Yeast two-hybrid screen
Two fragments of the KIF1C cDNA encoding amino acids 435–622 (corresponding to the U104 domain) or amino acids 261–800 were cloned into the LexA fusion protein vector pBTM116 and transformed into the Saccharomyces cerevisiae strain L40 (MATa trp1 leu2 his3 LYS2::lexA-HIS3 URA3::lexA-lacZ ), generating the L40 LexA-KIF1CU104 and L40 LexA-KIF1C298–840 strains, respectively. A brain cDNA library fused to the GAL4 activation domain in the pACT2 vector (Clontech) was transformed into the L40 LexA-KIF1CU104 and L40 LexA-KIF1C298–840 strains and 5 × 106 transformants were screened for interaction as described . Yeast plasmid DNA was isolated from His+ β-Gal+ colonies, rescued into Escherichia coli HB101, retransformed into L40 LexA-KIF1C298–840, and yeasts assayed for β-galactosidase activity and growth on medium with complete supplement lacking amino acids Trp, Leu, Ura, Lys, and His (Bio101). The specificity of the interaction between KIF1C298–840 fragment and potential candidates was proven by transforming the candidate plasmid also into a L40 LexA and a L40 LexA-laminin strain.
DNA sequence analysis
Clones interacting specifically with KIF1C in the two-hybrid screen were subjected to sequencing by the dideoxynucleotide chain termination method. Non-redundant databases (GenBank, EMBL, DDBJ and PDB) were searched for homologous sequences using the BLAST program [39, 40].
Northern blot analysis
A multiple tissue Northern blot was obtained from Clontech. Hybridization was performed at 67°C with 5 × 106 cpm/ml of 32P-random primed DNA probe as described by the manufacturer.
cDNA cloning and protein purification
For transient expression in 293 and NIH3T3 cells, all cDNAs were cloned by standard procedures into the cytomegalovirus immediate early promoter-based expression plasmid pRK5 . To clone a KIF1Bα encoding cDNA, the 3'-part of the open reading frame was received from clones FH12320 (1941–3721 bp of this clone) and HJ02790 (1–685 bp of this clone) known in HUGE Protein Database as genes KIAA1448  and KIAA0591 , respectively. The 5'-part was amplified in a PCR reaction using a human brain cDNA library as a template. A cDNA fragment encoding the amino acids 265–639 of the human version of KIF5B was amplified with specific primers in a similar way. The corresponding region of KIF1Bα (amino acids 270–641) was cut out from the KIF1Bα cDNA using restriction enzymes, and both fragments were cloned into a pGEX vector. The corresponding fusion proteins were produced in Escherichia coli BL21 (Novagen) or 298 F' strains and purified as described .
The KIF1B and KBP rabbit polyclonal antisera were raised against glutathione-S-transferase (GST) fusion proteins of KIF1B (amino acids 743–1153) and KBP (full length), respectively. Monoclonal antibodies used were derived from the following hybridoma: kinesin heavy chain – H2; phosphotyrosine – 4G10; β-tubulin – KMX1; vesicular stomatitis virus (VSV) epitope tag – p5d4; KIF5 – SUK4; hemagglutinin (HA) epitope tag – 12CA5.
Cell lines and cell culture
293 cells were grown in F12/Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 2 mM glutamine. BHK, HeLa, NIH3T3 and C2C12 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 2 mM glutamine.
Transient expression, cell lysis, and immunoprecipitation
Transfection of 293 cells and NIH3T3 cells was performed using the method of Chen and Okayama . The cells were lysed in 1 ml of lysis buffer/10-cm plate (1% Triton X-100, 50 mM HEPES, pH 7.5, 10% glycerol, 150 mM NaCl, 1.5 mM EGTA, 10 mM sodium pyrophosphate, 100 mM NaF, 1 mM sodium orthovanadate, 10 μg/ml aprotinin, 1 mM phenylmethylsulfonylfluoride) and the lysates were precleared by centrifugation at 13,000 g for 15 minutes at 4°C. The lysates were adjusted for equal protein concentration, the appropriate antibody (3 μl of serum or 2 μg of purified antibody) and either Protein A- or Protein G-Sepharose were added, and the lysates were incubated for at least 3 hours at 4°C on a turning wheel. The immunoprecipitates were washed with HNTG buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, 10 mM NaF, 1 mM sodium orthovanadate), separated on an 8 or 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and incubated with the appropriate primary antibody. After three washes, the membranes were incubated with secondary horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit antibodies (Sigma). After three washes they were visualized using the ECL system. Before reprobing, blots were incubated for 30 minutes in 62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 0.1% β-mercaptoethanol at 55°C.
In vitro binding assays
For in vitro binding assays, 293 cells transiently overexpressing KBP were lysed and incubated for 4 hours at 4°C with GST or GST-KIF1Bα/KIF5B fusion proteins immobilized on glutathione-Sepharose. After washing with HNTG buffer, the proteins were separated by SDS-PAGE and analysed by Western blotting.
NIH3T3 cells were grown on uncoated glass coverslips, fixed for 20 minutes in ice-cold methanol at -20°C, washed with phosphate-buffered saline and incubated for 10 minutes with 0.1% NaBH4 and 0.1 M glycine in phosphate-buffered saline to block autofluorescence. Nonspecific antibody binding was blocked for 45 minutes with PBS with 0.045% fish gelatin containing 5% normal goat serum and 1% bovine serum albumine. Incubation with primary antibody was done for 1.5 hours at 37°C after dilution in PBS/gelatin containing 5% normal goat serum. The affinity purified KBP and KIF1Bα antibodies were used at concentrations of 10 ng/μl and 20 ng/μl, respectively. The β-tubulin antibodies were diluted as recommended by the manufacturer, and the monoclonal VSVantibodies at a concentrations of 20 ng/μl. After five washes with PBS/gelatin, primary antibody binding was detected with isotype-specific secondary antibody conjugated with either DTAF (goat anti-rabbit, Dianova) or Alexa (Alexa Fluor 546 goat anti-mouse and goat anti-rabbit and Alexa Fluor 488 goat anti-mouse, Molecular Probes). The coverslips were mounted in PermaFluor (Immunotech). To allow unbound protein to diffuse out, extraction of cells with 0.5% Triton X-100 was performed prior to fixation for 5–10 seconds [46, 47]. MitoTracker (Molecular Probes) staining was done according to manufacturer's recommendations.
Pictures were taken with a Leica TCS NT/SP Laser Scanning Confocal Microscope supplied with HCX PL APO immersion oil objective of magnification 63× and aperture 1.32–0.62. The pictures were prepared using TCS software supplied by the manufacturer.
Cell extracts and motility assays
Cell extracts were prepared in BRB80 buffer (80 mM Pipes, pH 6.8, 2 mM MgCl2, 1 mM EGTA) supplemented with protease inhibitors, through multiple passing through cell cracker (HGM, Heidelberg, Germany). The nuclei were spun down and the supernatant was spun again at 120,000 g to separate cytosol from membrane fraction The concentration of the cytosolic fraction was adjusted to 1 mg/ml and supplemented with 1 mM DTT, 20 μM taxol, 2 mg/ml BSA, 5 mg/ml casein and 1/20 volume energy mix (150 mM creatine phosphate, 20 mM ATP, 20 mM MgCl2, 2 mM EGTA, pH 7.7).
For motility assays with latex beads, 1 μl of beads (2.5% solid-latex, carboxylate 0.1 micron microspheres; Polysciences, Inc., Warrington) was left untreated or incubated for at least 30 minutes with 4 μl of KIF1B antibody solution (3 mg/ml). Then the antibody/beads suspension was diluted 1:6 in BRB80, 1 mM DTT, 20 μM taxol, 2 mg/ml BSA, 5 mg/ml casein and 1/20 volume energy mix. One μl of beads was mixed with 9 μl of cell extract and floated into a flow cell with coverslips coated with microtubules. The beads were left for 5 – 7 minutes in a humid chamber to sediment onto the microtubules. To inhibit unspecific KIF5B driven movements, SUK4 antibody was incubated with extracts for 10 minutes at a final concentration of 0.25 mg/ml, and then the extracts were used in the assay. Flow cells were prepared as described earlier , and microtubules were isolated from pig brain as described .
Motility was observed using video-enhanced differential interference contrast microscopy (VE-DIC) in real time (Olympus optics BX60 microscope, equipped with DIC optics; Japan, Tokyo). To determine the rates and distances, RETRAC software (Dr. N. Carter, Marie Curie Research Institute, Oxted, Surrey, UK) was used.