Two-photon fluorescence images of the neurons expressing Camuiα4 mutants in hippocampal slice culture.

<p><b>(A)</b> Representative images of soma of the CA1 pyramidal neuron expressing Camuiα-mGsR or Camuiα4m-mGsR. Either Camuiα-mGsR or Camuiα4m-mGsR was transfected using a gene gun, and 2 days after transfection, cells were imaged with a 2-photon fluorescence microscope. Scale bar = 20 μm. <b>(B)</b> Representative images of apical dendrites from CA1 pyramidal neurons expressing Camuiα-mGsR or Camuiα4m-mGsR. Scale bar = 10 μm. <b>(C)</b> Spine density in apical dendrites of neurons. The numbers of samples (spines/neurons) are 1541/7 for Camuiα-mGsR and 1827/7 for Camuiα4m-mGsR. There were no significant differences between the spine density in neurons expressing Camuiα and Camuiα4m (p > 0.05, <i>t</i>-test, n.s., not significant). Error bars indicate S.E.M.</p

Oligomerization status of CaMKIIα mutants in living cells.

<p><b>(A)</b> Schematic drawing of the experimental design. Inter-molecular FRET between mEGFP-fused CaMKIIα (or CaMKIIα4m mutant) and sREACh-fused CaMKIIα (or CaMKIIα4m mutant) were monitored by 2-photon fluorescence lifetime microscope. mEGFP-CaMKIIα (1–325 a.a.) lacking association domain was used as negative control. <b>(B)</b> Representative fluorescence lifetime images of HeLa cells expressing the pair of CaMKIIα. The ratio of FRET donor and acceptor DNA plasmids transfected was 1:2. The binding fractions were measured from fluorescence decay curves as described elsewhere [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121109#pone.0121109.ref022" target="_blank">22</a>]. Scale bar = 50 μm. <b>(C)</b> Comparison of the binding fractions. The number of the cells analyzed (#1–#4) are 83, 89, 82, and 103, respectively. Error bars indicate S.E.M. For the statistical test, one-way ANOVA with Scheffé’s post-hoc test with *p < 0.05 was used.</p

Native-PAGE analysis of association domain mutants.

<p><b>(A)</b> His-tagged association domain mutants (molecular weight; 19,800) expressed in HeLa cells were purified with Ni-sepharose resin as described in Materials and Methods. Subsequently, native-PAGE and silver staining were carried out. The two arrowheads indicate oligomer forms of the association domain. <b>(B)</b> SDS-PAGE of purified association domain mutants. The same batch of samples as in (A) was used.</p

Schematic diagram of CaMKIIα FRET sensor evolution.

<p><b>(A)</b> The CaMKIIα fluorescence resonance energy transfer (FRET) sensor (Camuiα) consists of FRET donor (mEGFP) and acceptor fluorophores (sREACh) and full-length CaMKIIα, which contains kinase, regulatory, and association domains. For improving the folding efficiency of the sensor, the association domain was amplified by error-prone PCR and subcloned into a bacterial expression vector. Subsequently, the ligated DNA was transformed in <i>Escherichia coli</i> to make a library. Bright red colonies were isolated from the library and analyzed by DNA sequencing. The identified mutations were then incorporated into the FRET sensor, leading to an increase in its folding efficiency. This scheme is based on an assumption that the folding efficiency of the association domain is coupled to that of mCherry. <b>(B)</b> The crystal structure of the association domain of human CaMKIIβ7 (PDB code: 3SOA). Note that this sequence is identical to the corresponding region in rat CaMKIIα (345–474) used in this study. The positions of four mutations (F394L, I419V, A430T, and I434T) found in this study are shown in red.</p

Chemical unfolding and refolding of association domain mutant.

<p><b>(A, B)</b> Tryptophan fluorescence spectra of association domain (A) and its mutant (F394L/I419V/A430T/I434T) (B) in increasing urea concentrations (0–8 M with 0.5 M intervals) were recorded at 295 nm. <b>(C)</b> Equilibrium unfolding transitions defined as the ratio of fluorescence intensities at 335 nm and 353 nm are plotted as a function of denaturant concentration. Error bars indicate S.E.M. for three independent experiments. <b>(D, E)</b> Refolding of denatured association domain mutant was recorded by far-UV circular dichroism (CD) spectra. Native proteins (black) were denatured in 8 M urea and 1 mM DTT (gray), and refolded by 5-fold dilution (red). The lower spectral regions monitored with over 400 V of the photomultiplier tube voltage were cut out from respective spectra.</p

Folding parameters of the association domain (AD)-sfGFP mutants.

<p>T_1/2: Half-time for sfGFP recovery</p><p>% Recovery at ∞ minutes was estimated by exponential fitting</p><p>Folding parameters of the association domain (AD)-sfGFP mutants.</p

Camuiα4m-mGsR activation by single-spine stimulation.

<p><b>(A)</b> Fluorescence lifetime images of neurons expressing Camuiα4m-mGsR together with tandem mCherry during the induction of spine enlargement by 2-photon glutamate uncaging. The arrowhead indicates the stimulated spine. <b>(B)</b> Time courses of Camuiα mutants activities measured as a change in the fluorescence lifetime of Camuiα-mGsR, Camuiα4m-mGsR, Camuiα4m (T286A)-mGsR in the stimulated spines are overlaid with the spine volume change measured as a change in the mCherry fluorescence, respectively. The numbers of samples (spines/neurons) are 6/4 for wild-type, 9/5 for 4m, and 10/6 for 4m with T286A, respectively. Error bars are S.E.M. in <b>B</b>-<b>E</b>. <b>(C)</b> Peak Camuiα mutants activation (averaged over 0–24 s). Stars denote statistical significance (p < 0.05, ANOVA followed by the least significant difference (LSD) test). (<b>D)</b> Averaged time courses of spine volume change in the same experiments in <b>B</b>. (<b>E)</b> Transient (volume change averaged over 2–4 min subtracted by that over 15–18 min) and sustained volume change (volume change averaged over 15–18 min). Stars denote statistical significance (p < 0.05, ANOVA followed by the LSD tests).</p

Expression patterns of the Camuiα-mGsR mutants in HeLa cells.

<p><b>(A)</b> Camuiα mutants were expressed in HeLa cells and imaged under an epifluorescence microscope. Scale bar = 50 μm. The arrowheads highlight cells that exhibit aggregation of the FRET sensor in a dotted pattern. The arrows indicate the cells evenly expressing the Camuiα mutant. <b>(B)</b> The number of cells with aggregated Camuiα mutants in the dotted pattern were counted (wild-type, 16.6% (141/848 cells); F394L, 12.1% (97/800 cells); I419V, 17.4% (139/800 cells); A430T, 12.1% (87/717 cells); I434T, 12.8% (108/844 cells); F394L/I434T, 10.8% (81/748 cells); A430T/I434T, 12.9% (110/850 cells); F394L/A430T/I434T, 10.3% (79/765 cells); Camuiα4m, 0.8% (8/1038 cells)). <b>(C)</b> The fluorescence intensity distributions of the individual cells expressing Camuiα-mGsR and Camuiα4m-mGsR. The cells evenly expressing Camuiα mutant are designated EV cells, and the cells exhibiting aggregated Camuiα mutant are designated AG cells. Thick black horizontal bars show average intensities. The number of the cells analyzed (Camuiα (EV), Camuiα (AG), Camuiα4m) are 250, 62, and 401, respectively. For the statistical test, one-way ANOVA with Scheffé’s post-hoc test with *p < 0.05 was used. The cell exhibiting the aggregated form of Camuiα4m was not observed in this experiment.</p

Characterization of Camuiα4m-mGmC mutant activation in living cells.

<p>The activities of mEGFP- and mCherry-based Camuiα mutants (Camuiα-mGmC, Camuiα4m-mGmC) were monitored. The experimental conditions, analyses, and Fig. captions are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121109#pone.0121109.g006" target="_blank">Fig. 6</a>, except for the replacement of sREACh by mCherry in Camuiα mutants.</p

Comparative characterization of the folding properties of sfGFP fused to the association domain mutants.

<p>Refolding of the denatured sfGFP in the association domain mutants was initiated by a 100-fold dilution with renaturation buffer at room temperature. Fluorescence recovery of sfGFP in the association domain mutants was monitored by a fluorescence spectrometer (excitation at 488 nm, emission at 510 nm). Wild-type (no mutations), orange; 3 mutations (F394L, A430T, and I434T), green; 4 mutations (F394L, I419V, A430T, and I434T), blue.</p