Squid, Loligo pealeii, were obtained from the Marine Resources Department at the Marine Biological Laboratory at Woods Hole, Massachusetts. Giant axons were dissected from the squid as previously described (Vale et al., 1985a
) and were frozen in liquid nitrogen. Freshly removed squid optic lobes were also frozen in liquid nitrogen. Fresh bovine brain was obtained from a slaughter house, placed on ice, and used within 1 hr of slaughtering. Carboxylated latex beads (0.15 μ
m diameter; 2.5% solid/solution) were obtained from Polyscience Inc. (Warrington, Pennsylvania) and glass coverslips (type 0) from Clay Adams (Parisippany, New Jersey). Taxol was a gift from Dr. Matthew Suffness at the National Cancer Institute. Bid-Gel A5m and hydroxyapatite were obtained from Bio-Rad Inc. (Richmond, New York). All other reagents were purchased from Sigma Chemical Co. (St. Louis, Missouri).
Preparation of Axoplasmic Supernatant and Organelles
S2 axoplasmic supernatant and organelles were prepared as previously described (Vale et al., 1985b
) with the following modification in the sucrose gradient. A low speed (40,000 × g/min) supernatant (about 150 μ
l) from a homogenate of seven to ten axoplasms was incubated with 20 μ
M taxol and 1 mM GTP, and applied to a sucrose gradient in a 5 × 41 mm Ultra-Clear centrifuge tube (Beckman Inst., Palo Alto, California) consisting of 7.5% (125 μ
l), 15% (125 μ
l), and 35% (200 μ
l) sucrose layers. Sucrose solutions were made in motility buffer (175 mM potassium asparate, 65 mM taurine, 85 mM betaine, 25 glycine, 20 mM Hepes [pH 7.2], 6.4 mM MgCl2
, and 5 mM EGTA) containing 20 μ
M taxol and 1 mM GTP (this buffer is referred to as MTG buffer) and 2 mM ATP. The sucrose gradient was centrifuged at 135,000 × g for 70 min at 4°C in a SW50.1 rotor. The organelle band at the 15%–35% sucrose interface as well as 75–100 μ
l of the S2 supernatant above the gradient were collected.
Assays for Microtubule, Bead, and Organelle Movement
These assays (Vale et al., 1985b
), as well as a complete account of the procedure for visualizing objects by video-enhanced
differential interference contrast microscopy (B. Schnapp, submitted), will be described elsewhere. To measure microtubule movement, a 1 μ
l suspension of microtubules essentially free of microtubule-associated proteins (0.5–1 mg/ml solution; see below for preparation) was combined with 3–4 μ
l of a test sample on a glass coverslip. In some instances, a microtubule pellet was directly assayed for movement by placing an aliquot of the microtubules directly on a coverslip in an ATP-containing buffer. Microtubule movement on glass or microtubule movement relative to other microtubules in solution was visualized with the video microscope.
A sample was scored positive for microtubule movement if movement was observed on glass or in solution. The presence or absence of movement in different parts of a preparation was so consistent that only a minute or two of observation was required to evaluate a sample. To determine the amount of movement-inducing activity of a sample, the maximum dilution that still supported microtubule movement was determined.
For determining movement of carboxylated latex beads, glass coverslips were treated overnight with a 1 mg/ml solution of poly-D-lysine, which prevented microtubules from moving along the glass; bead movements on microtubules could then be examined without the complication of simultaneous microtubule movement. A test sample (10 μl) was combined with beads (4 μl of a 200-fold dilution of a 2.5% solid stock solution in sample buffer) for 5 min at 4°C. Then 3–4 μl of this mixture was combined with 1 μl of microtubules on a poly-D-lysine-coated coverslip, and movement of beads along the microtubules was evaluated.
Organelle movement was assayed by combining 1 μl of microtubules with 3 μl of organelles (in motility buffer and 2 mM ATP) and 3 μl of test sample. The purified protein sample in motility buffer plus 2 mM ATP or in 100 mM KCl, 50 mM Tris (pH 7.6), 5 mM MgCl2, 0.5 mM EDTA, 2 mM ATP yielded equivalent results with regard to organelle movement. High concentrations of phosphate, however, inhibited organelle movement, so fractions from the hydroxyapatite column were equilibrated with motility buffer using a Centricon filter (molecular weight cutoff of 30 kd; Amicon Corp., Massachusetts) precoated with bovine serum albumin. A video recording of a 20 × 20 μm field of microtubules was made over 2–5 min, and the number of different organelles moving per minute was determined. Velocities of microtubule, bead, and organelle movements were determined over distances of 3–10 μm.
ATPase activity was measured according to the procedure of Clarke and Spudich (1974)
. Protein samples (22.5 μ
l) in 0.1 M KCl, 50 mM Tris (pH 7.6), 5 mM MgCl2
, 0.5 mM EDTA were combined with 2.5 μ
l of 1–10 mM ATP containing 32
P-ATP (approximately 150,000 cpm) in 1.5 ml microfuge tubes at 23°C for 5 to 40 min.
Microtubule Affinity Purification of Squid Kinesin
Squid optic lobes (40 gm wet weight; either freshly dissected or previously frozen in liquid N2) were homogenized in 1½–2 vol of motility buffer containing 1 mM ATP, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 μg/ml leupeptin, 10 μg/ml N-a-p-tosyl-L-arginine methyl ketone (TAME), 100 μg/ml soybean trypsin inhibitor, and 0.5 mM DTT with 40 strokes in a Dounce homogenizer. Homogenization and all subsequent steps were performed at 4°C unless specified. The homogenate was centrifuged for 30 min at 40,000 × g, and the supernatant was collected and recentrifuged at 150,000 × g for 60 min. The supernatant from this centrifugation (S2) was incubated with 20 μM taxol and 1 mM GTP for 25 min at 23°C to polymerize tubulin into microtubules (see Preparation of Microtubules).
The mixture was then layered over a 15%/50% (2–4 ml of each) sucrose gradient made in MTG buffer, and the gradient was centrifuged at 100,000 × g for 60 min in a SW27 rotor. The supernatant (S3, approximately 40 ml) was collected, taking care to avoid the first interface. S3 supernatant then was incubated with microtubules (100 μg/ml) in the presence of 5 mM AMP-PNP for 15 min at 23°C, and microtubules were sedimented at 40,000 × g for 30 min. The following conditions proved best for releasing the translocator in a relatively pure form, although different conditions were used in some experiments, as described in the figure legends. The microtubule pellet was resuspended in 1–2 ml of MTG containing 10 mM AMP-PNP for 15 min at 4°C to release proteins that dissociate from microtubules by dilution. Microtubules were pelleted at 37,000 × g for 30 min, and the pellet was washed once with MTG to remove AMP-PNP. The pellet was then resuspended in 1 ml of MTG containing either 10 mM ATP or 5 mM ATP and 0.1 M KCl for 30–40 min at 23°C. Microtubules were centrifuged as before, and the supernatant containing proteins that induced microtubule movement was removed (typically containing 1–4 mg/ml total protein) and, if not used that day, was stored in liquid nitrogen without significant loss of activity. The amount of the 110 kd polypeptide in the microtubule pellet and supernatant was assayed by polyacrylamide gel electrophoresis, and when necessary, the pellet was extracted again with 1 ml of MTG containing 10 mM ATP to retrieve additional translocator protein.
Microtubule Affinity Purification of Bovine Translocator
White matter (70 gm) was obtained from brains of freshly killed cows and homogenized at 4°C in a weight:volume ratio of 1:1 of 50 mM Pipes, 50 mM Hepes, 2 mM MgCl2, 1 mM EDTA, 0.5 mM DTT, 1 mM PMSF, 10 μg/ml leupeptin, 10 μg/ml TAME, and 0.5 mM ATP (pH 7.0) with five bursts of 5 sec in a Polytron homogenizer. The homogenate was centrifuged at 25,000 × g for 30 min at 4°C, and the supernatant was collected and recentrifuged at 150,000 × g for 60 min at 4°C. GTP (1 mM) and taxol (10 μM) were added to the supernatant for 30 min at 25°C, and the polymerized microtubules were pelleted at 37,000 × g for 30 rain at 20°C. The supernatant was removed and the microtubule pellet was further processed as described below. The supernatant (comparable to squid S3) was then incubated with squid or bovine microtubules (100 μg/ml) and AMP-PNP (5 mM) for 15 min at 23°C, and the microtubules were pelleted at 37,000 × g for 20 min at 20°C. The supernatant was discarded, and the pellet was resuspended in 5 ml of homogenization buffer containing 5 mM AMP-PNP without ATP for 15 min at 23°C. The microtubules were pelleted as described above, and the pellet was washed with 2 ml of homogenization buffer without AMP-PNP or ATP. The bovine translocator was released by incubation in 1–3 ml of homogenization buffer containing 10 mM ATP for 30–40 min at 23°C. Microtubules were pelleted as before, and the supernatant containing released bovine translocator was collected (1–2 mg/ml). Additional translocator was obtained in some instances by further extraction with 0.1 M KCl/10 mM ATP.
Preparation of Microtubules
Taxol-polymerized microtubules essentially free of microtubule-associated proteins were prepared from squid optic lobes either as previously described (Vale et al., 1985b
) or in conjunction with the preparation of squid translocator. In the latter instance, S2 supernatant from squid optic lobes (see Microtubule Affinity Purification of Squid Kinesin) was incubated with 20 μ
M taxol and 1 mM GTP for 25 min at 23°C to polymerize soluble tubulin into microtubules. The mixture was layered over a 15%/50% (2–4 ml of each) sucrose gradient made in MTG buffer and centrifuged for 60 min at 100,000 × g in a SW27 rotor. The microtubule pellet was then washed once with 2 ml of 100 mM Pipes (pH 6.6), 5 mM EGTA, 1 mM MgSO4
(PEM) containing 20 μ
M taxol and 1 mM GTP and resuspended in 4 ml of the same buffer. To extract microtubule-associated proteins, 2 ml of PEM-taxol-GTP containing 3 M NaCl was added to the microtubules, and the sample incubated at 23°C for 30 min. Microtubules were centrifuged at 37,000 × g for 30 min at 10°C, and the pellet was washed once with PEM-taxol-GTP. Microtubules were then resuspended in 2 ml of PEM-taxol-GTP at a concentration of 2–8 mg/ml and stored in liquid N2
. Microtubules obtained during the purification of translocator from bovine brain (see above) were further processed by the protocol of Regula et al. (1981)
. Bovine microtubules were also prepared according to Vallee (1982)
Polyacrylamide Gel Electrophoresis
Electrophoresis was performed in polyacrylamide gels (7.5%) under denaturing and reducing conditions according to the method of Laemmli (1970)
and stained with Coomassie blue. Densitometry was performed using a Zenith Soft Laser Scanning densitometer equipped with a helium-neon laser.