Yeast spindle poles were prepared by a modification of the earlier method (Rout and Kilmartin, 1990
). The isolation of nuclei was scaled up threefold and the spindle poles were enriched by sucrose velocity and equilibrium gradients followed by a modified Percoll gradient. Nuclei from 40 liters of Saccharomyces uvarum
cells harvested at 2 × 107
cells/ml were pelleted (each Beckman Ty 70 tube [Becton Instruments, Inc., Palo Alto, CA] contained 250 OD260nm
of nuclei which, for example, would correspond to 25 ml of nuclei with an OD of 10). Lysis buffer (Rout and Kilmartin, 1990
) with 20 μg/ml RNase A was added (2.5 ml per tube) and vigorously whirlimixed to lyse the nuclei and release spindle poles. The pH was raised by addition of 0.25 ml of 0.1 M bis-tris (bt)-Cl, pH 6.5, buffer and then the tube was warmed to room temperature for 2–3 min to digest DNA and RNA. The tubes were spun at 2,000 rpm for 6 min in the Beckman Ty 70 rotor (Beckman Instruments, Inc.
) and the supernatant was removed avoiding the floppy part of the pellet. A sucrose gradient was prepared from sucrose-bt solutions (Rout and Kilmartin, 1990
) containing 0.01% Tween 20 (Sigma Chemical Co.
, St. Louis, MO) and DMSO supplements. The gradient was made by overlaying 10 ml of 0.8 M (20% DMSO), 1.2 M (15% DMSO), 1.6, and 2.0 M (10% DMSO) sucrose-bt in a SW28 tube (Beckman Instruments, Inc.
), sealing the top with Parafilm (American National Can, Chicago, IL) and then placing it horizontally for 4 h at 4°C. Sufficient sucrose was removed from the top of this gradient to accommodate the supernatant from two Ty 70 tubes (Beckman Instruments, Inc.
). The gradient was spun at 28,000 rpm for 50 min at 4°C. The spindle poles were visible as a broad band straddling the former 1.6/2.0 M sucrose interface. The sample was further purified and concentrated by a sucrose equilibrium gradient. The sample was incorporated into the solutions for the gradient, which, if there were no sample present, was prepared from overlaying 3.2 ml of 1.75, 2.0, 2.25, and 2.5 M sucrose-bt (with 0.01% Tween 20) in SW40 tubes (Beckman Instruments, Inc.
), which were left horizontal for 4 h at 4°C to prepare the gradient. The velocity gradient sample was incorporated into this gradient by adjusting its sucrose concentration to replace one or more of the layers. The equilibrium gradient was spun at 38,900 rpm for 21 h at 4°C. The spindle poles banded at 2.22 M sucrose. They were further enriched on a modified Percoll gradient as previously described (Rout and Kilmartin, 1990
), except that for each 2 ml of spindle poles, 9.7 ml 2.5 M sucrose, 2.4 ml Percoll, l and 1.5 ml DMSO were added. The concentrations of GTP, EGTA, solution P
(90 mg phenylmethylsulfonylfluoride and 2 mg pepstatin in 5 mL absolute ethanol), and DTT were as before (Rout and Kilmartin, 1990
). The gradient was spun in an angle head Ty 70 rotor (Beckman Instruments, Inc.
) at 35,000 rpm for 3 h at 4°C.