The role of CaMKII as an F-actin-bundling protein
crucial for maintenance of dendritic spine structure
Ken-Ichi Okamoto*, Radhakrishnan Narayanan*, Sang H. Lee†, Kazuyoshi Murata‡, and Yasunori Hayashi*§
*RIKEN-MIT Neuroscience Research Center, The Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences, Massachusetts
Institute of Technology, Cambridge, MA 02139;†Department of Pharmacology, BSB 608, Medical College of Wisconsin, 8701 Watertown Plank Road,
Milwaukee, WI 53226; and‡Whitehead Institute, Department of Biology and Division of Biological Engineering, Massachusetts Institute
of Technology, Cambridge, MA 02142
Communicated by Susumu Tonegawa, Massachusetts Institute of Technology, Cambridge, MA, February 27, 2007 (received for review October 25, 2006)
Ca2?-calmodulin-dependent protein kinase II (CaMKII) is a serine/
threonine protein kinase critically involved in synaptic plasticity in
the brain. It is highly concentrated in the postsynaptic density
fraction, exceeding the amount of any other signal transduction
molecules. Because kinase signaling can be amplified by catalytic
reaction, why CaMKII exists in such a large quantity has been a
mystery. Here, we provide biochemical evidence that CaMKII is
capable of bundling F-actin through a stoichiometric interaction.
Consistent with this evidence, in hippocampal neurons, RNAi-
mediated down-regulation of CaMKII leads to a reduction in the
volume of dendritic spine head that is mediated by F-actin dynam-
ics. An overexpression of CaMKII slowed down the actin turnover
in the spine head. This activity was associated with ? subunit of
CaMKII in a manner requiring its actin-binding and association
domains but not the kinase domain. This finding indicates that
CaMKII serves as a central signaling molecule in both functional
and structural changes during synaptic plasticity.
cytoskeleton ? plasticity ? synapse
This phenomenon, long-term potentiation (LTP), has been con-
sidered as a cellular counterpart of memory, and its molecular
mechanisms have been intensively studied. A strong postsynaptic
depolarization caused by high-frequency input induces a Ca2?
influx through postsynaptic NMDA receptors, which then triggers
a series of biochemical processes, including an activation of a
serine/threonine protein kinase, Ca2?/calmodulin-dependent pro-
tein kinase II (CaMKII). The activation of CaMKII is followed by
a series of autophosphorylation events, which enables active
CaMKII to stay activated until all subunits are dephosphorylated
(2). This activated CaMKII phosphorylates AMPA receptor and
various other postsynaptic proteins, which triggers a mechanism
synaptic transmission (2–9).
In addition to the functional change typically measured by
electrophysiological recordings, recent works revealed another
aspect of synaptic plasticity: the structural changes associated with
the functional modifications (10–13). During LTP of hippocampal
CA1 pyramidal cells, a dendritic spine, the tiny protrusion where
most of excitatory synapses are harbored in this class of neurons,
expands within ?60 s after the LTP-inducing stimulation (12, 13).
Antagonists for both NMDA receptor and CaMKII blocked the
effect of tetanic stimulation; therefore, the activation of NMDA
receptor and CaMKII are both important for structural plasticity
(12, 13). This observation encouraged us to explore the role that
CaMKII activity plays in the regulation of postsynaptic structure.
CaMKII is found in a large quantity in postsynaptic density
fraction much more than any other signal transduction molecules,
almost comparable with cytoskeletal components (14, 15). This
knowledge led us and others to speculate that CaMKII may serve
not only as a signal transduction molecule but also as a structural
element at synapses (2, 14). In relation to this speculation, it has
n hippocampal CA1 pyramidal cells, a transient burst of synaptic
input potentiates the efficiency of subsequent transmission (1).
sequence present in the ? subunit (16–20), but this interaction has
been considered to anchor CaMKII to the postsynaptic sites.
However, considering that CaMKII is an oligomeric protein com-
posed of 12–14 subunits with rotational symmetry (2), it is possible
that one oligomer can simultaneously bind to multiple F-actin,
thereby acting as an F-actin-bundling protein at postsynaptic sites.
Here, we show evidence that CaMKII is in fact not only a
signaling protein, but also a structural protein that bundles F-actin
through a specific and stoichiometric interaction mediated by the ?
subunit, and that such capacity is required for the maintenance of
the postsynaptic architecture of the dendritic spine. This work
ascribes a specific, previously undescribed role for CaMKII as a
cytoskeletal component in the postsynaptic structure.
F-Actin Is Bundled by CaMKII. To demonstrate the F-actin-bundling
activity of CaMKII, we first used the conventional F-actin cosedi-
mentation assay (21). Purified non-muscle actin was polymerized
and coincubated with purified CaMKII (heteromeric complex of ?
and ?). The resultant complex was centrifuged at 10,000 ? g, at
which linear, unbundled F-actin stays in supernatant but bundled
F-actin precipitates (21). Upon separation of both fractions on an
was absent, but in the pellet when CaMKII was present (Fig. 1a).
CaMKII without F-actin did not form a pellet, indicating that
CaMKII itself did not form complexes that precipitate, and that an
interaction between F-actin and CaMKII was indeed needed to
form the pellet. This activity was associated with ? subunit of
CaMKII, but not with the ? subunit, a finding consistent with the
presence of an F-actin binding sequence in CaMKII? but not in ?.
The concentration response curve of CaMKII? at a fixed concen-
tration of actin indicates that the maximum effect was attained at
a molar ratio of actin to CaMKII? of higher than 1:1 (or 1:?0.08,
as CaMKII? holoenzyme; Fig. 1b). This stoichiometry of actin and
CaMKII is reasonable in view of the amount of these two proteins
present in the postsynaptic density fraction (15).
We next attempted to visualize the F-actin bundling by CaMKII.
We negatively stained F-actin filaments formed in the presence or
absence of CaMKII and observed them using electron microscopy
In contrast, in the presence of CaMKII, F-actin formed clear
bundles. Again, this activity was associated with CaMKII?; homo-
Author contributions: K.-I.O. and R.N. contributed equally to this work; K.-I.O., R.N., S.H.L.,
and Y.H. designed research; K.-I.O., R.N., and S.H.L. performed research; S.H.L. and K.M.
contributed new reagents/analytic tools; K.-I.O. and R.N. analyzed data; and K.-I.O., R.N.,
and Y.H. wrote the paper.
The authors declare no conflict of interest.
Abbreviations: LTP, long-term potentiation; CaMKII, Ca2?-calmodulin-dependent protein
kinase II; FRAP, fluorescent recovery after photobleaching; shRNA, short hairpin RNA; CBB,
Coomassie Brilliant Blue.
§To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
April 10, 2007 ?
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we added varying concentrations of CaMKII to 3 ?M F-actin. The
CBB-stained gels were scanned, and the band intensities were
measured by using Metamorph (Molecular Devices).
Electron Microscopy. Actin and CaMKII were allowed to react for
was adsorbed onto freshly glow-discharged, carbon-coated copper
grids for 30 s and the excess sample was removed by wicking with
filter paper. The bound particles were stained with 2% uranyl
acetate for 30 s, and the excess stain was removed with filter paper.
Images were recorded on a slow-scan CCD camera with a JEOL
electron microscope operating at 200 kV at a magnification of
Actin Assembly and Disassembly. In vitro actin assembly and disas-
was monitored by the increase in fluorescence of 5% pyrenyl-
labeled nonskeletal muscle actin (Cytoskeleton). Nonskeletal mus-
cle actin (50 ?M) in G-buffer (10 mM Tris?HCl/0.2 mM CaCl2/0.2
mM DTT/0.2 mM ATP, pH 7.5) was thawed overnight at 4°C,
diluted to 22.5 ?M in G-buffer and precleared by centrifugation at
200,000 ? g for 60 min at 4°C. Actin (2 ?M) was mixed with 2 ?M
CaMKII? and the reaction was initiated by transfer to a quartz
fluorometer cuvette (3-mm light path) containing 20? initiation
mix (40 mM MgCl2/1 M KCl/10 mM ATP). Pyrene fluorescence
was recorded at 407 nm with an excitation at 365 nm at room
temperature. The amounts of sedimentable F-actin at 100,000 ? g
were similar in the presence or absence of CaMKII? (data not
shown). For actin disassembly assays, 2 ?M CaMKII? was incu-
bated for 5 min in the presence of preassembled actin filaments (2
was plotted. Disassembly was induced by dilution in F-buffer
(G-buffer containing 1? initiation mix) to a final concentration of
0.2 ?M. The initial F-actin fluorescence at the beginning of the
reaction was used as 100% to plot the disassembly reaction.
mM Hepes, 1 mM Mg-acetate, 0.1 mM EGTA, 0.2 mM Ca2?, 50
?M ATP, and 10 ?M (for Fig. 5b) or 1 ?M (others) calmodulin
adding 1 mM EGTA at the indicated time after start. Phosphory-
lation of CaMKII in the presence or absence of F-actin during
kinase reaction did not change the sedimentation characteristics of
Neuronal Imaging. Organotypic slice cultures of hippocampus were
prepared from postnatal day 6–8 rats as described (5, 7, 12). They
were biolistically transfected at days in vitro (DIV)7 days (for
experiment described in Fig. 3) or DIV 4 days (for Fig. 4), and the
imaging experiments were carried out from CA1 pyramidal neu-
rons 4 days (for Fig. 3) or 3–5 days after the transfection (for Fig.
4). For RNAi experiment, the expression vectors of GFP and
shRNA were transfected at 1:5 ratio; for rescue experiments, the
expression vectors for GFP, RNAi, and the original or resistant
cDNA of CaMKII? were transfected at 1:5:5 ratio; for FRAP
experiments, GFP-actin and untagged CaMKII were transfected at
a ratio of 1:5. Neurons were imaged with a custom-made two-
photon microscope 3–5 days after transfection in a solution con-
mM NaHCO3, 1 mM NaH2PO4, and 11 mM glucose, pH 7.4, and
gassed with 95% O2and 5% CO2(12). GFP was imaged at 860 nm
(excitation) and 570 nm short-pass (emission). For analyses, image
stacks typically composed of 15–20 sections taken at 0.5-?m
intervals were z-projected (summation), median-filtered, and back-
ground-subtracted by using Metamorph. For FRAP experiments,
photobleaching was achieved by scanning a spine of interest at
maximal excitation power (?1.4 W of Ti-sapphire laser output) 10
times. The FRAP data were analyzed as described (12). For
morphological analyses, dendritic spines separated well from den-
dritic shaft by at least 0.75 ?m from the edge of the dendritic shaft
were chosen. To measure the size of spine heads, the fluorescence
profile across a line drawn on spines, and adjacent dendritic shafts
were measured in ?10 zoomed images; the ratio of fluorescent
peaks of spine head and dendritic shaft were obtained as an index
of spine head size. To normalize the thickness of dendritic shafts,
the images were obtained at a consistent distance from the soma.
Spine length was defined as the distance from the edge of the
dendritic shaft to the tip of the spine. Spine density was obtained
from 5 x zoomed images. All analyses were done blind to the DNA
construct used. Results are reported as mean? SEM. Statistical
significance was determined by using Dunnett’s test (37) and
defined at P ? 0.05.
We thank Drs. Mariko Hayashi, Avital Rodal, Atsuhiko Ishida, Bernardo
Sabatini, J. Troy Littleton, Morgan Sheng, Susumu Tonegawa, Neal Wax-
of resources; Mr. Travis Emery for editing; and Ms. Cortina McCurry for
participating in the early part of this study. This work was supported by
grants from RIKEN and the Ellison Medical Foundation and by National
Institutes of Health Grant RO1 DA017310 (to Y.H.).
1. Bliss TV, Collingridge GL (1993) Nature 361:31–39.
2. Lisman J, Schulman H, Cline H (2002) Nat Rev Neurosci 3:175–190.
3. McGlade-McCulloh E, Yamamoto H, Tan SE, Brickey DA, Soderling TR (1993)
4. Benke TA, Lu ¨thi A, Isaac JT, Collingridge GL (1998) Nature 393:793–797.
5. Hayashi Y, Shi SH, Esteban JA, Piccini A, Poncer JC, Malinow R (2000) Science
6. Malinow R, Malenka RC (2002) Annu Rev Neurosci 25:103–126.
7. Shi SH, Hayashi Y, Petralia RS, Zaman SH, Wenthold RJ, Svoboda K, Malinow R
(1999) Science 284:1811–1816.
8. Derkach V, Barria A, Soderling TR (1999) Proc Natl Acad Sci USA 96:3269–3274.
9. Barria A, Muller D, Derkach V, Griffith LC, Soderling TR (1997) Science
10. Lamprecht R, LeDoux J (2004) Nat Rev Neurosci 5:45–54.
11. Yuste R, Bonhoeffer T (2001) Annu Rev Neurosci 24:1071–1089.
12. Okamoto K, Nagai T, Miyawaki A, Hayashi Y (2004) Nat Neurosci 7:1104–1112.
13. Matsuzaki M, Honkura N, Ellis-Davies GC, Kasai H (2004) Nature 429:761–766.
14. Erondu NE, Kennedy MB (1985) J Neurosci 5:3270–3277.
15. Peng J, Kim MJ, Cheng D, Duong DM, Gygi SP, Sheng M (2004) J Biol Chem
16. Shen K, Teruel MN, Subramanian K, Meyer T (1998) Neuron 21:593–606.
17. Urushihara M, Yamauchi T (2001) Eur J Biochem 268:4802–4808.
18. Ohta Y, Nishida E, Sakai H (1986) FEBS Lett 208:423–426.
19. Shen K, Meyer T (1999) Science 284:162–166.
20. O’Leary H, Lasda E, Bayer KU (2006) Mol Biol Cell 17:4656–4665.
21. Ba ¨hler M, Greengard P (1987) Nature 326:704–707.
22. Sagot I, Rodal AA, Moseley J, Goode BL, Pellman D (2002) Nat Cell Biol
23. Hayashi Y, Majewska AK (2005) Neuron 46:529–532.
24. Capani F, Martone ME, Deerinck TJ, Ellisman MH (2001) J Comp Neurol
25. Star EN, Kwiatkowski DJ, Murthy VN (2002) Nat Neurosci 5:239–246.
26. Butz S, Okamoto M, Su ¨dhof TC (1998) Cell 94:773–782.
28. Thiagarajan TC, Piedras-Renteria ES, Tsien RW (2002) Neuron 36:1103–1114.
29. Otmakhov N, Tao-Cheng JH, Carpenter S, Asrican B, Dosemeci A, Reese TS,
Lisman J (2004) J Neurosci 24:9324–9331.
30. Hudmon A, Lebel E, Roy H, Sik A, Schulman H, Waxham MN, De Koninck P
(2005) J Neurosci 25:6971–6983.
31. Bayer KU, De Koninck P, Leonard AS, Hell JW, Schulman H (2001) Nature
32. Dhavan R, Greer PL, Morabito MA, Orlando LR, Tsai LH (2002) J Neurosci
33. Walikonis RS, Oguni A, Khorosheva EM, Jeng CJ, Asuncion FJ, Kennedy MB
(2001) J Neurosci 21:423–433.
34. Robison AJ, Bass MA, Jiao Y, MacMillan LB, Carmody LC, Bartlett RK, Colbran
RJ (2005) J Biol Chem 280:35329–35336.
35. Takao K, Okamoto K, Nakagawa T, Neve RL, Nagai T, Miyawaki A, Hashikawa
T, Kobayashi S, Hayashi Y (2005) J Neurosci 25:3107–3112.
36. Waxham MN, Tsai AL, Putkey JA (1998) J Biol Chem 273:17579–17584.
37. Dunnett CW (1955) J Am Stat Assoc 50:1096–1121.
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