Cell cultures and transfections
Rat hippocampal neurons were isolated from 1-day-old pups (Sprague Dawely, Samtako, Osan, Republic of Korea). Cultures were maintained in Neurobasal–A (Life Technologies, Carlsbad, CA, USA) supplemented with B27 supplement (Life Technologies) at 37 °C and in 5% CO2, as previously described [31]. The cultures were performed in accordance with the approved animal protocols and the guidelines of the Institutional Animal Care and Use Committee of Chungbuk National University (CBNUA-1049-17-01). Human embryonic kidney (HEK) 293 T cells were maintained in Dulbecco’s Modified Eagle’s Medium (Life Technologies) supplemented with 10% fetal bovine serum (Biowest, Nuaillé, France). With regards to transfection of the neurons, target genes were subcloned to a pSinRep5 vector for Sindbis viral expressions and packaged into Sindbis virion particles according to Invitrogen’s user manual (Invitrogen, Carlsbad, CA, USA). The Sindibis virion was directly added to the cultured neurons and incubated for 6–12 h. Transfection of the HEK cells was carried out by transferring DNA constructs using a calcium phosphate method.
DNA constructs
Complete cDNA of mouse PSD-95 (disks large homolog 4, NM_007864) were amplified using PCR (mPSD95-R1-S → mPSD95-Xho-A) and inserted at the EcoRI/XhoI site of the pCS4-3xHA vector. To construct the mutant form of the PSD-95, full length or partial PSD-95 fragments were amplified using PCR and inserted at the EcoRI/XhoI site of the pCS4-3xHA vector (full length: mPSD95-R1-S → mPSD95-Xho-A; PDZ1: mPSD95-R1-S → PZD1-Xho-A; PDZ1–2: mPSD95-R1-S → PZD2-Xho-A; PDZ1–3: mPSD95-R1-S → PZD3-Xho-A; ∆GK: mPSD95-R1-S → SH3-Xho-A; GK: GMPK-R1-S → mPSD95-Xho-A). To allow fine mapping, PSD-95 fragments were amplified using PCR (ADPDZ3: mPSD95-ADPDZ3-R1-S → PDZ3-Xho-A2; PDZ3: mPSD95-PDZ3-R1-S → PDZ3-Xho-A2; AD: mPSD95-ADPDZ3-R1-S2 → AD-Xho-A). In the case of ADPD3 and PDZ3, the fragments were inserted at the EcoRI/XhoI site of the pCS4-3xHA vector. While in the case of AD, they were inserted at the EcoRI/XhoI site of the pCMV-myc vector. To allow Sindbis viral expression, GFP-tagged PSD-95 (PSD-95-GFP) and GFP-tagged ADPDZ3 (GFP-PDZ3) were amplified using PCR (PSD-95-GFP: mPSD95-Mlu-S → GFP-Sph-A; GFP-ADPDZ3: GFP-Mlu-S → PDZ3-Sph-A) and inserted at the MluI/SphI site of the pSinRep5 vector.
The complete cDNA of mouse Kif5a (NM_008447) was amplified using PCR (mKIF5A-Bam-S → mKIF5A-Apa-A) and inserted at the BamHI/ApaI site of the pCMV-tag2B vector. To construct mutants, the KIF5A fragments were amplified using PCR and inserted at the BamHI/ApaI site of the pCMV-tag2B vector (full length: mKIF5A-Bam-S → mKIF5A-Apa-A; 636: mKIF5A-Bam-S → mKIF5A-636-Apa-A; 826: mKIF5A-Bam-S → mKIF5A-826-Apa-A; 906: mKIF5A-Bam-S → mKIF5A-906-Apa-A; ∆MD: mKIF5A-330-Bam-S → mKIF5A-Apa-A). To allow Sindbis viral expression, wild type and ∆MD mutant cDNA was amplified using PCR (mKIF5A-Sph-S → mKIF5A-Apa-A; ∆MD: mKIF5A-330-Sph-S → mKIF5A-Apa-A) and inserted at the SphI/ApaI site of pSinRep5-mRFP, yielding the DNA constructs encoding for mRFP-tagged WT KIF5A or mRFP-tagged ∆MD. The complete cDNA of mouse Kif5b (NM_008448) was amplified using PCR (mKIF5B-Bam-S → mKIF5B-Apa-A) and inserted at the BamHI/ApaI site of the pCMV-tag2B vector. The complete cDNA of human Kif5c (NM_004522) was amplified using PCR (hKIF5C-R1-S → hKIF5C-Sal-A) and inserted at the EcoRI/SalI site of the pCMV-tag2B vector. The cDNA clones of KIF5s were provided by Dr. EY Shin (Chungbuk National University). All PCR primers for PCR were purchased from Bioneer (Daejeon, Republic of Korea). Restriction enzymes used in our experiments were purchased from New England Biolabs (NEB, Ipswich, MS, USA).
mPSD95-R1-S: 5′- ggaattcaatggactgtctctgtatagtg-3′,
mPSD95-Xho-A: 5′-ccgctcgagtcagagtctctctcgggctg-3′
PDZ1-Xho-A: 5′-ccgctcgagtcacttctcagctgggggttt-3′
PDZ2-Xho-A: 5′-ccgctcgagtcaggccacctttaggtacac-3′
PDZ3-Xho-A: 5′-ccgctcgagtcaccgcttggggttgcttcg-3′
SH3-Xho-A: 5′-ccgctcgagtcagcgagcgtagtgcacttc-3′
GMPK-R1-S: 5′-ggaattcacccatcatcatccttggg-3′
mPSD95-ADPDZ3-R1-S: 5′-ggaattcaaagcccagcaatgcctacc-3′
PDZ3-Xho-A2: 5′-ccgctcgagtcagatgatcgtgaccgtctg-3′
mPSD95-PDZ3-R1-S: 5′-ggaattcaaggcggatcgtgatccatc-3′
AD-Xho-A: 5′-ccgctcgagtcaccttggttcccggggaa-3′
mPSD95-Mlu-S: 5′-cgacgcgtatggactgtctctgtatagtg-3′
GFP-Sph-A: acatgcatgcttacttgtacagctcgtcca-3′
GFP-Mlu-S: 5′-cgacgcgtgtcgccaccatggtgagc-3′
PDZ3-Sph-A: 5′-acatgcatgctcagatgatcgtgaccgtctg-3′
mKIF5A-Bam-S: 5′-cgggatccatggcggagactaacaac-3′
mKIF5A-Apa-A: 5′: 5′-tgggcccccttagctggctgctgtctc-3′
mKIF5A-636-Apa-A: 5′-tgggggcccttaatgctgtgagatgagcag-3′
mKIF5A-826-Apa-A: 5′-tgggggcccttaggaatgaatccccccac-3′
mKIF5A-906-Apa-A: 5′-tgggggcccttagtaccgcacggcttcttt-3′
mKIF5A-330-Bam-S: 5′-cgggatccgcctcagtgaatctggag-3′
mKIF5A-Sph-S: 5′-acatgcatgctcgaccaccatggcgga-3′
mKIF5A-330-Sph-S: 5′-acatgcatgcgcctcagtgaatctggag-3′
mKIF5B-Bam-S: 5′-cgggatccatggcggacccggcggag-3′
mKIF5B-Apa-A: 5′-agggggcccttacgactgcttgcctccac-3′
hKIF5C-R1-S: 5′-ggaattctatggcggatccagccgaa-3′
hKIF5C-Sal-A: 5′-cgacgtcgacttatttctggtagtgagtgg-3′
Co-immunoprecipitation
For co-immunoprecipitation (co-IP), cell lysates were prepared by adding lysis buffer (150 mM NaCl, 1% IGEPAL® CA-630, 50 mM Tris·Cl; pH 8.0) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland). The lysate was immunoprecipitated using 2–3 μg of antibody (specificity indicated in the figures), mouse immunoglobulin G (IgG; Sigma-Aldrich, St. Louis, MO, USA), and incubated with 50 μL of Protein G-Sepharose (GE Healthcare, Chicago, IL, USA). The immunoprecipitates were washed three times in 1 mL of ice-cold lysis buffer, followed by additional wash an additional time with 1 mL of 50 mM Tris·Cl (pH 8.0). The precipitated proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (8%–12%). For western blot analysis, the blots were incubated using the antibody indicated in the figures. All co-IPs and western blot analyses were performed more than twice to confirm that the data were reproducible. The following antibodies were used in the co-IPs and western blot analyses: monoclonal anti-FLAG antibody (1:2000, Clone M2; Sigma-Aldrich), monoclonal anti-HA antibody (1:2000, Clone HA-7; Sigma-Aldrich), and monoclonal anti-Myc antibody (1:2000, Clone 9E10; Sigma-Aldrich).
Immunocytochemistry and proximity ligation assay
For the immunocytochemistry, cultures were fixed using a fixative (4% paraformaldehyde, 4% sucrose, pH 7.2) and permeabilized using PBT (0.1% TritonX-100, 0.1% BSA in PBS). In the case of surface GluA1 immunocytochemistry, no permeabilization step was performed. The cultures were pretreated using the preblock solution (2% BSA, 0.08 TritonX-100 in PBS) for 1 h and each antibody was directly added to the preblock solution for 2 h. The following antibodies were used for staining, each at a dilution of 1:50; monoclonal anti-PSD-95 antibody (clone 6G6-1C9; Affinity Bioreagents, Golden, CO, USA), polyclonal anti-PSD-95 antibody (Cell Signaling, Danvers, MA, USA), monoclonal anti-kinesin antibody (Clone: H2; Millipore, Temecula, CA, USA), polyclonal anti-synapsin I antibody (Millipore), polyclonal anti-GluA1 antibody (Upstate, Lake Placid, NY), polyclonal anti-GluA1 antibody (Alomone Labs, Jerusalem, Israel) for surface GluA1.The following antibodies were used for secondary staining, each at a dilution of 1:200: Alexa Fluor® 488 anti-rabbit IgG antibody (Molecular Probes, Eugene, OR, USA), Cy3-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), Cy3-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories), and Alexa Fluor® 647 anti-rabbit IgG antibody (Molecular Probes).
For PLA using Duolink® In Situ-Fluorescence (Sigma-Aldrich), the cultures were infected with Sindbis viruses encoding GFP to visualize whole dendritic structures and then fixed as described above; rabbit polyclonal anti-PSD-95 antibodies (Cell Signaling) and mouse monoclonal anti-KIF5 antibodies (Clone H2, Millipore) were then used. All procedures were performed according to the manufacturers’ instructions. The nucleus of each neuron was stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). Immunostaining and PLA were visualized using confocal microscopy (Zeiss 710; Carl Zeiss, Oberkochen, Germany).
Image analysis
Secondary or tertiary dendrites with a similar diameter were selected from acquired neuron images and straightened using a plugin of ImageJ program (ver 1.47; National Institute of Health, Bethesda, VA, USA). The images of straightened dendrites were transited to threshold images. The number and size of PSD-95 or GluA1 particles were measured using the particle analysis plugin. Colocalization was measured from the threshold images using colocalization plugins and represented using either Pearson’s correlation coefficient (R − value) or a percentage. All image analyses were performed by blind experiment.
Statistical analysis
Normality of the data was assessed using either the Kolmogorov-Smirnov test or the D’Agostino and Pearson omnibus normality tests. If the data followed Gaussian distribution, a Student’s t-test was performed to determine statistical significance between two groups, while analysis of variance (ANOVA) was performed among three or more groups, with Newman Keul’s analysis used as a post hoc analysis. If the data did not follow Gaussian distributions, the non-parametric Mann-Whitney test was performed to determine statistical significance between two groups, while the Kruskal-Wallis test combined with Dunn’s multiple comparison test was performed among three or more groups. All statistical analyses were performed using GraphPad prism (ver 5.02; GraphPad Software, San Diego, CA, USA).