DCLK-immunopositive. Bars, 100 µm for B, 50 µm for C.

Transcription

1 Supplementary Figure S1. Characterization of rabbit polyclonal anti-dclk antibody. (A) Immunoblotting of COS7 cells transfected with DCLK1-GFP and DCLK2-GFP expression plasmids probed with anti-dclk antibody and anti-gfp antibody. DCLK immunoreactivity of two samples was comparable. (B) Immunoblotting of COS7 cells transfected with DCX-GFP and DCLK1( KD)-GFP expression plasmids probed with anti-dclk antibody and anti-gfp antibody. Anti-DCLK antibody did now show cross-reactivity with DCX protein. (C) Immunoblotting of the cerebrum, hippocampus, and cerebellum prepared from wild type mice (WT), DCLK1 KO mice, and DCLK2 KO mice with anti-dclk antibody. Significant reduction of the band intensity at 85 kda and 43 kda in samples from DCLK1 KO mice indicates higher expression level of DCLK1 in comparison with DCLK2 and the specificity of the antibody. 1

2 Supplementary Figure S2. Immunohistochemistry of DCLKs in the adult brain. (A) Double staining of the adult hippocampus with anti-dclk antibody and anti-map2 antibody. Comparable immunoreactivity could be detected in the area CA1, CA3, and the dentate gyrus (DG). Bar, 100 µm. (B and C) Double staining of the adult neocortex with anti-dclk antibody and anti-map2 antibody. MAP2-positive apical dendrites of pyramidal neurons were also DCLK-immunopositive. Bars, 100 µm for B, 50 µm for C. 2

3 Supplementary Figure S3. Microtubule organization in COS7 cells. COS7 cells were transfected with GFP, DCLK1-GFP, DCLK2-GFP, DCX-GFP, DCLK1 ( MT)-GFP, DCLK1 ( KD)-GFP, DCLK1 (K435R)-GFP, DCLK1 (K435A)-GFP, and DCLK1 (MAP2 swap)-gfp. The extent of microtubule bundling by DCLK1-GFP, DCLK2-GFP, DCX-GFP, DCLK1 ( KD)-GFP, DCLK1 (K435R)-GFP, DCLK1(K435A)-GFP and DCLK1 (MAP2 swap)-gfp was similar, suggesting their ability to bind and stabilize microtubules. Bar, 10 µm. 3

4 Supplementary Figure S4 Correlation between the advancement of dendritic tips and extension of MT bundles within the distal dendrites. (A) White bars indicate the distal end of the dendrite and MT bundle. (B) A plot of the distal end positions indicates tight temporal correlation. Bar, 5 µm. 4

5 Supplementary Figure S5 Characterization of DCLK1 and 2 shrna and their influence on DCX expression. (A) Specific suppression of DCLK1-GFP and DCLK2-GFP expression by respective shrnas in COS7 cells. COS7 cells were co-transfected with DCLK1-GFP or DCLK2-GFP together with DCLK1 or DCLK2 shrna constructs and an expression plasmid for β-galactosidase. The protein extracts from transfected cells were analyzed by immunoblotting. (B) DCX immunocytochemistry of primary hippocampal neurons transfected with plasmids for the expression of DCLK1 and 2 shrna. Expression of these shrna did not affect the expression level of endogenous DCX. Bar, 50 µm. 5

6 Supplementary Figure S6 Effects of DCLK1 overexpression and shrna-mediated knockdown on the density of PSD-95 puncta and VGLUT1 puncta. (A) Immunocytochemistry of control neurons and neurons expressing DCLK1 with antibodies against PSD-95 and VGLUT1. Bar, 5 µm. (B-D) Quantification of the densities of PSD-95 puncta, VGLUT1 puncta, and VGLUT1 puncta colocalized with PSD-95 in neurons expressing DCLK1. (control: n = 14 cells, DCLK1: n = 18 cells, t-test: *p < 0.05) All numeric data are mean + SEM. (E) Immunocytochemistry of control neurons and neurons expressing shrna for DCLK1 with antibodies against PSD-95 and VGLUT1. Bar, 5 µm. (F-H) Quantification of the densities of PSD-95 puncta, VGLUT1 puncta, and VGLUT1 puncta colocalized with PSD-95 in neurons expressing DCLK1 shrna. (control shrna: n = 19 cells, DCLK1 shrna: n = 19 cells, t-test: **p < 0.01) All numeric data are mean + SEM. 6

7 Supplementary Figure S7 Amount of PSD-95 in neurons infected with adenoviruses for the expression of either GFP or DCLK1-GFP. (A) Immunoblot of hippocampal neuronal culture infected with either GFP or DCLK1-GFP and probed with anti-gfp antibody. (B and C) Anti-PSD95 and anti-tubulin immunoblot of hippocampal neuronal culture infected with either GFP or DCLK1-GFP. Quantification of the data indicates that the amount of PSD-95 is indistinguishable. (GFP: n = 3, DCLK1-GFP: n = 3 independent cultures, N.S. = no statistical difference) All numeric data are mean + SEM. 7

8 Supplementary Figure S8 Reduced synaptic content of Homer in neurons overexpressing DCLK1. (A-D) Full-length DCLK1 or truncated forms of DCLK1 (doublecortin domain: DCLK1( KD) or kinase domain: DCLK1( MT)) tagged with GFP were expressed in dissociated hippocampal neurons by using recombinant adenoviruses (cells were observed at 20 DIV). Cells were immunoreacted with anti-homer antibody. GFP adenovirus was used as control. Bar, 5 µm. (E) Quantification of the immunoreactivity measured from culture dishes infected with each type of adenoviruses. DCLK1( MT)-GFP showed significant suppression of Homer content in dendrites compared with the control.(gfp: n = 8 cells, DCLK1-GFP: n = 8 cells, DCLK1( KD)-GFP: n = 8 cells, DCLK1( MT)-GFP: n = 8 cells, one-way ANOVA followed by Tukey Kramer multiple comparison tests: *p < 0.05) All numeric data are mean + SEM. 8

9 Supplementary Figure S9 Effect of DCLK1 kinase domain overexpression and the role of kinase activity. (A) Anti-β-galactosidase staining and anti-psd-95 staining of neurons overexpressing GFP, the GFP-tagged kinase domain of DCLK1 (DCLK1( MT)-GFP) or the GFP-tagged kinase domains with a single amino acid substitution to eliminate kinase activity (DCLK1( MT K435R)-GFP and DCLK1( MT K435A)-GFP). Bar, 5 µm. (B and C) Dendritic protrusion lengths and relative PSD-95 immunoreactivity of neurons expressing GFP, DCLK1( MT)-GFP, DCLK1( MT K435R)-GFP, or DCLK1 ( MT K435A)-GFP. Elimination of kinase activity selectively affected the ability of DCLK1 ( MT) to reduce the content of PSD-95 at the postsynaptic sites. (GFP: n = 26 cells, DCLK1 ( MT)-GFP: n = 29 cells, DCLK1( MT K435R)-GFP: n = 19 cells, DCLK1( MT K435A)-GFP: n = 17 cells, one-way ANOVA followed by Tukey Kramer multiple comparison tests: *p < 0.05, **p < 0.01, ***p < 0.001). All numeric data are mean + SEM. 9

10 Supplementary Figure S10 Suppression of AMPA receptor subunit contents in the postsynaptic sites by overexpression of DCLK1. (A) Immunocytochemistry of AMPA receptor subunit GluA2 and NMDA receptor subunit GluN1 in neurons expressing DCLK1-GFP. (B) Relative fluorescence intensity of GluA2 and GluN1 staining in neurons expressing GFP or DCLK1-GFP. (GFP: n = 11 cells, DCLK1-GFP: n = 10 cells, t-test: ***p< 0.001) Bar, 5 µm. 10

11 Supplementary Figure S11 In vivo phenotypes of DCLK1 knockout in the cortex and its knockdown in the hippocampus. (A-C) Morphological analyses of cortical pyramidal neuron dendrites in vivo after introduction of GFP by in utero electroporation of wild type and DCLK1 -/- embryos (A) revealed reduction of dendritic complexity by Sholl analyses (B) and a smaller number of terminal dendritic branches (C). (control: n = 22 cells, DCLK1 -/- : n = 19 cells, t-test: ***p< 0.001) (D-F) Morphological analyses of hippocampal CA1 pyramidal neuron dendrites in vivo after introduction of DCLK1 shrna by in utero electroporation (D) revealed reduction of dendritic complexity by Sholl analyses (E) and a smaller number of terminal dendritic branches (F). (control: n = 13 cells, DCLK1 shrna - : n = 14 cells, t-test: ***p< 0.001) (G, H) Introduction of DCLK1 shrna plasmids by in utero electroporation into hippocampal CA1 pyramidal neurons affected spine morphology in vivo. GFP fluorescence of dendritic protrusions (G) revealed an increase in spine width (H). (control: n = 17 cells, DCLK1 shrna - : n = 15 cells, t-test: ***p< 0.001) Bars, 50 µm for A and D; 5 µm for G. 11

12 Supplementary Table S1 DCLK1 DCLK1(ΔKD) DCLK1(ΔMT) DCLK1(MAP2 swap) DCLK1(K435A/R) DCLK1(ΔMT K435A/R) DCLK2 DCX Amino acids of mouse DCLK1 (NM_019978) Truncated mutant of DCLK1 corresponding to amino acids of DCLK1. This mutant has similar domain organization with DCLK1 (DCX-like) Truncated mutant of DCLK1 corresponding to amino acids This mutant has similar domain organization with DCLK1 (CPG16) The MT-binding domain of DLCK1 (amino acids 1-270) was replaced with the MT-binding domain of MAP2C (amino acids ) Single amino acid substitution of DCLK1 in the catalytic domain of the kinase. Lysine at 435 was replaced with Alanine or Arginine. Truncated mutant of DCLK1 corresponding to amino acids This mutant has similar domain organization with DCLK1 (CPG16) with a mutation at Lysine at 435, which was replaced with either Alanine or Arginine. Amino acids of mouse DCLK2 (NM_027539) Amino acids of mouse DCX (NM_ ) 12

13 Supplementary Methods Other Antibodies Antibodies used in this study are: mouse monoclonal antibodies: MAP2: Sigma-Aldrich, PSD-95: Thermo Scientific Pierce Antibodies, GluA2: NeuroMab, tau1:chemicon/millipore, α-tubulin: Sigma-Aldrich, β-galactosidase: Promega, rabbit polyclonal antibodies: VGLUT1, spinophilin: Chemicon/Millipore, β-galactosidase: Cappel, GFP: MBL, synaptophysin: Boehringer Mannheim Biochemica, GluN1: Chemicon/Millipore, rat polyclonal antibodies: Homer: Chemicon/Millipore, guinea pig polyclonal antibodies: DsRed2, Secondary antibodies: Alexa488 conjugated anti-mouse/rabbit/guinea pig IgG: Invitrogen, Cy3 conjugated anti-mouse/rabbit/guinea pig IgG:Jackson ImmunoResearch, Alexa633 conjugated anti-mouse/rabbit IgG: Invitrogen, and Alexa647 conjugated anti-rabbit IgG: Invitrogen. We previously found that mouse monoclonal anti-psd-95 antibody (clone 6G6-1C9; Thermo Scientific Pierce Antibodies) recognizes four MAGUK proteins (PSD-95, Chapsyn-110, SAP102, SAP97) with roughly equal sensitivity 53. Because this antibody is widely used in previous publications as a reagent to detect PSD-95 by 13

14 immunocytochemistry and the most abundant MAGUK protein in the PSD is indeed PSD-95, we described the signal detected by this antibody as PSD-95 immunoreactivity in this manuscript. Western blotting Samples were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% skim milk in TBS for 1 hour and probed with antibodies overnight at 4 o C. The primary antibodies were rabbit polyclonal anti-dclk antibody, mouse monoclonal anti-α-tubulin antibody, mouse monoclonal anti-psd-95 antibody, and rabbit polyclonal anti-synaptophysin antibody. Secondary antibodies were peroxidase labeled goat antibodies against mouse or rabbit IgG (Amersham). Biochemical purification of PSDs The brain tissue was homogenized in 10 volumes of HEPES-buffered sucrose (0.32 M sucrose, 4 mm HEPES, ph 7.4). The homogenate was centrifuged at 1,000X g for 10 min to remove the nuclear fraction. The supernatant was spun at 10,000 X g for 15 min to yield the crude synaptosomal fraction. The pellet was resuspended in 10 14

15 volumes of HEPES-buffered sucrose and then respun at 10,000 X g for another 15 min. The resulting pellet was resuspended in 4 mm HEPES (ph 7.4), homogenized, and mixed constantly for 30 min at 4 o C. After centrifugation of the lysate at 25,000 X g for 20 min, the supernatant was saved as crude synaptic vesicle fraction, and the pellet was resuspended in HEPES-buffered 0.32 M sucrose. The resuspended membrane was then carefully layered on top of a discontinuous gradient containing layers of 0.8 M, 1.0 M and 1.2 M sucrose and centrifuged at 150,000 X g for 120 min. Synaptic plasma membranes were recovered in the layer between 1.0 M and 1.2 M sucrose and resuspended in HEPES solution (ph 7.4) to the final concentration of 0.32 M sucrose. Sample was spun at 150,000 X g for 30 min and the resulting pellet was resuspended in 50 mm HEPES and 2 mm EDTA (ph 7.4) with protease inhibitors. Further purification of PSDs was performed by adding concentrated stock solution of Triton X-100 to the final concentration of 0.5%, gentle agitation at 4 o C for 15 min, and subsequent centrifugation at 32,000 X g for 20 min. The resulting pellet was resuspended in 50 mm HEPES and 2 mm EDTA (ph 7.4) (PSD-1T fraction). Next round of wash with Triton X-100 and sedimentation of PSDs with a stronger centrifugation condition (200,000 X g for 20 min) was 15

16 performed to obtain PSD-2T fraction. To obtain PSD-sarkosyl fraction, PSD-1T fraction was washed with 3% sarkosyl and subsequently centrifuged at 200,000 X g for 20 min. To obtain synaptic vesicle fraction, saved crude synaptic vesicle fraction was centrifuged at 165,000 X g for 120 min and the pellet was resuspended in PBS (ph 7.4). Golgi-Cox impregnation Golg-Cox impregnation of hippocampal pyramidal neurons was performed by using FD Rapid Golgi StainII kit (FD NeuroTechnologies). Spines were analyzed by X100 oil objective lens (NA1.3). Dendritic protrusions with their widths larger than half of their lengths were classified as mushroom spines. The other spines were classified as thin spines

17 Supplementary Reference 53. Sugiyama, Y., Kawabata, I., Sobue, K. & Okabe, S. Determination of absolute protein numbers in single synapses by a GFP-based calibration technique. Nat Methods2, (2005). 17