Rho family GTPases regulate the actin and adhesion dynamics that control cell migration. Current models postulate that Rac promotes membrane protrusion at the leading edge and that RhoA regulates contractility in the cell body1,2. However, there is evidence that RhoA also regulates membrane protrusion3,4. Here we use a fluorescent biosensor, based on a novel design preserving reversible membrane interactions, to visualize the spatiotemporal dynamics of RhoA activity during cell migration. In randomly migrating cells, RhoA activity is concentrated in a sharp band directly at the edge of protrusions. It is observed sporadically in retracting tails, and is low in the cell body. RhoA activity is also associated with peripheral ruffles and pinocytic vesicles, but not with dorsal ruffles induced by platelet-derived growth factor (PDGF). In contrast to randomly migrating cells, PDGF-induced membrane protrusions have low RhoA activity, potentially because PDGF strongly activates Rac, which has previously been shown to antagonize RhoA activity5,6. Our data therefore show that different extracellular cues induce distinct patterns of RhoA signalling during membrane protrusion.
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We are grateful to G. Bokoch, P. Sun, M. Schwartz, R. Tsien, C. Der and E. Sahai for reagents, and to F. Shen for help with image analysis. This work was supported by grants from the Swiss National Science Foundation, Roche Research Foundation, Novartis and Philip Morris to O.P., and from the National Institutes of Health to K.M.H. and R.L.K.
K.M.H. is a paid consultant of Panomics Corporation, which markets reagents for high-content microscopy screening.
This file contains Supplementary Methods and Supplementary Figures 1–6. (PDF 582 kb)
Phase contrast timelapse movie comparing MEFs with or without biosensor expression (induced versus non-induced). (MOV 5924 kb)
Timelapse movie showing YFP and ratio images of a MEF fibroblast randomly migrating on fibronectin (as in Fig. 2a). (MOV 3638 kb)
Timelapse movie showing ratio images of a MEF fibroblast randomly migrating on fibronectin (as in Fig. 2b). (MOV 1028 kb)
Timelapse movie of a peripheral ruffle. (MOV 3633 kb)
Timelapse movie of PDGF-stimulated pinocytosis. (MOV 7683 kb)
Timelapse movie showing robust PDGF stimulation of protrusions in the MEF cells, as shown in Fig.4a. (MOV 5446 kb)
Timelapse movie showing PDGF induction of membrane protrusions in MEF cells expressing T19N DN biosensor. (MOV 7177 kb)
Timelapse movie of a MEF migrating out of a wounded monolayer, then stimulated with PDGF. (MOV 5463 kb)
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Pertz, O., Hodgson, L., Klemke, R. et al. Spatiotemporal dynamics of RhoA activity in migrating cells. Nature 440, 1069–1072 (2006). https://doi.org/10.1038/nature04665
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