Abstract
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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1038%2Fnature09823/MediaObjects/41586_2011_Article_BFnature09823_Fig1_HTML.jpg)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1038%2Fnature09823/MediaObjects/41586_2011_Article_BFnature09823_Fig2_HTML.jpg)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1038%2Fnature09823/MediaObjects/41586_2011_Article_BFnature09823_Fig3_HTML.jpg)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1038%2Fnature09823/MediaObjects/41586_2011_Article_BFnature09823_Fig4_HTML.jpg)
Similar content being viewed by others
References
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)
Luo, L. Rho GTPases in neuronal morphogenesis. Nature Rev. Neurosci. 1, 173–180 (2000)
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)
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)
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)
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)
Asrar, S. et al. Regulation of hippocampal long-term potentiation by p21-activated protein kinase 1 (PAK1). Neuropharmacology 56, 73–80 (2009)
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)
Nadif Kasri, N. & Van Aelst, L. Rho-linked genes and neurological disorders. Pflugers Arch. 455, 787–797 (2008)
Hotulainen, P. & Hoogenraad, C. C. Actin in dendritic spines: connecting dynamics to function. J. Cell Biol. 189, 619–629 (2010)
Yasuda, R. et al. Supersensitive Ras activation in dendrites and spines revealed by two-photon fluorescence lifetime imaging. Nature Neurosci. 9, 283–291 (2006)
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)
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)
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)
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)
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)
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)
McAllister, A. K. Biolistic transfection of neurons. Sci. STKE 2000, pl1 (2000)
Harvey, C. D. & Svoboda, K. Locally dynamic synaptic learning rules in pyramidal neuron dendrites. Nature 450, 1195–1200 (2007)
Patterson, G. H. & Lippincott-Schwartz, J. A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297, 1873–1877 (2002)
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)
Wilde, C., Genth, H., Aktories, K. & Just, I. Recognition of RhoA by Clostridium botulinum C3 exoenzyme. J. Biol. Chem. 275, 16478–16483 (2000)
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)
Nikolić, M. The Pak1 kinase: an important regulator of neuronal morphology and function in the developing forebrain. Mol. Neurobiol. 37, 187–202 (2008)
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)
Iden, S. & Collard, J. G. Crosstalk between small GTPases and polarity proteins in cell polarization. Nature Rev. Mol. Cell Biol. 9, 846–859 (2008)
Tamura, M. et al. Development of specific Rho-kinase inhibitors and their clinical application. Biochim. Biophys. Acta 1754, 245–252 (2005)
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)
Sabatini, B. L., Oertner, T. G. & Svoboda, K. The life cycle of Ca(2+) ions in dendritic spines. Neuron 33, 439–452 (2002)
Stoppini, L., Buchs, P. A. & Muller, D. A simple method for organotypic cultures of nervous tissue. J. Neurosci. Methods 37, 173–182 (1991)
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)
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)
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)
O'Brien, J. A. & Lummis, S. C. Biolistic transfection of neuronal cultures using a hand-held gene gun. Nature Protocols 1, 977–981 (2006)
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)
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)
Pologruto, T. A., Sabatini, B. L. & Svoboda, K. ScanImage: flexible software for operating laser scanning microscopes. Biomed. Eng. Online 2, 13 (2003)
Lakowicz, J. R. Principles of Fluorescence Spectroscopy (Plenum, 2006)
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)
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)
Acknowledgements
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.).
Author information
Authors and Affiliations
Contributions
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.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Information
The file contains a Supplementary Note, Supplementary References, Supplementary Table 1 and Supplementary Figures 1-16 with legends. (PDF 1142 kb)
Rights and permissions
About this article
Cite this article
Murakoshi, H., Wang, H. & Yasuda, R. Local, persistent activation of Rho GTPases during plasticity of single dendritic spines. Nature 472, 100–104 (2011). https://doi.org/10.1038/nature09823
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature09823
This article is cited by
-
Functional and phosphoproteomic analysis of β-adrenergic receptor signaling at excitatory synapses in the CA1 region of the ventral hippocampus
Scientific Reports (2023)
-
Linking spontaneous and stimulated spine dynamics
Communications Biology (2023)
-
Generalized extinction of fear memory depends on co-allocation of synaptic plasticity in dendrites
Nature Communications (2023)
-
Involvement of Paired Immunoglobulin-Like Receptor B in Cognitive Dysfunction Through Hippocampal-Dependent Synaptic Plasticity Impairments in Mice Subjected to Chronic Sleep Restriction
Molecular Neurobiology (2023)
-
Genome-wide characterization of the Rho family in cotton provides insights into fiber development
Journal of Cotton Research (2022)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.