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A genetically encoded photoactivatable Rac controls the motility of living cells


The precise spatio-temporal dynamics of protein activity are often critical in determining cell behaviour, yet for most proteins they remain poorly understood; it remains difficult to manipulate protein activity at precise times and places within living cells. Protein activity has been controlled by light, through protein derivatization with photocleavable moieties1 or using photoreactive small-molecule ligands2. However, this requires use of toxic ultraviolet wavelengths, activation is irreversible, and/or cell loading is accomplished via disruption of the cell membrane (for example, through microinjection). Here we have developed a new approach to produce genetically encoded photoactivatable derivatives of Rac1, a key GTPase regulating actin cytoskeletal dynamics in metazoan cells3,4. Rac1 mutants were fused to the photoreactive LOV (light oxygen voltage) domain from phototropin5,6, sterically blocking Rac1 interactions until irradiation unwound a helix linking LOV to Rac1. Photoactivatable Rac1 (PA-Rac1) could be reversibly and repeatedly activated using 458- or 473-nm light to generate precisely localized cell protrusions and ruffling. Localized Rac activation or inactivation was sufficient to produce cell motility and control the direction of cell movement. Myosin was involved in Rac control of directionality but not in Rac-induced protrusion, whereas PAK was required for Rac-induced protrusion. PA-Rac1 was used to elucidate Rac regulation of RhoA in cell motility. Rac and Rho coordinate cytoskeletal behaviours with seconds and submicrometre precision7,8. Their mutual regulation remains controversial9, with data indicating that Rac inhibits and/or activates Rho10,11. Rac was shown to inhibit RhoA in mouse embryonic fibroblasts, with inhibition modulated at protrusions and ruffles. A PA-Rac crystal structure and modelling revealed LOV–Rac interactions that will facilitate extension of this photoactivation approach to other proteins.

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Figure 1: Engineering and in vivo characterization of a photoactivatable Rac1 (PA-Rac1).
Figure 2: Localized activation or inactivation of PA-Rac1 induces myosin-dependent migration.
Figure 3: Inhibition of RhoA by PA-Rac1.
Figure 4: Crystallization and structural modelling of PA-Rac1.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The structural coordinates of PA-Rac1 and its mutants have been submitted to the Protein Data Bank under accessions 2wkp (wild type), 2wkq (C450A) and 2wkr (C450M).


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We are grateful for help and constructs from W. Briggs, K. Moffat, A. Tripathy, G. Bokoch and K. Jacobson. Diffraction data were collected at the Swiss Light Source, beamline X10SA, Paul Scherrer Institute, Villigen, Switzerland. We thank the Dortmund-Heidelberg team for data collection, and A. Pauluhn and M. Fuchs for their support in setting up the beamline. This research was supported by the American Heart Association (Y.I.W.) and the National Institutes of Health (K.M.H. grants GM057464 and GM64346).

Author Contributions Y.I.W. initiated the project, demonstrated the validity of caging Rac with LOV, and performed the studies of Rac biological function. He was assisted by A.J. in cloning and protein expression. D.F. and I.S. determined and analysed the crystal structures. O.I.L. performed molecular modelling and isothermal calorimetry studies under the direction of B.K. K.M.H. coordinated the study and wrote the final version of the manuscript, based on contributions from all authors.

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Correspondence to Yi I. Wu or Klaus M. Hahn.

Supplementary information

Supplementary Information

This file contains Supplementary Notes, Supplementary Figures S1-S14 with Legends, Supplementary Tables S1-S5, Legends for Supplementary Movies S1-S16 and Supplementary References. (PDF 1507 kb)

Supplementary Movie S1

This movie file shows ruffles and protrusions induced in a HeLa cell expressing PA-Rac1 - see file s1 for full Legend. (MOV 5158 kb)

Supplementary Movie S2

This movie file shows localized illumination of PA-Rac1 - see file s1 for full Legend. (MOV 4219 kb)

Supplementary Movie S3

This movie file shows actin reorganization induced by sequential activation of PA-Rac1 at multiple locations - see file s1 for full Legend. (MOV 5105 kb)

Supplementary Movie S4

This movie file shows translocation of PAK to protrusions upon PA-Rac1 activation - see file s1 for full Legend. (MOV 3337 kb)

Supplementary Movie S5

This movie file shows reversible induction of ruffling at different locations - see file s1 for full Legend. (MOV 9874 kb)

Supplementary Movie S6

This movie file shows complex control of cell morphology guided by a moving laser beam - see file s1 for full Legend. (MOV 12170 kb)

Supplementary Movie S7

This movie file shows diffusion of PA-Rac1 observed using FRAP (fluorescence recovery after photobleaching) - see file s1 for full Legend. (MOV 6159 kb)

Supplementary Movie S8

This movie file shows diffusion of PA-Rac1 observed using PA-GFP - see file s1 for full Legend. (MOV 3954 kb)

Supplementary Movie S9

This movie file shows polarization induced by localized activation of PA-Rac1 – see file s1 for full Legend. (MOV 975 kb)

Supplementary Movie S10

This movie file shows that localized irradiation of PA-Rac1-T17N generates reversed polarity – see file s1 for full Legend. (MOV 1057 kb)

Supplementary Movie S11

This movie file shows that irradiation of PA-Rac1-T17N reverses polarization – see file s1 for full Legend. (MOV 1685 kb)

Supplementary Movie S12

This movie file shows directional migration induced by repeated irradiation at the cell edge – see file s1 for full Legend. (MOV 14014 kb)

Supplementary Movie S13

This movie file shows directional migration induced by PA-Rac1 in HEK293 cells – see file s1 for full Legend. (MOV 10906 kb)

Supplementary Movie S14

This movie file shows downregulation of RhoA activity propagates from the site of PA-Rac1 activation - see file s1 for full Legend. (MOV 3611 kb)

Supplementary Movie S15

This movie file shows ruffle translocation upon activation of PA-Rac1 - see file s1 for full Legend. (MOV 3844 kb)

Supplementary Movie S16

This movie file shows contrasting morphological changes produced by activation of PACdc42 versus PA-Rac1– see file s1 for full Legend. (MOV 4226 kb)

Supplementary Data 1

This zipped file contains the data for PA-RAC1(C450A). (ZIP 62 kb)

Supplementary Data 2

This zipped file contains the data for PA-RAC1(C450M). (ZIP 59 kb)

Supplementary Data 3

This zipped file contains the data for PA-RAC1(WT). (ZIP 58 kb)

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Wu, Y., Frey, D., Lungu, O. et al. A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461, 104–108 (2009).

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