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Local, persistent activation of Rho GTPases during plasticity of single dendritic spines


The Rho family of GTPases have important roles in the morphogenesis of the dendritic spines1,2,3 of neurons in the brain and synaptic plasticity4,5,6,7,8,9 by modulating the organization of the actin cytoskeleton10. Here we used two-photon fluorescence lifetime imaging microscopy11,12,13 to monitor the activity of two Rho GTPases—RhoA and Cdc42—in single dendritic spines undergoing structural plasticity associated with long-term potentiation in CA1 pyramidal neurons in cultured slices of rat hippocampus. When long-term volume increase was induced in a single spine using two-photon glutamate uncaging14,15, RhoA and Cdc42 were rapidly activated in the stimulated spine. These activities decayed over about five minutes, and were then followed by a phase of persistent activation lasting more than half an hour. Although active RhoA and Cdc42 were similarly mobile, their activity patterns were different. RhoA activation diffused out of the stimulated spine and spread over about 5 µm along the dendrite. In contrast, Cdc42 activation was restricted to the stimulated spine, and exhibited a steep gradient at the spine necks. Inhibition of the Rho–Rock pathway preferentially inhibited the initial spine growth, whereas the inhibition of the Cdc42–Pak pathway blocked the maintenance of sustained structural plasticity. RhoA and Cdc42 activation depended on Ca2+/calmodulin-dependent kinase (CaMKII). Thus, RhoA and Cdc42 relay transient CaMKII activation13 to synapse-specific, long-term signalling required for spine structural plasticity.

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Figure 1: Spatiotemporal dynamics of RhoA activation during long-term structural plasticity induced in single spines
Figure 2: Spatiotemporal dynamics of Cdc42 activation during long-term structural plasticity induced in single spines.
Figure 3: Spatial profile of RhoA and Cdc42 activities.
Figure 4: The effect of Rho GTPase inhibition for structural plasticity of spine head enlargement.


  1. 1

    Tashiro, A. & Yuste, R. Regulation of dendritic spine motility and stability by Rac1 and Rho kinase: evidence for two forms of spine motility. Mol. Cell. Neurosci. 26, 429–440 (2004)

    CAS  Article  Google Scholar 

  2. 2

    Luo, L. Rho GTPases in neuronal morphogenesis. Nature Rev. Neurosci. 1, 173–180 (2000)

    ADS  CAS  Article  Google Scholar 

  3. 3

    Saneyoshi, T., Fortin, D. A. & Soderling, T. R. Regulation of spine and synapse formation by activity-dependent intracellular signaling pathways. Curr. Opin. Neurobiol. 20, 108–115 (2009)

    Article  Google Scholar 

  4. 4

    Fortin, D. A. et al. Long-term potentiation-dependent spine enlargement requires synaptic Ca2+-permeable AMPA receptors recruited by CaM-kinase I. J. Neurosci. 30, 11565–11575 (2010)

    CAS  Article  Google Scholar 

  5. 5

    O’Kane, E. M., Stone, T. W. & Morris, B. J. Activation of Rho GTPases by synaptic transmission in the hippocampus. J. Neurochem. 87, 1309–1312 (2003)

    Article  Google Scholar 

  6. 6

    Rex, C. S. et al. Different Rho GTPase-dependent signaling pathways initiate sequential steps in the consolidation of long-term potentiation. J. Cell Biol. 186, 85–97 (2009)

    CAS  Article  Google Scholar 

  7. 7

    Asrar, S. et al. Regulation of hippocampal long-term potentiation by p21-activated protein kinase 1 (PAK1). Neuropharmacology 56, 73–80 (2009)

    CAS  Article  Google Scholar 

  8. 8

    Wang, H. G. et al. Presynaptic and postsynaptic roles of NO, cGK, and RhoA in long-lasting potentiation and aggregation of synaptic proteins. Neuron 45, 389–403 (2005)

    CAS  Article  Google Scholar 

  9. 9

    Nadif Kasri, N. & Van Aelst, L. Rho-linked genes and neurological disorders. Pflugers Arch. 455, 787–797 (2008)

    CAS  Article  Google Scholar 

  10. 10

    Hotulainen, P. & Hoogenraad, C. C. Actin in dendritic spines: connecting dynamics to function. J. Cell Biol. 189, 619–629 (2010)

    CAS  Article  Google Scholar 

  11. 11

    Yasuda, R. et al. Supersensitive Ras activation in dendrites and spines revealed by two-photon fluorescence lifetime imaging. Nature Neurosci. 9, 283–291 (2006)

    CAS  Article  Google Scholar 

  12. 12

    Harvey, C. D., Yasuda, R., Zhong, H. & Svoboda, K. The spread of Ras activity triggered by activation of a single dendritic spine. Science 321, 136–140 (2008)

    ADS  CAS  Article  Google Scholar 

  13. 13

    Lee, S. J., Escobedo-Lozoya, Y., Szatmari, E. M. & Yasuda, R. Activation of CaMKII in single dendritic spines during long-term potentiation. Nature 458, 299–304 (2009)

    ADS  CAS  Article  Google Scholar 

  14. 14

    Matsuzaki, M., Honkura, N., Ellis-Davies, G. C. & Kasai, H. Structural basis of long-term potentiation in single dendritic spines. Nature 429, 761–766 (2004)

    ADS  CAS  Article  Google Scholar 

  15. 15

    Honkura, N., Matsuzaki, M., Noguchi, J., Ellis-Davies, G. C. & Kasai, H. The subspine organization of actin fibers regulates the structure and plasticity of dendritic spines. Neuron 57, 719–729 (2008)

    CAS  Article  Google Scholar 

  16. 16

    Heasman, S. J. & Ridley, A. J. Mammalian Rho GTPases: new insights into their functions from in vivo studies. Nature Rev. Mol. Cell Biol. 9, 690–701 (2008)

    CAS  Article  Google Scholar 

  17. 17

    Okamoto, K., Nagai, T., Miyawaki, A. & Hayashi, Y. Rapid and persistent modulation of actin dynamics regulates postsynaptic reorganization underlying bidirectional plasticity. Nature Neurosci. 7, 1104–1112 (2004)

    CAS  Article  Google Scholar 

  18. 18

    McAllister, A. K. Biolistic transfection of neurons. Sci. STKE 2000, pl1 (2000)

    CAS  Article  Google Scholar 

  19. 19

    Harvey, C. D. & Svoboda, K. Locally dynamic synaptic learning rules in pyramidal neuron dendrites. Nature 450, 1195–1200 (2007)

    ADS  CAS  Article  Google Scholar 

  20. 20

    Patterson, G. H. & Lippincott-Schwartz, J. A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297, 1873–1877 (2002)

    ADS  CAS  Article  Google Scholar 

  21. 21

    Michaelson, D. et al. Differential localization of Rho GTPases in live cells: regulation by hypervariable regions and RhoGDI binding. J. Cell Biol. 152, 111–126 (2001)

    CAS  Article  Google Scholar 

  22. 22

    Wilde, C., Genth, H., Aktories, K. & Just, I. Recognition of RhoA by Clostridium botulinum C3 exoenzyme. J. Biol. Chem. 275, 16478–16483 (2000)

    CAS  Article  Google Scholar 

  23. 23

    Elliot-Smith, A. E., Mott, H. R., Lowe, P. N., Laue, E. D. & Owen, D. Specificity determinants on Cdc42 for binding its effector protein ACK. Biochemistry 44, 12373–12383 (2005)

    CAS  Article  Google Scholar 

  24. 24

    Nikolić, M. The Pak1 kinase: an important regulator of neuronal morphology and function in the developing forebrain. Mol. Neurobiol. 37, 187–202 (2008)

    Article  Google Scholar 

  25. 25

    Kreis, P. et al. The p21-activated kinase 3 implicated in mental retardation regulates spine morphogenesis through a Cdc42-dependent pathway. J. Biol. Chem. 282, 21497–21506 (2007)

    CAS  Article  Google Scholar 

  26. 26

    Iden, S. & Collard, J. G. Crosstalk between small GTPases and polarity proteins in cell polarization. Nature Rev. Mol. Cell Biol. 9, 846–859 (2008)

    CAS  Article  Google Scholar 

  27. 27

    Tamura, M. et al. Development of specific Rho-kinase inhibitors and their clinical application. Biochim. Biophys. Acta 1754, 245–252 (2005)

    CAS  Article  Google Scholar 

  28. 28

    Deacon, S. W. et al. An isoform-selective, small-molecule inhibitor targets the autoregulatory mechanism of p21-activated kinase. Chem. Biol. 15, 322–331 (2008)

    CAS  Article  Google Scholar 

  29. 29

    Sabatini, B. L., Oertner, T. G. & Svoboda, K. The life cycle of Ca(2+) ions in dendritic spines. Neuron 33, 439–452 (2002)

    CAS  Article  Google Scholar 

  30. 30

    Stoppini, L., Buchs, P. A. & Muller, D. A simple method for organotypic cultures of nervous tissue. J. Neurosci. Methods 37, 173–182 (1991)

    CAS  Article  Google Scholar 

  31. 31

    Zacharias, D. A., Violin, J. D., Newton, A. C. & Tsien, R. Y. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002)

    ADS  CAS  Article  Google Scholar 

  32. 32

    Shaner, N. C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nature Biotechnol. 22, 1567–1572 (2004)

    CAS  Article  Google Scholar 

  33. 33

    Pédelacq, J. D., Cabantous, S., Tran, T., Terwilliger, T. C. & Waldo, G. S. Engineering and characterization of a superfolder green fluorescent protein. Nature Biotechnol. 24, 79–88 (2005)

    Article  Google Scholar 

  34. 34

    O'Brien, J. A. & Lummis, S. C. Biolistic transfection of neuronal cultures using a hand-held gene gun. Nature Protocols 1, 977–981 (2006)

    CAS  Article  Google Scholar 

  35. 35

    Yasuda, R. Imaging spatiotemporal dynamics of neuronal signaling using fluorescence resonance energy transfer and fluorescence lifetime imaging microscopy. Curr. Opin. Neurobiol. 16, 551–561 (2006)

    CAS  Article  Google Scholar 

  36. 36

    Murakoshi, H., Lee, S. J. & Yasuda, R. Highly sensitive and quantitative FRET-FLIM imaging in single dendritic spines using improved non-radiative YFP. Brain Cell Biol. 36, 31–42 (2008)

    Article  Google Scholar 

  37. 37

    Pologruto, T. A., Sabatini, B. L. & Svoboda, K. ScanImage: flexible software for operating laser scanning microscopes. Biomed. Eng. Online 2, 13 (2003)

    Article  Google Scholar 

  38. 38

    Lakowicz, J. R. Principles of Fluorescence Spectroscopy (Plenum, 2006)

    Book  Google Scholar 

  39. 39

    Lee, S. J. & Yasuda, R. Spatiotemporal regulation of signaling in and out of dendritic spines. CaMKII and Ras Open Neurosci. J. 3, 117–127 (2010)

    Article  Google Scholar 

  40. 40

    Zhao, J., Wang, W. N., Tan, Y. C., Zheng, Y. & Wang, Z. X. Effect of Mg(2+) on the kinetics of guanine nucleotide binding and hydrolysis by Cdc42. Biochem. Biophys. Res. Commun. 297, 653–658 (2002)

    CAS  Article  Google Scholar 

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We thank K. Hahn, G. Bokock, A. Aplin, S. Soderling, M. Matsuda and Y. Hayashi for plasmids and reagents, S.-J. Lee for CaMKII activation data, S. Soderling, S. Raghavachari, L. van Aelst, Y. Hayashi, H. Kasai and M. Ehlers for discussion, and H. Hedrick and M. Patterson for comments on the manuscript. We also thank A. Wan for preparing cultured slices and D. Kloetzer for laboratory management. This study was funded by the Howard Hughes Medical Institute, the National Institute of Mental Health, the National Institute of Neurological Disorders and Stroke, the National Institute of Drug Abuse, the Alzheimer’s Association and the Japan Society for the Promotion of Science (H.M.).

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H.M. and R.Y. designed the experiments. H.M. performed the experiments and data analysis. H.W. performed electrophysiological experiments. H.M. and R.Y. wrote the paper.

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Correspondence to Ryohei Yasuda.

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The authors declare no competing financial interests.

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Murakoshi, H., Wang, H. & Yasuda, R. Local, persistent activation of Rho GTPases during plasticity of single dendritic spines. Nature 472, 100–104 (2011).

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