

This movie shows YFP point recruitment - see file s1 for full Legend. (MOV 185 kb)

Supplementary Movie 5

This movie shows patterned recruitment of YFP - see file s1 for full Legend. (MOV 583 kb)

Supplementary Movie 6

This movie shows titrated recruitment of YFP - see file s1 for full Legend. (MOV 755 kb)

Supplementary Movie 7

This movie shows cell extrusion by Tiam(Rac GEF) recruitment - see file s1 for full Legend. (MOV 764 kb)

Supplementary Movie 8

This movie shows dynamic lamellipodia by Tiam(Rac GEF - see file s1 for full Legend. (MOV 7019 kb)

Supplementary Movie 9

This movie shows periodic contractions by Tim(Rho GEF) recruitment - see file s1 for full Legend. (MOV 481 kb)

Supplementary Movie 10

This movie shows PIF-YFP only recruitment control - see file s1 for full Legend. (MOV 806 kb)

Supplementary Movie 11

This movie shows monitoring membrane-bound Cdc42-GTP dynamics via recruited mCherry-WASP-GBD in TIRF - see file s1 for full Legend. (MOV 4621 kb)

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Levskaya, A., Weiner, O., Lim, W. et al. Spatiotemporal control of cell signalling using a light-switchable protein interaction. Nature 461, 997–1001 (2009). https://doi.org/10.1038/nature08446

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