Xenopus egg extracts, spindle assembly, immunodepletion, and antibody addition
M-phase egg extracts (M-phase extracts; cytostatic factor arrested) were prepared as described previously (Murray, 1991
). Cycled spindle assembly, immunodepletion, and antibody addition were performed as described previously (Wittmann et al., 2000
; Hannak and Heald, 2006
). In antibody addition experiments, rabbit IgG (Sigma-Aldrich) or anti–full-length Cdk11 antibody was added to extracts at 0.2–0.5 mg/ml.
RanGTP-dependent MT stabilization assay
A standard assay reaction contained 10 μl of M-phase extract, 1 μM Cy3-labeled tubulin, 0.15 mg/ml anti–full-length TPX2 antibody, and 2,000 isolated centrosomes per microliter in the presence or absence of 12–32 μM RanQ69L-GTP. Samples were incubated at 20°C for 30 min, fixed with 1 ml 0.25% glutaraldehyde, 10% glycerol, and 0.1% Triton X-100 in BRB80 (80 mM K-Pipes, 1 mM MgCl2, and 1 mM EGTA, pH 6.8), and spun down onto 12-mm round coverslips through a cushion of 25% glycerol in BRB80. The coverslips were postfixed with cold methanol for 10 min at −20°C, washed with PBS, and mounted on slides. Images were acquired using a microscope (Axiovert 200M; Carl Zeiss, Inc.), a plan-Neofluar 40× NA 1.3 oil objective lens (Carl Zeiss, Inc.), a Cy3 emission filter, a camera (AxioCam HRm; Carl Zeiss, Inc.), and AxioVision software (Carl Zeiss, Inc.). The mean MT length of centrosomal asters was quantified using a macro written in Matlab (The MathWorks; Fig. S1).
Preparation of recombinant proteins and affinity beads
His-RanQ69L, His-RanT24N, His–importin β, His–importin α, and His-ED mutant of importin α were expressed in bacteria and purified with talon beads (BD Biosciences). Loading of GTP on RanQ69L was described previously (Weis et al., 1996
). Cyclin B Δ90 was prepared as described previously (Glotzer et al., 1991
Saturating amounts of z tag-RanQ69L or GST–importin β bacterial lysate were incubated with IgG Sepharose or glutathione Sepharose, respectively. The beads were washed with cytostatic factor extract buffer (CSF-XB; 10 mM K-Hepes, 100 mM KCl, 3 mM MgCl2, 0.1 mM CaCl2, 5 mM EGTA, and 50 mM sucrose, pH 7.7). To prepare GST–importin β/α or GST–importin β/ED beads, excess purified recombinant His–importin α or His-ED mutant was incubated with the GST–importin β lysate at 4°C for 1 h. Then, the mixture was incubated with glutathione Sepharose at 4°C for 1 h. The beads were washed with CSF-XB.
Preparation of the NLS protein fraction and the depleted extract
M-phase extracts with 10 μg/ml cyclin B Δ90 were incubated with a 40% wet bead volume of z-RanQ69L beads in a Mobicol column (MoBiTec) at 4°C for 1 h with rotation. The beads were collected by centrifugation, and the remaining extract (activated extract) was incubated with a 120% wet bead volume of GST–importin β beads at 4°C for 1 h with rotation. After centrifugation, the extract (depleted extract) was frozen in 50-μl aliquots in liquid nitrogen and stored at −80°C. The GST–importin β beads were washed five times with wash buffer (CSF-XB, 100 mM KF, 80 mM β-glycerophosphate, 0.1 mM sodium vanadate, 0.03% digitonin, and 1 mM DTT) and eluted with elution buffer (20 μM His-RanQ69L-GTP, 500 mM NaCl, 1 mM GTP, 1 mM ATP, and 10% glycerol in the wash buffer) at 4°C overnight with rotation. The NLS protein fraction was recovered as the supernatant by centrifugation and dialyzed to buffer A (CSF-XB, 10% glycerol, and 1 mM DTT). The fraction was directly used for experiments or stored in aliquots at −80°C.
Purification of MT stabilization activity from the NLS protein fraction
40 ml of the NLS protein fraction prepared from 100 ml of M-phase extracts was applied to a 1-ml Mono S column. The bound proteins were eluted with 10 ml of a 100–1,000-mM KCl gradient in buffer A and fractionated into 10 fractions. The MT stabilization activity of each fraction, which had been dialyzed to buffer A, was assayed in the depleted extract supplemented with centrosomes, anti-TPX2 antibodies, and Cy3-labeled tubulin at 20°C for 30 min. The pooled Mono S active fractions (fractions 3–7; 5 ml; ~300 mM KCl) were adjusted to 350 mM KCl and applied to a 0.1-ml Mono Q column. The bound proteins were eluted with 1 ml of a 350–1,000-mM KCl gradient in buffer A and fractionated into 10 fractions. The pooled Mono Q active fractions (fractions 3–6; 0.4 ml; ~400 mM KCl) were concentrated by Centricon 10 (Millipore), applied to a 2.4-ml Superdex 200 column, eluted with buffer A + 100 mM KCl, and fractionated (40 μl). The Superdex 200 fractions were separated on SDS-PAGE, and the gel was stained with Cypro ruby. Selected bands in the active fractions were cut and digested with trypsin (Shevchenko et al., 1996
). The resulting peptide mixture was analyzed by matrix-assisted laser desorption/ionization time of flight (Uraflex; Bruker Daltonics) and liquid chromatography tandem mass spectrometry (liquid chromatography, Eksigent NanoLC_1D; mass spectrometry, Qstar Pulsar I; Applied Biosystems). Proteins were identified using the Mascot search tool.
Cloning and expression of Xenopus Cdk11
A cDNA clone (IMAGE:5073384; RZPD) covering the complete Xenopus Cdk11 cDNA was amplified by PCR and cloned into NcoI–XhoI sites of pFastBac HTa (Invitrogen). Baculoviruses were prepared according to the manufacturer's instructions. Recombinant Cdk11 was expressed in Sf9 insect cells and purified on Talon beads and the Mono Q column. The K460R mutant of Cdk11, in which lysine 460 was replaced with arginine, was generated by site-directed mutagenesis using PfuTurbo DNA polymerase (Stratagene). The K460R mutant, N-terminal Cdk11 (1–349 aa), and p58 Cdk11 (350–788 aa) were constructed, expressed, and purified as described for Cdk11.
Rabbit polyclonal antibodies against Xenopus full-length TPX2, full-length Cdk11 (1–788 aa), N-terminal Cdk11 (1–349 aa), and p58 Cdk11 (350–788 aa) were prepared and purified against recombinant proteins according to a standard protocol. Antinucleoplasmin monoclonal antibody was purchased from Iowa University.
Online supplemental material
Fig. S1 shows the quantification method to determine astral MT length used in the purification assay, identification of the TPX2 antibody that blocks MT nucleation, and specific inhibition of the MT stabilization activity by the importin α/β heterodimer. Fig. S2 shows that recombinant Cdk11 has RanGTP-dependent MT stabilization activity but that a kinase-dead mutant does not. Fig. S3 shows the localization of Cdk11 at spindle poles and on spindle MTs in Xenopus
tissue culture cells and egg extracts. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200706189/DC1