Identification of cDNA
Degenerate PCR was performed on a mouse retinal cDNA (oligo-dT primed) library using primers to conserved motor domain amino acids, MGKTY/FTM (EcoRI · GGG/A/T/CAAA/GACG/A/T/CTA/TT/ CACG/A/T/CATG) and DLAGSE (BamHI · TCA/GCTG/A/T/CCCG/ A/T/CGCC/TAAA/GTC). PCR products were cloned into pBluescript (Stratagene) using the EcoRI and BamHI sites from the oligos and sequenced. One novel cDNA (KIF21A) was identified and used to screen the retinal cDNA library from which it was isolated. Five overlapping clones containing the entire coding sequence of KIF21A were identified from the dT primed retinal library, as well as a random primed retinal library (B6 strain mouse retina), and sequenced. A second gene, KIF21B, was isolated due to its cross hybridization to a KIF21A cDNA probe. The entire KIF21B coding sequence is contained on two overlapping clones. The GCG sequence analysis software package (Devereux et al., 1984
) was used to align the KIF21A and KIF21B amino acids sequences (gap program) as well as produce a dendrogram comparing the motor domain core amino acids of many KLPs (from IFAY to LAGSE).
Mapping of KIF21A and KIF21B to Chromosome Location
The murine chromosomal locations of KIF21A and KIF21B genes were determined using an interspecific backcross DNA panel obtained from The Jackson Laboratories
(Rowe et al., 1994
). The panel consists of 94 F2 progeny from a (C57BL/6J × SPRET/Ei) F1 female mated to a SPRET/Ei male and DNA from parental C57BL/6J and Mus spretus
. ScaI polymorphisms between SPRET/Ei and C57BL/6J mouse strains were identified by Southern blotting for KIF21A (using bp 640–1800) and KIF21B (bp 2880–3900). The distribution of the C57BL/6J allele among the F1 progeny was used to estimate gene locus position and linkage distance by The Jackson Laboratories
. Human chromosomal locations that are syntenic to the mouse regions were determined by using the map generated by DeBry and Seldon (1996).
Northern Blot Analysis
Total RNA was isolated from various mouse tissues using the guanidinium isothiocyanate extraction method as previously described (Chomczynski and Sacchi, 1987
). RNA amount and purity was determined by absorbance at 260 nm and the 260/280 nm ratio using a spectrometer. 30 μg of total RNA was separated on a 1% formaldehyde agarose gel, transferred to GeneScreen Plus membrane (New England Nuclear
), and fixed by baking. The blots were probed with random primed DNA (KIF21A; bp 3400–5000) and (KIF21B; bp 3000–5400) with α[32
P]dATP incorporation in a buffer of 0.5 M NaPO4
and 7% SDS at 65°C for 16 h. Blots were washed in 0.1× SSC/1% SDS at 65°C, twice for 30 min. Washed blots were exposed to Biomax MS film (Kodak
Preparation of Antibodies
A KIF21A-HIS fusion protein construct which contained amino acids 1124–1355 (KLCG to QINQ) was generated in pET-23b (Novagen, Inc.). A comparable KIF21B-HIS fusion protein containing amino acids 1135– 1419 (KFKG to QINQ) was also generated in pET-23b. These fusion proteins were grown in pLysS BL21(DE3) bacteria, induced with 0.5 mM IPTG, and purified using Ni-NTA–agarose (Qiagen, Inc.). Each fusion protein was further purified by SDS-PAGE. Gel slices (containing 300 μg of fusion protein) were injected into three rabbits to produce polyclonal sera against KIF21A or KIF21B. To generate antisera that recognize only KIF21A or KIF21B, each antiserum was incubated with the conflicting Affigel (Bio-Rad Laboratories) bound fusion protein to immunodeplete antibodies that recognize both KIF21 proteins. Immunodepletion was confirmed by Western immunoblot analysis of a dilution series of known amounts of KIF21A and KIF21B fusion proteins. Affinity-purified antibody was generated by incubating each antiserum with its Affigel-bound antigen used in its generation. The antibody was then released from the column by lowering the pH.
Western Blot Analysis
Tissue was homogenized in 25 mM sodium phosphate, 5 mM EDTA, 1% SDS, pH 7.5, buffer using a polytron. Protein concentrations were determined using the Bio-Rad Dc protein assay kit. Protein samples were separated on a SDS-PAGE gel using standard Laemmli method and then transferred to PVDF membrane (Bio-Rad), dried, and blocked in 5% dry milk in 1× TBS/0.05% Tween for 1 h. Primary antibodies were incubated 1 h in the same solution at room temperature, washed, incubated with an HRP-conjugated secondary antibody, and then washed again. ECL (Nycomed-Amersham Inc.) was used for detecting the antibodies by exposing the blots to X-OMAT-XAR5 (Kodak) film. Antibody concentrations used: polyclonals α-KIF21A and α-KIF21B whole serum, 1:1,000; α-KIF3A, 1:5,000; nKHC, 1:2,000 (a gift from Dr. Ron Vale, UCSF); monoclonals α-synaptotagmin, 1:500; Stressgen α-SV2, 1:50 (Hybridoma Bank, University of Iowa).
Microtubule Binding Assay
A mouse brain was homogenized in 3 ml microtubule binding buffer (0.1 M Pipes, 0.9 M glycerol, 5 mM EGTA, 0.5 mM EDTA, 2.5 mM MgSO4, pH 6.4) using a glass dounce homogenizer (Kontes). The homogenate was centrifuged at 25,000 rpm and 25°C in a Sorvall 1270 rotor for 20 min. The supernatant was recentrifuged for 30 min to remove vesicles and endogenous microtubules. 90 μl of supernatant was supplemented with 10 μl of purified taxol stabilized mouse microtubules (3 mg/ml). To this, 10 μl of either 0.1 M Mg · ATP, or 1 μl 0.5 M Mg · AMP-PNP was added for 15 min at room temperature. Then the samples were layered onto 100 μl of a 40% sucrose cushion in the same buffer and centrifuged for 30 min at 25,000 rpm and 25°C in a Sorvall 42.2 TI rotor. Taxol was added to 10 mM in all the buffers. Pellets were resuspended in 1× SDS-PAGE sample buffer (10% glycerol, 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 700 mM β-mercaptoethanol) and analyzed by Western blotting.
A mouse brain was homogenized in 4 ml cell fractionation buffer (20 mM Hepes, 100 mM sodium aspartate, 40 mM KCl, 5 mM EGTA, 5 mM MgCl2, 2 mM Mg-ATP, 1 mM DTT, pH 7.2) supplemented with protease inhibitors (to 1 mM PMSF, 0.7 ng/ml pepstatin A, 10 μg/ml leupeptin, 10 μg/ml soybean trypsin inhibitor; final concentrations). The homogenate was centrifuged twice at 8,800 rpm 10 min each in a Sorvall SS34 rotor. 1.2 ml of lysate was preabsorbed with 100 μl of protein A–Sepharose beads (Pharmacia Biotech, Inc.). 5 μl of immunodepleted KIF21A or KIF21B whole serum was added to the lysate and incubated 1.5 h at 4°C on a rotator. 20 μl of protein A beads were added to lysate for 2 h at 4°C on a rotator. Proteins were eluted by boiling 5 min with 75 μl of 1× SDS-PAGE sample buffer without β-mercaptoethanol and spun in microfuge. To the supernatant, 25 μl of 4× buffer + β-mercaptoethanol was added and boiled for 5 min more. 15 μl of each sample was analyzed by Western immunoblotting.
Cell fractionation was performed using a modified version of the protocol described by Okada et al. (1995)
. In brief, one mouse brain was homogenized on ice with a glass dounce homogenizer in 3 ml of CF buffer (see Immunoprecipitations section). The homogenate was spun at 3,000 gavg
for 5 min, 9,000 gavg
for 10 min, then centrifuged in a Sorvall 1270 rotor at 100,000 gavg
for 1 h. 50 μg of total protein from each fraction, as determined using Bio-Rad Dc
protein assay kit, was separated on 7.5% polyacrylamide gels and transferred to PVDF membrane (Bio-Rad Laboratories) for Western immunoblotting. P3 pellets were extracted by homogenization with a dounce homogenizer and recentrifuged at 100,000 gavg
for 1 h. The pellet was resuspended in the starting volume and equal volumes of the pellet and supernatant were analyzed by Western immunoblotting.
Construction of KIF21B Motor Protein
A KIF21B motor construct (amino acids 1–750) was generated by PCR with the following primers that contained either a NdeI or XhoI restriction enzyme site (5′-CTG GTG CCG GAG CAT ATG GCT GGC CAG GGC, and 3′-CGC TTG TAG CTT CTC GAG CTC CCT TTC ATA). The PCR product was cloned into the NdeI and XhoI sites of pET-23b (Novagen Inc.). The construct was introduced into BL21 (DE3) bacteria and cells were grown at 37°C until an OD600 ~1.5 and then induced with 0.5 mM IPTG overnight at room temperature. Cells were harvested by centrifugation and resuspended in lysis buffer (300 mM NaCl, 50 mM sodium phosphate, 0.5 mM MgCl2, 0.01% NP-40, 10 μg/ml soybean trypsin inhibitor, 0.7 μl/ml β-ME, 1 mM PMSF, 0.1 μM ATP, pH 7.4) at 1 g/5 ml. Cells were lysed three times with a French press and then spun for 45 min at 30,000 rpm in a 647.5 Sorvall rotor at 4°C. KIF21B-HIS protein was isolated by incubating the high speed supernatant with 0.5 ml of Ni-NTA– agarose beads (Qiagen Inc.) for 2 h. The beads were washed three times with lysis buffer supplemented with 25 mM imidazole and 1 μM ATP, and protein was eluted with lysis buffer + 200 mM imidazole and 1 μM ATP. Protein was concentrated by centrifuging the protein in a Millipore-4 concentrator.
In Vitro Motility Assay
Polarity marked microtubules were generated by the method described in Howard and Hyman (1993)
, using unlabeled bovine tubulin, rhodamine-labeled tubulin (Cytoskeleton), and N
-ethyl maleimide tubulin (Hyman et al., 1991
). 5 μl of 0.5 mg/ml KIF21B motor protein was absorbed onto a coverslip for 3 min and then washed with PEM40 buffer (40 mM Pipes, pH 6.9, 1 mM EGTA, 1 MgCl2
) for 1 min. The motility assay was performed by adding the following components: 1:1,000 polarity marked microtubules; 1 mM MgATP; 10 μM taxol; and an oxygen scavenging system (0.1 mg/ml catalase, 0.03 mg/ml glucose oxidase, 10 mM glucose, 0.1% β-ME; Kishino and Yanagida, 1988
) to PEM40 buffer. Images were gathered using a Zeiss
Axioplan fluorescence microscope, a Princeton Instruments
cooled CCD, and the MetaMorph software package (Universal Imaging Corp.
Animals were killed by suffocation with carbon dioxide and their circulatory system flushed by intracardial perfusion of 1× PBS, followed by fixation with 4% paraformaldehyde in 0.1 M PO4. Tissue was dissected and cryoprotected by overnight incubation in 20% sucrose/1× PBS at 4°C. Tissue was then embedded in OCT compound and 10–15 μm sections were cut on a cryostat. Sections were dried 1 h at room temperature and then blocked with 1× PBS/0.1% Triton X-100/1% BSA for 1 h. Tissue was incubated in 1° antibody for 1 h at room temperature in incubation buffer (1× PBS/0.1% Triton X-100/1% BSA), washed, incubated with the appropriate 2° antibody for 1 h, washed, and then mounted with Vectashield (Vector Labs, Inc.). Tissue was observed using a Bio-Rad MRC-1000 scanning confocal imaging system. Antibody concentrations used: affinity-purified KIF21A and KIF21B, 1 μg/ml; SMI-31, 1:1,000 (Sternberger and Sternberger); MAP2, 1:400 (Boehringer Mannheim); goat α–rabbit Cy5 1:200 (Jackson ImmunoResearch Laboratories); and goat α–mouse Texas red, 1:200 (Jackson ImmunoResearch Laboratories).
In Situ Hybridization
Adult BALB/C mice were killed by cervical dislocation and isolated brains were frozen using Tissue-Tek OCT (Miles Inc.) and Histofreeze (Fisher Scientific Co.
). Parasagittal cryostat sections (20 μm) were cut, thaw-mounted onto charged microscope slides (Superfrost Plus; Fisher Scientific Co.
), fixed, and processed as previously described (Chun et al., 1991
). Digoxigenin-labeled riboprobes were transcribed in the sense and antisense orientations from linearized plasmids containing: KIF21A, nucleotide (nt) 2720 (BamHI) to nt 4260 (EcoO109); KIF21B, nt 3020 (HindIII) to nt 4430 (EcoRV); and est W46113 for MAP2 cDNAs using standard protocols (Boehringer Mannheim Corp.
). Hybridization and color reaction were carried out as previously described (Weiner and Chun, 1997