Supplemental Information. Caldendrin Directly Couples. Postsynaptic Calcium Signals. to Actin Remodeling in Dendritic Spines

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1 Neuron, Volume 97 Supplemental Information Caldendrin Directly Couples Postsynaptic Calcium Signals to Actin Remodeling in Dendritic Spines Marina Mikhaylova, Julia Bär, Bas van Bommel, Philipp Schätzle, PingAn YuanXiang, Rajeev Raman, Johannes Hradsky, Anja Konietzny, Egor Y. Loktionov, Pasham Parameshwar Reddy, Jeffrey Lopez-Rojas, Christina Spilker, Oliver Kobler, Syed Ahsan Raza, Oliver Stork, Casper C. Hoogenraad, and Michael R. Kreutz

Figure S1 Figure S1 refers to Figure 1. Caldendrin and cortactin co-localize in hippocampal primary neurons and their interaction is modulated by Ca 2+.

2 Figure S1 Figure S1 refers to Figure 1. Caldendrin and cortactin co-localize in hippocampal primary neurons and their interaction is modulated by Ca 2+. (A) Co-localization of endogenous caldendrin and cortactin in mature DIV17 hippocampal primary neurons. Dendritic spines in which caldendrin and cortactin co-localize are indicated by arrows. (B) Caldendrin and cortactin are enriched in the same subcellular compartments. Immunoblots for subcellular fractionation of rat hippocampus and cortex. PSD-95 blot is included as quality control of the preparation. H: homogenate, P1: nuclear pellet, S2: supernatant 2, P2: crude membrane fraction, SS: synaptosomes, SJ: synaptic junction, PSD: postsynaptic density. (C) N-terminal PxxP motifs of caldendrin mediate the interaction with cortactin in heterologous coimmunoprecipitation assays. (D) Isotermal titration calorimetry (ITC) for Mg 2+ -bound caldendrin in comparison to the calcium binding mutant of caldendrin indicates that the latter one does not bind Ca 2+. (E) Heterologous co-immunoprecipitation shows reduced binding of cortactin to the caldendrin Ca 2+ binding mutant. (F) SPR sensorgrams of titration of caldendrin and GST-cortactin indicate a high association constant and affinity of the interaction in the presence of physiological Ca 2+ concentrations. 1

3 Figure S2 Figure S2 refers to Figure 2. Measurement of the Ca 2+ -binding kinetics by UV flash photolysis of Ca 2+ loaded DM-nitrophen. (A) Coomassie blue stained SDS-PAGE demonstrating the integrity of purified caldendrin before lyophilization and after reconstitution. (B) Fluorescence spectra of caldendrin incubated with different concentrations of Ca 2+ and Mg 2+. Spectra were recorded before lyophilization and after reconstitution. (C) Cartoon demonstrating the experimental system for measuring Ca 2+ on binding kinetics and affinity by UV flash photolysis of Ca 2+ loaded DM-nitrophen. (D) Loading of 10 mm DM-nitrophen with different concentrations of Ca 2+ (concentrations indicated). Intensity profiles were recorded in the presence of 10 µm OGB-5N. (E) UV flash-induced transients in OGB-5N fluorescence in the presence of 59 µm, 29 µm CaM and 60 µm or 30 µm Mg 2+ -bound caldendrin in comparison to control without Ca 2+ binding proteins. Comparable affinities can be seen for both calcium binding proteins. (F) Titration of caldendrin concentration to estimate the amount of protein that is sufficient for interception of uncaged Ca 2+. (G) Immunoblot showing the caldendrin abundance in adult rat and mouse brain. Recombinant purified fulllength caldendrin was used as quantification standard. hom: homogenate, Cx: cortex. (H) Quantification of caldendrin abundance in (G). Data are represented as mean + S.E.M. n = number of experimental replications. 2

Figure S3 Figure S3 refers to Figure 3. Analysis of actin dynamics in vitro. (A) Back-folding of the SH3-domain of cortactin is competed by caldendrin.

4 Figure S3 Figure S3 refers to Figure 3. Analysis of actin dynamics in vitro. (A) Back-folding of the SH3-domain of cortactin is competed by caldendrin. Spectral FRET is measured in extracts of HEK293T cells transfected with CFP-cortactin-YFP in the presence or absence of full-length caldendrin. CFP-YFP tandem or separate CFP and YFP are used as positive and negative controls, respectively. (B) Replating assay in COS-7 cells. Overexpressed caldendrin-gfp and mcherry-cortactin co-localize at the cells edges 4 h after plating. (C) Coomassie blue stained SDS PAGE gel showing purified proteins used for in vitro actin polymerization and depolymerization assays. (D) Controls for in vitro pyrene-labelled actin polymerization assay. n=3 experimental replications. (E) Cofilin-induced actin severing assay analyzed by TIRF microscopy. Indicated groups were imaged before and 300 s after addition of cofilin-1. See also Movie S1. 3

5 Figure S4 Figure S4 refers to Figure 4. Effect of caldendrin overexpression and knockdown on dendritic morphology and synapses in primary neurons. 4

6 (A) Effect of overexpression of different caldendrin-gfp-tagged constructs co-transfected with the volume marker β-galactosidase. Analysis of spine width and length. Data are represented as mean + S.E.M. n numbers of dendritic segments are indicated. 1-way ANOVA with Bonferroni post hoc test. ** p<0.01, *** p< (B) Immunoblot demonstrating the efficiency of different caldendrin knockdown constructs tested in COS-7 cells co-transfected with caldendrin-gfp and shrnas. (C) Representative images of primary hippocampal neurons demonstrating the effect of caldendrin knockdown on dendritic morphology. The simplification of dendritic arbors can be rescued by chronic application of JPK. (D) Sholl analysis for (C). Data are represented as mean ± S.E.M. n numbers of neurons are indicated. Repeated measures 2-way ANOVA with Bonferroni post hoc test. * p<0.05, ** p<0.01, *** p<0.001 (for shrna_scr vs. shrna_cald#5 * for 48 h, # for 72 h; shrna_cald#5 DMSO vs. 50nM JPK: *). (E) Expression of an shrna resistant construct of caldendrin can rescue the knockdown phenotype following 48 h of transfection. (F) Sholl analysis for (E). Data are represented as mean ± S.E.M. n numbers of neurons are indicated. Repeated measures-2-way ANOVA with Bonferroni post hoc test. * p<0.05, ** p<0.01. (G) Expression of an shrna resistant construct of caldendrin can rescue the spine phenotype. (H) Quantification of basal spine and filopodia densities of (G). Data are represented as mean + S.E.M. n numbers of dendritic segments are indicated. 1-way ANOVA with Bonferroni post hoc test. * p<

7 Figure S5 Figure S5 refers to Figure 4. Basic characterization of caldendrin knockout mouse brain morphology. (A) Schematic showing the generation of gene trap caldendrin knockout mice. 6

8 (B) RT-PCR confirming the lack of caldendrin expression in caldendrin knockout mouse brain. nt: no template, R: retina, Hc: hippocampus. (C) Immunoblot of hippocampus (Hc) and cortex (Cx) homogenates from cald +/+ and -/- mice confirming the lack of caldendrin protein expression. (D) Immunoblot showing that caldendrin knockout has no influence on total cortactin expression in brain. (E) Nissl stainings of frontal brain sections of adult mouse. Overview of total section and higher magnification of hippocampus reveal no structural difference. (F) Dendritic morphology of CA1 pyramidal neurons in organotypic hippocampal slice cultures of cald +/+ and - /- mice. Examples of single cell electroporated neurons with mruby2 and corresponding traces are shown. (G) Sholl analysis of (F). Data are represented as mean ± S.E.M. n numbers of neurons (from 8 slices from 4 cald -/- mice or 7 slices from 2 cald +/+ mice) are indicated. Repeated measures-2-way ANOVA. (H) Analysis of dendritic fields in (F). Data are represented as mean + SEM. n numbers as in (G). Unpaired, 2- tailed Student s t-test, ** p<0.01. For further analysis see also Figure 4G+H. (I) Analysis of total dendritic length of CA1 pyramidal neurons depicted in (F). Data are represented as mean + S.E.M. n numbers as in (G). Unpaired, 2-tailed Student s t-test. (J+K) Application of BDNF or bicuculline to primary cultures induces synaptic potentiation measured as increase in surface AMPA receptors. Analysis of surface GluR1 expression in primary hippocampal cultures at DIV17 under basal conditions and after application of 100 ng/ml BDNF or 50 µm bicuculline. n= numbers of images. 1-way ANOVA with Dunnett s post hoc test. * p<0.05, ** p<

9 Figure S6 Figure S6 refers to Figure 5. Further characterization of spines upon caldendrin knockdown and BDNF stimulation. 8

10 (A) Schematic demonstrating the lentiviral knockdown construct with doxicycline-inducible expression of membrane label MARCKS-GFP and constitutive shrna knockdown. (B) Immunoblot showing the knockdown of endogenous caldendrin in hippocampal primary neurons 5 days after infection with lentiviral shrna. (C) Representative overlay images (0 and 60 min) of dendrites of neurons infected with caldendrin, cortactin of control shrna. Red arrows indicate appearing and green arrows disappearing protrusions. (D) Quantification of basal spine number and dynamics (ctr n=19, shrna_cort n=35, shrna_cald n=21; n= number of dendritic segments). Data are represented as mean + S.E.M. 2-way (left graphs) or 1-way ANOVA (right graphs) with Bonferroni post hoc test. * p<0.05, *** p< (E) Quantification of basal spine number of DIV19 primary hippocampal cultures upon knockdown of caldendrin or cortactin (ctr n=15, shrna_cort n=15, shrna_cald n=13; n= number of dendritic segments). Data are represented as mean + S.E.M. 1-way ANOVA with Bonferroni post hoc test. *** p< (F+G) Analysis of spine length and spine head diameter. n= number of spines. 2-way ANOVA with Bonferroni post hoc test. * p<0.05, ** p<0.01, *** p< # comparison to BDNF treated control group. (H) Analysis of BDNF induced spine potentiation in hippocampal primary neurons. Relative ratio of spine types. (I-K) NMDA/AMPA current ratio (I) as well as AMPA-mediated mepsc frequency (J) and amplitude (K) are unchanged in neurons of cald -/- mice. n= number of cells from slices of 2 (I) or 3 (J+K) animals per genotype. (L) Positive and negative controls for FRET AB in individual spines of hippocampal primary cultures (DIV14-17) transfected with indicated constructs upon silencing with TTX and stimulation with bicuculline. See also Figure 6B. 9

11 Figure S7 10

12 Figure S7 refers to Figure 6. Cortactin localization in dendritic spines. (A) GFP-actin fills complete spines and can be used as volume marker. Double transfection of cald +/+ and -/- hippocampal primary cultures DIV12 with mruby2 and GFP-actin under basal conditions. Individual frames from time-lapse imaging and corresponding individual and averaged graphs showing increased fluctuations of GFP-actin and mruby2 in cald -/- neurons. Data are represented as mean ± S.E.M. n= number of spines. (B) Group analysis shows no differences between GFP-actin and mruby2 in either genotype. Both readouts correlate (left). Correlations coefficients are even significantly higher in cald -/- neurons (right). n= number of spines. Unpaired, 2-tailed Student s t-test. *** p< (C) Total deviation from GFP-actin average per spine during 3 min of acquisition is significantly higher in cald - /- than in cald +/+. n=66 (cald +/+), n=75 (cald -/-) spines. Unpaired, 2-tailed Student s t-test, *** p< (D) Bicuculline induced redistribution of endogenous cortactin from dendritic spines in mature rat primary cultures. Representative maximum projections of confocal images. (E) Quantification of synaptic cortactin under mask of bassoon of (D). Data are represented as mean + S.E.M. n numbers of images are indicated. 1-way ANOVA with Bonferroni post hoc test. * p<0.05, *** p< (F) Synaptic activity leads to caldendrin-dependent redistribution of cortactin from synaptic sites. Mature primary hippocampal cultures were stimulated by bath application of 50 µm bicuculline for 5 min and 15 min, fixed and stained for endogenous cortactin and bassoon. Cortactin staining intensities were measured under the mask of bassoon to assess synaptic cortactin levels. Bic.: bicuculline. (G) Quantification of (F). n numbers of analysed dendritic segments are indicated. Data are represented as mean + S.E.M. n numbers of images are indicated. 2-way ANOVA with Bonferroni post hoc test. * p<0.05, *** p< (H+I) Spinous calcium transients do not differ in slices of cald -/- and +/+ mice. Calcium influx was measured in spines of CA1 pyramidal neurons filled with Alexa Fluor 594 and Fluo 5F after a backpropagating action potential elicited by a short current injection to the soma. Representative images and individual ΔG/R are shown. n numbers of spines (from slices of 2 animals per genotype) are n=84 (cald +/+), n=109 (cald -/-). Unpaired, 2- tailed Student s t-test. 11

13 Figure S8 Figure S8 refers to Figure 8. Electrophysiological and behavioral characterization of caldendrin knockout mice. (A) Paired pulse facilitation ratio is unaffected in slices of cald-/- mice, indicating proper presynaptic function. Sample traces are shown in inlets. n numbers of slices from 13 (cald +/+) or 15 (cald -/-) animals are indicated. (B) Schematic representation of the injection cannulae position in right and left dorsal hippocampus. Histological plates illustrating the injection sites are adopted from Franklin and Paxinos Mouse Brain Atlas. JPK: jasplakinolide treatment, VEH: saline vehicle control. (C) Experimental protocol for analyzing the effect of JPK on novel memory recognition task. (D) Cald -/- mice show no differences in locomotion (right) or anxiety-related behavior (left) as assessed with the open field test. n numbers of animals are indicated. (E) Cald -/- mice exhibit no change in anxiety-related behavior in the elevated plus maze as compared to cald +/+ control mice. n numbers of animals are indicated. 12

14 Supplemental Tables Table S1 refers to Figure 1. Layout of the SH3 domain array I (Panomics) A Amphiphysin LCK SPCN Cortactin MLPK3 Yes1 Abl2 SJHUA Itk CRK-D2 B Dlg2 EMP55 FGR SLK Nebulin c-src FYB-D1 Hck VAV2-D2 NOF2-D1 C VAV-D1 NCK1-D3 Y124 PEXD BTK RasGAP PSD95 Tim HS1 Stam D BLK Abl PLCγ Riz PI3β ITSN-D1 ITSN-D2 TXK GST Table S2 refers to Figure 2. Maximum Binding Points and Stability Points of the intramolecular interaction of amino acids 1-60 of caldendrin with its second EF-hand domain measured by SPR. Maximum Binding Point Stability Point Mg 2+ Mg 2+ + Ca 2+ Mg 2+ Mg 2+ + Ca 2+ Mean (n=4) Standard Deviation Molar Binding 11x x10-2 6x x10-2 Activity Table S3 refers to Figure 6. FRAP recovery in caldendrin +/+ and -/- primary hippocampal cultures within 1 min after photobleaching. Mobile F-actin pool ± S.E.M. (%) Caldendrin +/+, basal 54.6 ± 3.6 Caldendrin -/-, basal 84.8 ± 5.8 Caldendrin +/+, bicuculline 71.5 ± 2.7 Caldendrin -/-, bicuculline 84.7 ± 6.0 Caldendrin -/-, with caldendrin wildtype expression 83.3 ± 4.6 Caldendrin -/-, with caldendrin wildtype expression, bicuculline 57.1 ± 4.4 Caldendrin -/-, with caldendrin mutant expression, bicuculline 76.6 ± 4.0 GFP-cortactin recovery ± S.E.M. (%) Caldendrin +/+, basal 80.9 ± 4.2 Caldendrin -/-, basal ± 12.2 CaMKIIβ-GFP recovery ± S.E.M. (%) Caldendrin +/+, basal 41.2 ± 4.8 Caldendrin -/-, basal 44.9 ±

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION DOI: 10.1038/ncb2566 Figure S1 CDKL5 protein expression pattern and localization in mouse brain. (a) Multiple-tissue western blot from a postnatal day (P) 21 mouse probed with an antibody against CDKL5.