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Protein kinase A governs a RhoA–RhoGDI protrusion–retraction pacemaker in migrating cells

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Abstract

The cyclical protrusion and retraction of the leading edge is a hallmark of many migrating cells involved in processes such as development, inflammation and tumorigenesis. The molecular identity of the signalling mechanisms that control these cycles has remained unknown. Here, we used live-cell imaging of biosensors to monitor spontaneous morphodynamic and signalling activities, and employed correlative image analysis to examine the role of cyclic-AMP-activated protein kinase A (PKA) in protrusion regulation. PKA activity at the leading edge is closely synchronized with rapid protrusion and with the activity of RhoA. Ensuing PKA phosphorylation of RhoA and the resulting increased interaction between RhoA and RhoGDI (Rho GDP-dissociation inhibitor) establish a negative feedback mechanism that controls the cycling of RhoA activity at the leading edge. Thus, cooperation between PKA, RhoA and RhoGDI forms a pacemaker that governs the morphodynamic behaviour of migrating cells.

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Figure 1: PKA controls the characteristic time and magnitude of protrusive activity.
Figure 2: Temporal correlation of PKA activity with cell protrusion.
Figure 3: PKA phosphorylation of RhoA regulates the dynamics of RhoA activity at the cell edge.
Figure 4: Increased RhoGDI complements the effect of overexpression of RhoAS188A on protrusion duration.
Figure 5: Increased RhoGDI does not complement the effect of overexpression of RhoAR68E on protrusion duration.
Figure 6: Overexpression of RhoGDI compensates for the effect of PKA inhibition on protrusion morphodynamics.
Figure 7: PKA activation requires both protrusion and adhesion of the leading edge to create a self-inhibiting feedback loop.

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Change history

  • 17 October 2011

    In the version of this article initially published online, supplementary Fig. 3A erroneously depicted the pmAKAR3 activity reporter as containing CyPet and YPet fluorophores, instead of ECFP and cpVenus E172.

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Acknowledgements

Supported by grants from the NIH (AR27214, M.H.G.; HL31950, M.H.G.; GM071868, G.D.; F32 HL094012-01, E.T.) and the Cell Migration Consortium (M.H.G. and G.D.).

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E.T. conceived the project, carried out experiments, analysed data and wrote the paper. M.S-G., C.K. and M.M. analysed data and wrote software. O.P. conceived experiments and provided critical reagents. A.G. and E.G. manufactured micropatterned substrates. G.D. conceived experiments, analysed data and edited the paper. M.H.G. conceived experiments, analysed data and wrote the paper.

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Correspondence to Mark H. Ginsberg.

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Tkachenko, E., Sabouri-Ghomi, M., Pertz, O. et al. Protein kinase A governs a RhoA–RhoGDI protrusion–retraction pacemaker in migrating cells. Nat Cell Biol 13, 660–667 (2011). https://doi.org/10.1038/ncb2231

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