Chemicals were from Sigma-Aldrich unless stated otherwise. The following reaction buffers were used: BRB80 (80 mM Pipes 6.8, 1 mM EGTA, 1 mM MgCl2); SRB1 (BRB80, 80 mM KCl, 0.5 mg/ml casein, 1 mM GTP, 1 mM DTT); SRB2 (SRB1, 0.25% Brij-35); and SRB3 (BRB80, 100 mM KCl, 0.25% Brij-35, 5% glycerol).
Stu2p antibody production
Polyclonal Stu2p antibodies were raised against a COOH-terminal peptide of Stu2p in rabbits and subsequently affinity purified (Eurogentec).
Purification of tubulin and centrosomes
Tubulin was purified to homogeneity from porcine brain by three cycles of microtubule assembly before phosphocellulose chromatography (Ashford et al., 1998
). Rhodamine or Oregon green labeling of tubulin was done as described previously (Hyman et al., 1991
). Centrosomes were purified from human KE37 cells using a low ionic strength lysis step and subsequent sucrose gradients (Moudjou and Bornens, 1998
Microtubules were spotted at appropriate dilutions on poly-lysine microscope slides (Sigma-Aldrich), fixed for 3 min in −20°C methanol, and incubated in PBN (PBS, 2% BSA, 0.1% NP-40) for 5 min at RT. Microtubules were subsequently incubated directly with FITC-labeled DM1α tubulin antibody (Sigma-Aldrich) in PBN for 20 min at RT. After several washes, the slides were mounted in antifade-mounting medium. Images were acquired using a Axioplan 2 microscope (Carl Zeiss MicroImaging, Inc.) with a 63× Plan-Apochromat lens (NA 1.40; Carl Zeiss MicroImaging, Inc.) together with the MetaMorph Imaging System (version 1.4.2.) and processed using Adobe Photoshop®.
Preparation of yeast extracts
Yeast extracts were prepared in SRB3, 1 mM DTT, and protease inhibitor cocktail (Complete Mini, EDTA free; Roche) by bead beating using a W303 (mating type a) strain. Extracts were spun in a Beckman Coulter TLA 100 rotor at 100 krpm for 5 min at 4°C, and aliquots of the supernatant were snap frozen in liquid nitrogen and kept at −80°C. The protein concentration was between 10 and 20 mg/ml.
Purification of recombinant Stu2p as baculovirus-derived protein
A 500-ml suspension culture of Sf+ cells was infected at a density of 106 cells per ml with 2 ml of recombinant baculovirus at ~108 pfu/ml. Recombinant baculovirus expressing full-length Stu2p was a gift from Peter Sorger (Massachusetts Institute of Technology, Cambridge, MA) and prepared using the pFAST Bac system (Invitrogen). Cells were harvested 52 h after infection. Cell extracts were prepared by resuspension of the cell pellet in 50 mM Hepes, pH 7.5, 5% glycerol, 0.1% Triton X-100, 150 mM NaCl, and 1 mM DTT, and supplemented with protease inhibitors. The extracts, cleared by ultracentrifugation and ultrafiltration, were loaded onto a cation exchange column (HiTrap SP, 5 ml; Amersham Biosciences) equilibrated in CB (6.7 mM Hepes, 6.7 mM MES, 6.7 mM sodium acetate, pH 7.2) and 150 mM NaCl. A linear gradient from CB, 150 mM NaCl to CB, 1 M NaCl was developed over 10 column vol. Stu2p eluted between 420 and 490 mM NaCl. These fractions were pooled and run on a gel filtration column (Superose 6, prep grade; Amersham Biosciences) in BRB80, 75 mM KCl, 1 mM DTT, and 10 μM ATP, and supplemented with protease inhibitors. Fractions containing the Stu2p peak were pooled and loaded onto a cation exchange column (HiTrap SP, 1 ml; Amersham Biosciences), equilibrated, and run in BRB80, 100 mM KCl. Stu2p was eluted in BRB80, 450 mM KCl and desalted using a PD10 column equilibrated and run in SRB3 supplemented with protease inhibitors and (optionally) 0.25% Brij-35. This preparation was aliquoted, snap frozen, and kept in liquid nitrogen.
Direct labeling of Stu2p
7.5 μM recombinant Stu2p in SRB3 was incubated with 180 μM monoreactive Cy3 (Amersham Biosciences) for 1 h on ice. After quenching the reaction by addition of potassium glutamate to 220 mM, labeled Stu2p was desalted into SRB3 and spun at 100 krpm for 5 min at 4°C in a Beckman Coulter TLA 100 rotor. DTT was added to 1 mM, and the supernatant was aliquoted, snap frozen, and kept in liquid nitrogen. The labeling stoichiometry (dye to protein) was determined spectrophotometrically and found to be 1.05.
Gel filtration was done on a Superose 6 PC 3.2/30 column (2.4 ml; Amersham Biosciences), equilibrated, and run in SRB3 at a flow rate of 0.02 ml/min. The column was calibrated in several runs with the following markers (Amersham Biosciences) of known Stokes radius: albumin, thyroglobulin, chymotrypsinogen A, ferritin, ovalbumin, catalase, aldolase, ribonuclease A. The void volume was determined using Blue Dextran. Recombinant Stu2p or yeast extracts cleared by ultrafiltration were subsequently run, 100-μl fractions were collected, and the peak position of Stu2p was determined using the chromatogram (absorption at 280 nm) or by SDS-PAGE and subsequent Western blotting of the collected fractions using a polyclonal Stu2p antibody.
Sucrose step gradients were prepared from 5 to 40% sucrose (in SRB3) in five equal steps and allowed to diffuse into a linear gradient overnight at 4°C. Precleared (100 krpm in a Beckman Coulter TLA 100, at 4°C for 5 min) recombinant Stu2p or yeast extracts, and in parallel a mixture of BSA, aldolase, catalase, and thyroglobulin (Amersham Biosciences) as markers of known S value, were spun through the gradients (4°C for 4 h at 50 krpm in a Beckman Coulter TLS 55 rotor), and gradients were fractionated by taking 16 equal fractions from the top. The position of proteins within the gradients was determined on Coomassie-stained SDS-PAGE gels (markers) or by Western blotting using a polyclonal Stu2p antibody (recombinant Stu2p and yeast extracts).
Microtubule nucleation assay
All protein preparations were precleared before usage (100 krpm in a Beckman Coulter TLA 100, at 4°C for 5 min). 22 μM tubulin was incubated in SRB2 with varying concentrations of Stu2p (0–2.5 μM) for 10 min at 29°C. Nucleated microtubules were separated from soluble tubulin by centrifugation through a 40% glycerol cushion (70 krpm for 5 min at 29°C in a Beckman Coulter TLA 100 rotor). Equivalent amounts of supernatant and pellet were analyzed by SDS-PAGE and Coomassie staining.
Determination of microtubule length by a fixed time point assay
Microtubules were grown from purified centrosomes at 26 μM tubulin (1:10 labeled with rhodamine tubulin) and various concentrations of Stu2p (0–1 μM Stu2p) in SRB2 at 29°C for 10 min. Prior to usage, protein preparations were precleared by centrifugation (100 krpm at 4°C for 5 min in a Beckman Coulter TLA 100 rotor). Reactions were fixed with 0.75% glutaraldehyde for 3 min and subsequently quenched for 5 min with 0.1% sodium borohydride. Part of the reaction was squashed under a coverslip and analyzed microscopically. Images were acquired using a Axioplan 2 Microscope (Carl Zeiss MicroImaging, Inc.) with a 63× Zeiss Plan-Apochromat lens (NA 1.40; Carl Zeiss MicroImaging, Inc.) together with the MetaMorph Imaging System (version 1.4.2.). The radii of at least 39 asters were measured for each Stu2p concentration using the MetaMorph Imaging software, and images were subsequently processed using Adobe Photoshop®.
Measurement of cytoplasmic microtubule length in vivo
Wild-type cells or stu2–10 cells (both GFP-tub1–His3 and derivatives of W303) were grown in YP, 2% raffinose at 25°C to an OD600
of 0.4, arrested with 100 mM HU at 25°C for 140 min, and shifted to 37°C for another 140 min. A FACScan®
was used as described (Epstein and Cross, 1992
). Cells were prepared for indirect immunofluorescence as published (Piatti et al., 1996
) using the mouse monoclonal antitubulin antibody DM1α (Sigma-Aldrich) and a FITC-conjugated secondary antibody. DNA was visualized by DAPI. Imaging was done using a 100×, 1.35 NA objective on an Olympus DeltaVision microscope using SoftWorx (Applied Precision). 40 0.2-μm stacks were taken for each field, and cytoplasmic microtubule length in single cells was measured three-dimensionally from the tip of the microtubule to the spindle pole body as multiple segments (SoftWorx; Applied Precision). Between 153 and 175 cytoplasmic microtubules were quantitated. Images depicted in this paper were projected using SoftWorx (Applied Precision) and processed using Adobe Photoshop®
. To determine cell sizes, DIC images of at least 68 cells were acquired using a Axioplan 2 microscope (Carl Zeiss MicroImaging, Inc.) with a 63× Plan-Apochromat lens (NA 1.40; Carl Zeiss MicroImaging, Inc.) together with the MetaMorph Imaging System (version 1.4.2.). Cells were analyzed by measuring the length of a line drawn from the tip of the mother cell through the bud neck to the tip of the bud using the MetaMorph Imaging software. The average mother cell–bud distance was for the cells arrested at the permissive temperature with HU before the shift to the restrictive temperature: 6.5 ± 1.1 μm (standard deviation) for the wild-type and 6.5 ± 1.0 μm for the stu2–10 cells. After the temperature shift, this distance was 7.5 ± 1.1 μm for the wild-type and 7.5 ± 1.1 μm for the stu2–10 cells. Thus, stu2–10 and wild-type cells did not differ in their size.
The VE-DIC assay was adapted from Kinoshita et al. (2001)
. Small (~12 μl) home made perfusion chambers were put on a metal block on ice, and purified centrosomes were perfused into the chambers and allowed to adsorb to the coverslips for 5 min. The glass surface was blocked three times for 5 min with 5 mg/ml casein in BRB80, and free casein was removed by four washes with 20 μl 0.25% Brij-35 in BRB80. 30-μl mixtures of prespun (4°C for 5 min at 100 krpm in a Beckman Coulter TLA 100 rotor) tubulin (21.5 μM) and various levels of prespun Stu2p (0–1 μM) in SRB2 was perfused at 4°C into the chambers. Reactions were started by transferring the chambers to a metal block at RT and then immediately onto the stage of a VE-DIC microscope kept in a temperature control box at 29°C. For each experiment, one aster was analyzed between 5 and 12 min after the start of the reaction. At least four experiments were done at each concentration of Stu2p. Videos were acquired on an Olympus BX50 microscope using an Olympus PlanApo 60× lens (NA 1.40) and a Hamamatsu 2400 Newvicon video-camera. Images were enhanced with an Argus 20 (Hamamatsu). Microtubules were tracked using ScionImage (version 1.62). The parameters of microtubule polymerization were subsequently determined from the entire dataset at each concentration of Stu2p. Rescue events occurred too rarely to be reported as a rescue frequency.
To determine the relationship of growth rate to catastrophe frequency, we used the same set-up with minor changes: smaller chambers (~6 μl) were employed and axonemes (gifts from Denis Chrétien, Université de Rennes, Rennes, France) instead of centrosomes were used as microtubule nucleators. The tubulin concentration ranged from 14.3 to 27.5 μM in SRB2. Three films from ~1.5 to ~13 min after the start of the reaction were obtained at each concentration of tubulin. Microtubule growth rates were linear with tubulin concentration. Microtubules were grouped according to their growth rates in groups of 0.06-μm/min increments, and the average growth rate and the catastrophe frequency of the groups were subsequently calculated. Groups contained between 9 microtubules with a total growth time of 19 min and 44 microtubules with a total growth time of 104 min.
Taxol-stabilized microtubules were sheared on ice 10 times for 1 s with 3 min on ice in between with a tip sonicator at 40% amplitude. The lengths of at least 91 microtubules were determined by immunofluorescence using the MetaMorph Imaging software. The average microtubule length was 16.6 μm for the unsheared (SEM [P < 0.05]; 2.5 μm) and 3.4 μm for the sheared microtubules (SEM [P < 0.05]; 0.5 μm).
Microtubule copelleting assay
Increasing amounts of taxol-stabilized microtubules were incubated in SRB2: 20 μM taxol with prespun (4°C for 5 min at 100 krpm in a Beckman Coulter TLA 100 rotor) Stu2p for 5 min at RT. Microtubule-bound protein was separated from unbound protein by centrifugation (25°C for 5 min at 70 krpm in a Beckman Coulter TLA 100 rotor). For the shearing experiment, the reactions were spun through a 40% glycerol BRB80 cushion. Equivalent amounts of supernatants and pellets were subsequently analyzed by Western blotting using a polyclonal Stu2p antibody, the monoclonal anti–α-tubulin antibody DM1α (Sigma-Aldrich), or the monoclonal anti–β-tubulin antibody TUB2.1 (Sigma-Aldrich). The percentage of binding was measured using the Histogram function in Adobe Photoshop®.
Production of polarity-marked microtubules
Bright microtubule seeds were made by incubating 75 μM of a prespun (4°C for 5 min at 100 krpm in a Beckman Coulter TLA 100 rotor) 1.8:1 mixture of Oregon green–labeled tubulin and unlabeled tubulin in BRB80, 1 mM DTT with 0.1 mM GMPCPP at 37°C for 15 min. The seeds were diluted 1:125 in a prewarmed mixture containing 20 μM of a 1:5 mixture of Oregon green–labeled and unlabeled tubulin in BRBR80, 1 mM GTP, 1 mM DTT, and 12.5 μM NEM-treated tubulin (to inhibit minus end polymerization). After a 30-min incubation at 37°C, the mixture was diluted in prewarmed BRB80, 20 μM taxol, and 1 mM DTT. Taxol-stabilized microtubules were reconcentrated by centrifugation (25°C for 5 min at 50 krpm in a Beckman Coulter TLA 100 rotor) and resuspended in BRB80, 20 μM taxol, and 1 mM DTT.
Removal of the COOH terminus of β-tubulin in microtubules
Taxol-stabilized microtubules were incubated in BRB80, 1 mM DTT, and 20 μM taxol for 15 min at 37°C with 30 μg/ml subtilisin (Fluka). The digestion was stopped by adding PMSF to 5 mM. Completion of digestion was checked by SDS-PAGE and Coomassie staining or by Western blotting using the β-tubulin–specific mAb TUB2.1 (Sigma-Aldrich) whose epitope is situated in the COOH terminus of β-tubulin.
Visualization of Stu2p binding on microtubules
Prespun (4°C for 5 min at 100 krpm in a Beckman Coulter TLA100 rotor) 16 nM directly labeled Stu2p in SRB3, 1 mM DTT, and 20 μM taxol was incubated for 5 min at RT with 11.5 μM taxol-stabilized polarity marked microtubules. The reaction was fixed with 0.75% glutaraldehyde for 3 min, quenched for 5 min with 0.1% sodium borohydride, and analyzed microscopically after a 1:200 dilution in SRB3, 20 μM taxol. Images were acquired using a Axioplan 2 microscope with a 63× Plan-Apochromat lens (NA 1.40; Carl Zeiss MicroImaging, Inc.) together with the MetaMorph Imaging System (version 1.4.2.). The intensities of the signals in the rhodamine (Stu2p) channel were scanned along the length of microtubules using the Linescan function of the MetaMorph Imaging software with a pixel width of 5. Peaks higher then 10% of the median intensity along the microtubule were scored with their location along the microtubule. Microtubules were divided in 10% intervals from their minus ends to their plus ends. 124 polarity marked microtubules were quantitated. In the absence of directly labeled Stu2p, no significant number of peaks were detected. The median intensity in the absence and presence of directly labeled Stu2p was found to be virtually identical. Images were processed using Adobe Photoshop®.
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