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[17] P u r i f i c a t i o n a n d C h a r a c t e r i z a t i o n o f D y n a m i n By H O W A R D S. SHPETNER a n d R I C H A R D B. VALLEE
Dynamin is a microtubule-associated mechanochemical ATPase recently identified in calf brain white matter cytosol) Like kinesin 2,3 and brain cytoplasmic dynein,4-7 it exhibits nucleotide-sensitive binding to microtubules, its ATPase activity can be stimulated severalfold by microtubules, and it has shown evidence of force production against microtubules in in vitro assays. It can be distinguished from kinesin and dynein by its nucleotide specificity, its polypeptide composition, and, most strikingly, by the structures it produces in combination with tubulin: unlike kinesin and cytoplasmic dynein, dynamin forms regular arrays of periodic crossbridges between microtubules, arranging them in bundles that fragment and elongate in the presence of ATP. Thus, dynamin may mediate in vivo sliding between microtubules, in processes as diverse as axonal transport and anaphase B of mitosis. Dynamin consists of a 100-kDa polypeptide and an activating factor which has been only partially purified. The purified 100-kDa species forms cross-bridges between microtubules that appear morphologically identical to those formed in the presence of the activating factor, which, however, is required for both microtubule-activated ATPase activity and bundle elongation. We describe here a method for obtaining both the purified 100-kDa protein and the activating factor in separate fractions from a single preparation of brain microtubules. Principle Taxol-stabilized microtubules prepared without nucleotides from calf brain cytosol are specifically enriched in dynein, kinesin, and dynamin, and all three proteins can be dissociated by ATP. However, unlike dynein, dynamin is also dissociable by GTP, and, unlike kinesin, dynamin is not
H. S. Shpetner and R. B. Vallee, Cell59, 421 (1989). 2 R. D. Vale, T. S. Reese, and M. P. Sheetz, Ce1142, 39 (1985). 3 S. A. Kuznetsov and V. I. Gelfand, Proc. Natl. Acad. Sci. U.S.A. 83, 8530 (1986). 4 B. M. Paschal, H. S. Shpetner, and R. B. Vallee, J. CellBioL 105, 1273 (1987). 5 B. M. Paschal and R. B. Vallee, Nature (London) 330, 181 (1987). 6 R. B. Vallee, J. S. Wall, B. M. Paschal, and H. S. Shpetner, Nature (London) 332, 561 (1988). 7 H. S. Shpetner, B. M. Paschal, and R. B. Vallee, J. CellBioL 107, 1001 (1988).
METHODS IN ENZYMOLOGY, VOL. 196
Copyright © 1991 by Academic Press, Inc. All fights of reproduction in any form reserved.
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induced to bind microtubules by AMP-PNP. Thus, dynamin can be specifically extracted from microtubules using a combination of AMP-PNP and GTP, leaving the other two proteins bound. The dynamin-enriched extract is further fractionated by centrifugation in the presence of exogenous microtubules. For reasons that are not yet understood, the activating factor remains dissociated from microtubules at this stage and is obtained in the supernatant, while the 100-kDa protein is quantitatively found in the microtubule pellet. In contrast to earlier stages of the preparation, extraction of the 100-kDa protein from the microtubules now requires elevated salt concentrations as well as nucleotide, apparently due to binding to microtubules via both nucleotide-insensitive and nucleotide-sensitive sites. The extracted 100-kDa protein is subsequently purified by anion-exchange chromatography. Materials Calf brain white matter, 75-100 g, dissected from five or six fresh brains Extraction buffer: 250 ml of 10 m M sodium phosphate, pH 7.0, 80 m M sodium glutamate, 1 m M MgSO4, 1 m M EGTA, 1 m M dithiothreitol (DTT), 1 m M phenylmethylsulfonyl fluoride, (PMSF), 4 pg/ml leupeptin, 10/2g/ml tosyl arginine methyl ester (TAME), and 1/tg/ml pepstatin Taxol, 350 #1 of a 10 m M stock in dimethyl sulfoxide, obtained as a dry powder from the National Cancer Institute, Drug Synthesis and Chemistry Branch, DTP, DCT, Bethesda, Maryland. Store at - 8 0 ° Sucrose underlayer solution: 40 ml extraction buffer containing 7.5% sucrose MgAMP-PNP: 0.3 ml ofa 0.1 M stock, made equimolar with MgSO4 and adjusted to pH 7.0 on ice using NaOH MgGTP [Sigma (St. Louis, MO) type IIS or equivalent]: 0.3 ml of a 0.1 M stock, made equimolar with MgSO4 and adjusted to pH 7.0 on ice using NaOH P/G buffer: 250 ml of 5 m M sodium phosphate, pH 7.0, 50 m M sodium glutamate, 1 m M MgSO4, 1 m M EGTA, and I m M dithiothreitol (DTT) 3 PD-10 gel-filtration columns (Pharmacia-LKB, Piscataway, NJ): Preequilibrated with P/G buffer DEAE-purified tubulins ( - 5 mg/ml): 0.3 ml in PEM buffer (100 m M PIPES, pH 6.6, 1 mMMgSO4, 1 mMEGTA) plus 0.1 m M G T P s R. B. Vallee,this series,Vol. 134,p. 89.
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[ 17]
MgATP (Sigma type IIS or equivalent): 2.0 ml ofa 0.1 Mstock, made equimolar with MgSO4 and adjusted to pH 7.0 on ice using NaOH Sodium phosphate (0.5 M) pH 7.0 Anion-exchange column: 0.3 ml DEAE-Sepharose CL-6B (Pharmacia-LKB), preequilibrated with P/G buffer containing 5 m M MgATP 1 NAP-10 gel-filtration column (Pharmacia-LKB): Preequilibrated with P/G buffer Purification A diagram of the purification procedure is shown in Fig. 1. Electrophoretic gels of all the preparative stages are shown in Figs. 2-4. A cytosolic extract (CE) is prepared from the white matter using the extraction buffer prescribed above, and microtubules are assembled with taxol and centrifuged, exactly as described in the preceding chapter? The microtubule pellet (P1) is resuspended to 30-40 ml in room-temperature extraction buffer containing 5/tM taxol and centrifuged in two tubes of a Beckman (Palo Alto, CA) 60 Ti rotor at 35,000 rpm for 30 min at room temperature. The microtubule pellets (P2) are resuspended and recentrifuged as in the preceding step. The pellets (P3) are taken up in a solution of 6.75 ml extraction buffer, 0.75 ml H20, and taxol at 5 ~tM. The slightly reduced ionic strength at this stage is to prevent nonspecific dissociation of the high-molecular-weight microtubule-associated proteins (MAPs) upon addition of nucleotides. MgAMP-PNP (0.1 M) is added to a final concentration of 3 m M and the microtubules are resuspended using a 10-ml serological pipette until homogeneous (20- 30 times). MgGTP (0.1 M; pH 7.0) is added to a final concentration of 3 mM, and the microtubules are resuspended as before, incubated for 5 min at room temperature, and centrifuged as in the preceding step. Also at this time, 3/A of taxol (final concentration = 10/tM) is added to a 300-/d aliquot of DEAE-purified tubulin ( - 5 mg/ml). The microtubules are incubated at 37 ° for 5 min and centrifuged in an SS-34 rotor at 18,000 rpm for 30 min at 37 °. The supernatant from the nucleotide extraction step (E', Fig. 3; total volume = 7.5 ml) should contain as its principal components the 100-kDa dynamin polypeptide, tubulin, and a nucleotide-dissociable 37-kDa species. The E' extract is applied at 4 ° in 2.5-ml aliquots to three prepacked PD-10 columns (Pharmacia-LKB), preequilibrated in P/G buffer. P/G buffer (0.5 ml) is applied to each column and the eluate discarded. P/G buffer (2.5 ml) is then applied to each column and an equivalent amount of the E' extract is collected. The desalted E' extract is added to the DEAE-tubulin microtubules, which are resuspended with a Pasteur pipette until homogeneous. The microtubules are centrifuged in an SS-34 rotor at
[ 17]
PURIFICATION AND CHARACTERIZATIONOF DYNAMIN
195
Cytosolic Extract Taxol~
$1
Buffer [ wash
p~,,...,~ $2
Buffer wash
~
p ~ ~ $3
PNP,
GTP
E'
$1' PI' ATP'~I
salt
P2'
~
$2' DEAE-Sepharose
Chromatography DEAE- ~urified lO0-kda }rotein
FIG. 1. Flow diagram of dynamin preparation. The heavier arrows trace the course of the purification of the 100-kDa protein. Preparation of the individual fractions is described in the text. Microtubules are assembled with taxol from calf brain cytosolic extract and pelleted (P1). The microtubules are twice resuspended in buffer and pelleted (P2, P3), and extracted with GTP and AMP-PNP, yielding a supematant (E') specifically enriched in dynamin. The E' extract is incubated with taxol-stabilized microtubules and centrifuged, yielding a pellet (Pl') enriched in the 100-kDa protein and a supematant (Sl') containing the dynamin-activating factor. The Pl' pellet is extracted with ATP and salt and centrifuged, and the supernarant ($2') chromatographed on DEAE-Sepharose, yielding the purified 100-kDa protein. (Reprinted with permission from Ref. 1.)
196
MICROTUBULES, MAPs, AND MICROTUBULEMOTORS
1
2
3
4
5
6
7
8
[ 17]
9
0
FIG. 2. Extraction of dynamin from white matter microtubules by GTP and AMP-PNP. Lane 1: Cytosolicextract. Lanes 2 and 3: Supernatant (S1) and pellet (P1) after taxol-assisted microtubule assemblywithout nucleotide. Dynein (D), kinesin (K), and the 100-kDasubunit of dynamin are prominent components of P1. Lanes 4 and 5: Supernatant ($2) and pellet (P2) after first buffer wash of microtubules. Lanes 6 and 7: Supernatant ($3) and pellet (P3) after second buffer wash of microtubules. Lanes 8 and 9: Supernatant (E') and pellet (P4) after extraction of microtubules with GTP and AMP-PNP. In addition to the 100-kDa protein, a 37-kDa polypeptide is specificallyextracted from the microtubules. Fractions were mixed with an equal volume of gel sample buffer and analyzed on an 8% polyacrylamidegel stained with Coomassie Blue. T, Tubulin. 18,000 rpm for 30 min at 30 °. The supernatant ($1'), which should be quantitatively depleted o f the 100-kDa polypeptide, is removed with a Pasteur pipette and saved for use in subsequent assays o f ATPase activity and force production. The pellet (PI') is resuspended in 1.3 ml P / G buffer plus 0.25 ml 0.1 M MgATP (pH 7.0) with a Pasteur pipette until homogeneous ( - 2 0 times). One milliliter o f a 0.5 M solution o f sodium phosphate (pH 7.0) is slowly added to the resuspended microtubules, which are further resuspended 1 0 - 2 0 times. The microtubules are centrifuged as in the preceding step, and the supernatant ($2'; volume = 2.5 ml) is desalted on a PD-10 column as described above. The $2' supernatant should contain the 100-kDa protein as its major component, in addition to tubulin, variable
[ 17]
PURIFICATION AND CHARACTERIZATION OF DYNAMIN
E' SI'
197
PI' $2' P2'
-100
m-T
-37 FIG. 3. Fractionation of the GTP/AMP-PNP extract (E') by microtubule affinity. Taxolstabilized microtubules composed of DEAE-purified tubulin were added to the E' extract and centrifuged. The pellet (PI') contained all of the 100-kDa protein and some of the 37-kDa species, while the supernatant (SI') contained the dynamin activating factor. The P I ' pellet was extracted with MgATP (10 raM) and sodium phosphate (0.2 M) and centrifuged; the 100-kDa protein and other nontubulin species were found in the supernatant ($2'), leaving only tubulin in the pellet (P2'). Fractions were mixed with an equal volume of gel sample buffer and analyzed on an 8% polyacrylamide gel stained with Coomassie Blue.
amounts of the 37-kDa species, and trace contaminants in the 150- to 300-kDa range. The desalted $2' supernatant is applied to a DEAE-Sepharose CL-6B column (volume--0.3 ml) preequilibrated in P/G buffer. P/G buffer (1.5 ml) containing 2 m M MgATP is applied to the column, which is then eluted with a 12-ml gradient of sodium phosphate (0-0.35 M; pH 7.0) in P/G buffer containing 2 m M MgATP, at a rate of 2.5 ml/hr, in 0.5-ml aliquots. The 100-kDa protein elutes from the column at the position of 125 m M sodium phosphate. One milliliter of the purified protein is applied to a prepacked NAP-10 column (Pharmacia-LKB). The column is washed with 0.35 ml P/G buffer and the DEAE-purified 100-kDa protein (total yield -- 100-200 ag) is collected by eluting with 1.0 ml P/G buffer.
198
$2' 5 7
MICROTUBULES, MAPs, ANI3 MICROTUBULEMOTORS
9
10
11
12
13 14
15
17
[17]
19 30
-100
-T
-37
FI~. 4. Purification of the 100-kDa protein by anion-exchange chromatography. Numbers above the lanes indicate the column fractions. The $2' supernatant was applied to a DEAESepharose column and eluted with a gradient of 0-0.6 M sodium glutamate in P/G buffer (pH 7). The 100-kDa protein eluted in fractions 10-13. A trace amount of tubulin eluted slightly afterward. Lane 5 shows part of the flow through from the column, which contained the 37-kDa polypeptide at low concentration. Fraction 30 contained a 1M NaCI wash. Only those fractions that contained detectable protein are shown. Fractions were prepared as described in the text, except that the column was eluted with sodium glutamate rather than sodium phosphate. Fractions were mixed with an equal volume of gel sample buffer and analyzed on an 8% polyacrylamide gel stained with Coomassie Blue. W h e n stored on ice, the microtubule-activated ATPase activity of dyna m i n generally declines by 1 0 - 2 0 % / d a y , although in a few preparations full activity was seen after 1 week. The extent o f cross-bridge formation appears to decline somewhat m o r e rapidly, possibly due to denaturation a n d / o r aggregation o f the protein, and both bundling and motility assays should be performed within 48 hr after purification. F u n c t i o n a l A s s a y s for D y n a m i n As noted earlier, d y n a m i n forms axially periodic cross-bridges between microtubules that exhibit distinctive structural and enzymatic properties. Cross-bridges between microtubules are formed by the DEAE-purified 100-kDa protein alone, and are assayed by mixing the DEAE-purified 100-kDA protein (final concentration = 5 0 - 1 0 0 / t g / m l ) with taxol-stabi-
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lized microtubules composed of DEAE-purified tubulin (final concentration = 0.2#g/ml). Microtubule bundles exhibiting numerous periodic striations (axial repeat = 13.0 __. 1.1 nm) should be readily detectable by negative stain electron microscopy. Bundling activity should be apparent from the E' stage of the preparation onward, and is dramatically reduced when ATP is added prior to reconstitution of the protein with microtubules. Microtubule bundling is also readily detectable by video-enhanced direct interference contrast (DIC) microscopy. However, the formation of cross-bridges must be confirmed by negative stain electron microscopy. Both microtubule-activated ATPase activity and bundle elongation require, in addition to the purified 100-kDa protein, an additional activating factor present in the S 1' supernatant. ATPase assay mixtures contain 20pl each of the purified 100-kDa protein (final concentration = 1530 pg/ml), the S 1' supernatant, and either microtubules in P/G buffer or P/G buffer alone, plus 15/tl of [y-32p]ATP (final concentration -- 1 raM). Microtubules are prepared from frozen stocks of DEAE-purified tubulin ( - 5 mg/ml)s stored in PEM buffer (100 m M PIPES, pH 6.6, 1.0 m M MgSO4, 1 m M EGTA) plus 0.1 m M GTP. The tubulin is polymerized by addition of taxol (100 pM) and centrifuged at 18,000 rpm in a Sorvall SS-34 rotor at 37 ° for 30 min. The microtubules are resuspended in P/G buffer containing an additional 100 m M sodium glutamate to minimize solubilization oftubulin. The microtubules are recentrifuged as above, and the pellet resuspended in P/G buffer to a final concentration o f - 1.5 mg/ml. Saturation of the microtubule-activated ATPase in P/G buffer has been seen at microtubule concentrations as low as 0.18 mg/ml, and we routinely assay our preparations at concentrations o f - 0.4 mg/ml microtubules. ATPase assays are performed for 20-25 min at 37 °, using the method described in the preceding chapter? When visualized by video-enhanced DIC microscopy, dynamin-induced bundles are seen to fragment and elongate upon perfusion with ATP. Bundles are prepared by mixing 20 ~1 each of the purified 100-kDa protein and the S 1' supernatant with 4/A of microtubules ( - 1.5 mg/ml) in P/G buffer, prepared as described above. Twenty to 25 ~1 of the mixture is placed on a glass slide, and two parallel 4-mm strips of Parafilm "M" (American National Can, Greenwich, CT) are placed on either side of the mixture. A glass coverslip is placed on the Parafilm strips so as to slightly overhang the front edge of the slide. The edges of the perfusion chamber are sealed with Valap B (a 1:1:1 mixture of petrolatum, lanolin, and beeswax), leaving small openings along the front and back edges through which solutions can be introduced and withdrawn. The perfusion chamber is mounted on a Zeiss (Thornwood, NY) IM 35 inverted microscope and 9 B. M. Paschal, H. S. Shpetner, and R. B. Vallee, this volume [16].
200
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visualized by video-enhanced DIC microscopy, as described previously.4 A number of bundles will be seen to be loosely attached to the slide. When perfused with a solution of 4 m M MgATP in the S 1' supernatant, bundles will be seen to partially detach from the slide and fragment along their longitudinal axis, in some cases elongating as microtubules are extruded from the ends of the bundle. Remarks The yield and quality ofdynamin preparations depend on a number of factors. As noted for dynein in the preceding chapter, optimal preparations of dynamin are obtained when the tissue is chilled immediately after slaughtering. Delay at this stage reduces the amount of dynamin in the initial microtubule pellet. Between slaughtering and homogenization, insofar as is possible, the tissue is kept in constant contact with an ice-cold solution of 1 m M sodium phosphate (pH 7), 1 m M MgSO4, and 0.1 m M EGTA. All procedures are done as rapidly as possible, and purification is usually completed 9 - 10 hr after homogenization. The relatively low ionic strength of the P/G extraction buffer is intended to prevent dissociation of the high-molecular-weight MAPs from the crude microtubule pellet (P1-P4). However, microtubules tend to pack somewhat loosely in this buffer, and, therefore, in the early stages of the preparation microtubules are centrifuged at higher g forces than are customary. If it is necessary to perform these steps at lower than the recommended g forces, considerable care should be taken when decanting the supernatants not to disturb the pellets. Dynamin is extremely sensitive to changes in both buffer and ionic strength. Phosphate/glutamate buffer has been chosen because it appears to optimally stabilize the microtubule-activated ATPase of dynamin (H. S. Shpetner and R. B. Vallee, unpublished observations). However, we have also obtained enzymatically active preparations using the HEPES/PIPES extraction buffer described in the preceding chapter,9 through the nucleotide extraction step. Incubation of dynamin at high ionic strength can result in irreversible loss of ATPase activity. ATP appears to mitigate this effect, and it has therefore been included at those stages of the preparation at which high concentrations of salt are required. We have found sodium phosphate, at high concentrations, to be less deleterious than sodium glutamate, and have used the former to elute dynamin from both the P 1' pellet and DEAE-Sepharose in the preparation described here (cf. Ref. 1). Potentially, significant amounts of protein can be lost by nonspecific adsorption to Sephadex G-25 during the desalting steps. We have found it useful to pretreat both the PD-10 and the NAP-5 desalting columns with a
[18]
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201
half-volume of bovine serum albumin (5 mg/ml) in P/G buffer, followed by four column volumes of P/G buffer. Pretreatment of the DEAE-Sepharose column does not seem necessary. We have occasionally noted proteolysis of the 100-kDa protein after the ion exchange step. Preliminary observations suggest that degradation is due to trace levels of a protease, possibly salt activated, in the $2' supernatant. While we do not routinely include protease inhibitors during the latter part of the preparation, such precautions could prove useful. It is recommended that the DEAE-Sepharose column not be reused, since it could contain residual protease. NOTE ADDED IN PROOF. Recently, primary sequence analysis of a cDNA encoding the 100-kDa dynamin polypeptide has indicated the presence of GTP-binding consensus elements20 Enzymological studies suggest that GTP is, in fact, the preferred substrate of dynamin, microtubule-activated hydrolysis requiring only the 100-kDa protein.~' 10R. Obar, C. A. Collins, J. A. Hammarback, H. S. Shpetner, and R. B. Vallee, Nature (London) 347, 256 (1990). " H. S. Shpetner and R. B. Vallee, J. Cell Biol. abstract, in press (1990).
[ 18] P u r i f i c a t i o n a n d C h a r a c t e r i z a t i o n o f Salmo gairdneri Outer Arm Dynein
By ANTHONY G. MOSS, JEAN-LUC GATTI, STEPHENM. KING, and GEORGE B. W I T M A N Introduction Dyneins are multimeric ATPases which make up the inner and outer arms that bridge the outer doublet microtubules of eukaryotic cilia and flagella. The dyneins are responsible for the generation of sliding between outer doublets, which in turn is the basis for the formation and propagation of bending waves in both cilia and flagella? The general biochemical and pharmacological properties of axonemal dynein are inferred from the characteristics of a limited phylogeny? the 22S outer arm dynein from Tetrahymena cilia, 3 sea urchin outer arm dynein from Tripneustes,4 and ~G. B. Witman, in "Structure and Function of Ciliary and Flageilar Surfaces" (R. A. Bloodgood, ed.), p. 1. Plenum, New York, 1990. 2 G. B. Witman, in "The Dynein ATPases" (F. D. Warner, P. Satir, and I. R. Gibbons, eds.), Vol. l, p. 25. Alan R. Liss, New York, 1989. 3 K. A. Johnson, Annu. Rev. Biophys. Biophys. Chem. 14, 161 (1985). 4 I. R. Gibbons and E. Frnnk, J. Biol. Chem. 254, 187 (1979).
METHODS IN ENZYMOLOGY, VOL. 196
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
MICROTUBULES, MAPs, ANDMICROTUBULEMOTORS
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[17] P u r i f i c a t i o n a n d C h a r a c t e r i z a t i o n o f D y n a m i n By H O W A R D S. SHPETNER a n d R I C H A R D B. VALLEE
Dynamin is a microtubule-associated mechanochemical ATPase recently identified in calf brain white matter cytosol) Like kinesin 2,3 and brain cytoplasmic dynein,4-7 it exhibits nucleotide-sensitive binding to microtubules, its ATPase activity can be stimulated severalfold by microtubules, and it has shown evidence of force production against microtubules in in vitro assays. It can be distinguished from kinesin and dynein by its nucleotide specificity, its polypeptide composition, and, most strikingly, by the structures it produces in combination with tubulin: unlike kinesin and cytoplasmic dynein, dynamin forms regular arrays of periodic crossbridges between microtubules, arranging them in bundles that fragment and elongate in the presence of ATP. Thus, dynamin may mediate in vivo sliding between microtubules, in processes as diverse as axonal transport and anaphase B of mitosis. Dynamin consists of a 100-kDa polypeptide and an activating factor which has been only partially purified. The purified 100-kDa species forms cross-bridges between microtubules that appear morphologically identical to those formed in the presence of the activating factor, which, however, is required for both microtubule-activated ATPase activity and bundle elongation. We describe here a method for obtaining both the purified 100-kDa protein and the activating factor in separate fractions from a single preparation of brain microtubules. Principle Taxol-stabilized microtubules prepared without nucleotides from calf brain cytosol are specifically enriched in dynein, kinesin, and dynamin, and all three proteins can be dissociated by ATP. However, unlike dynein, dynamin is also dissociable by GTP, and, unlike kinesin, dynamin is not
H. S. Shpetner and R. B. Vallee, Cell59, 421 (1989). 2 R. D. Vale, T. S. Reese, and M. P. Sheetz, Ce1142, 39 (1985). 3 S. A. Kuznetsov and V. I. Gelfand, Proc. Natl. Acad. Sci. U.S.A. 83, 8530 (1986). 4 B. M. Paschal, H. S. Shpetner, and R. B. Vallee, J. CellBioL 105, 1273 (1987). 5 B. M. Paschal and R. B. Vallee, Nature (London) 330, 181 (1987). 6 R. B. Vallee, J. S. Wall, B. M. Paschal, and H. S. Shpetner, Nature (London) 332, 561 (1988). 7 H. S. Shpetner, B. M. Paschal, and R. B. Vallee, J. CellBioL 107, 1001 (1988).
METHODS IN ENZYMOLOGY, VOL. 196
Copyright © 1991 by Academic Press, Inc. All fights of reproduction in any form reserved.
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induced to bind microtubules by AMP-PNP. Thus, dynamin can be specifically extracted from microtubules using a combination of AMP-PNP and GTP, leaving the other two proteins bound. The dynamin-enriched extract is further fractionated by centrifugation in the presence of exogenous microtubules. For reasons that are not yet understood, the activating factor remains dissociated from microtubules at this stage and is obtained in the supernatant, while the 100-kDa protein is quantitatively found in the microtubule pellet. In contrast to earlier stages of the preparation, extraction of the 100-kDa protein from the microtubules now requires elevated salt concentrations as well as nucleotide, apparently due to binding to microtubules via both nucleotide-insensitive and nucleotide-sensitive sites. The extracted 100-kDa protein is subsequently purified by anion-exchange chromatography. Materials Calf brain white matter, 75-100 g, dissected from five or six fresh brains Extraction buffer: 250 ml of 10 m M sodium phosphate, pH 7.0, 80 m M sodium glutamate, 1 m M MgSO4, 1 m M EGTA, 1 m M dithiothreitol (DTT), 1 m M phenylmethylsulfonyl fluoride, (PMSF), 4 pg/ml leupeptin, 10/2g/ml tosyl arginine methyl ester (TAME), and 1/tg/ml pepstatin Taxol, 350 #1 of a 10 m M stock in dimethyl sulfoxide, obtained as a dry powder from the National Cancer Institute, Drug Synthesis and Chemistry Branch, DTP, DCT, Bethesda, Maryland. Store at - 8 0 ° Sucrose underlayer solution: 40 ml extraction buffer containing 7.5% sucrose MgAMP-PNP: 0.3 ml ofa 0.1 M stock, made equimolar with MgSO4 and adjusted to pH 7.0 on ice using NaOH MgGTP [Sigma (St. Louis, MO) type IIS or equivalent]: 0.3 ml of a 0.1 M stock, made equimolar with MgSO4 and adjusted to pH 7.0 on ice using NaOH P/G buffer: 250 ml of 5 m M sodium phosphate, pH 7.0, 50 m M sodium glutamate, 1 m M MgSO4, 1 m M EGTA, and I m M dithiothreitol (DTT) 3 PD-10 gel-filtration columns (Pharmacia-LKB, Piscataway, NJ): Preequilibrated with P/G buffer DEAE-purified tubulins ( - 5 mg/ml): 0.3 ml in PEM buffer (100 m M PIPES, pH 6.6, 1 mMMgSO4, 1 mMEGTA) plus 0.1 m M G T P s R. B. Vallee,this series,Vol. 134,p. 89.
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MgATP (Sigma type IIS or equivalent): 2.0 ml ofa 0.1 Mstock, made equimolar with MgSO4 and adjusted to pH 7.0 on ice using NaOH Sodium phosphate (0.5 M) pH 7.0 Anion-exchange column: 0.3 ml DEAE-Sepharose CL-6B (Pharmacia-LKB), preequilibrated with P/G buffer containing 5 m M MgATP 1 NAP-10 gel-filtration column (Pharmacia-LKB): Preequilibrated with P/G buffer Purification A diagram of the purification procedure is shown in Fig. 1. Electrophoretic gels of all the preparative stages are shown in Figs. 2-4. A cytosolic extract (CE) is prepared from the white matter using the extraction buffer prescribed above, and microtubules are assembled with taxol and centrifuged, exactly as described in the preceding chapter? The microtubule pellet (P1) is resuspended to 30-40 ml in room-temperature extraction buffer containing 5/tM taxol and centrifuged in two tubes of a Beckman (Palo Alto, CA) 60 Ti rotor at 35,000 rpm for 30 min at room temperature. The microtubule pellets (P2) are resuspended and recentrifuged as in the preceding step. The pellets (P3) are taken up in a solution of 6.75 ml extraction buffer, 0.75 ml H20, and taxol at 5 ~tM. The slightly reduced ionic strength at this stage is to prevent nonspecific dissociation of the high-molecular-weight microtubule-associated proteins (MAPs) upon addition of nucleotides. MgAMP-PNP (0.1 M) is added to a final concentration of 3 m M and the microtubules are resuspended using a 10-ml serological pipette until homogeneous (20- 30 times). MgGTP (0.1 M; pH 7.0) is added to a final concentration of 3 mM, and the microtubules are resuspended as before, incubated for 5 min at room temperature, and centrifuged as in the preceding step. Also at this time, 3/A of taxol (final concentration = 10/tM) is added to a 300-/d aliquot of DEAE-purified tubulin ( - 5 mg/ml). The microtubules are incubated at 37 ° for 5 min and centrifuged in an SS-34 rotor at 18,000 rpm for 30 min at 37 °. The supernatant from the nucleotide extraction step (E', Fig. 3; total volume = 7.5 ml) should contain as its principal components the 100-kDa dynamin polypeptide, tubulin, and a nucleotide-dissociable 37-kDa species. The E' extract is applied at 4 ° in 2.5-ml aliquots to three prepacked PD-10 columns (Pharmacia-LKB), preequilibrated in P/G buffer. P/G buffer (0.5 ml) is applied to each column and the eluate discarded. P/G buffer (2.5 ml) is then applied to each column and an equivalent amount of the E' extract is collected. The desalted E' extract is added to the DEAE-tubulin microtubules, which are resuspended with a Pasteur pipette until homogeneous. The microtubules are centrifuged in an SS-34 rotor at
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PURIFICATION AND CHARACTERIZATIONOF DYNAMIN
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Cytosolic Extract Taxol~
$1
Buffer [ wash
p~,,...,~ $2
Buffer wash
~
p ~ ~ $3
PNP,
GTP
E'
$1' PI' ATP'~I
salt
P2'
~
$2' DEAE-Sepharose
Chromatography DEAE- ~urified lO0-kda }rotein
FIG. 1. Flow diagram of dynamin preparation. The heavier arrows trace the course of the purification of the 100-kDa protein. Preparation of the individual fractions is described in the text. Microtubules are assembled with taxol from calf brain cytosolic extract and pelleted (P1). The microtubules are twice resuspended in buffer and pelleted (P2, P3), and extracted with GTP and AMP-PNP, yielding a supematant (E') specifically enriched in dynamin. The E' extract is incubated with taxol-stabilized microtubules and centrifuged, yielding a pellet (Pl') enriched in the 100-kDa protein and a supematant (Sl') containing the dynamin-activating factor. The Pl' pellet is extracted with ATP and salt and centrifuged, and the supernarant ($2') chromatographed on DEAE-Sepharose, yielding the purified 100-kDa protein. (Reprinted with permission from Ref. 1.)
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MICROTUBULES, MAPs, AND MICROTUBULEMOTORS
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2
3
4
5
6
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8
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9
0
FIG. 2. Extraction of dynamin from white matter microtubules by GTP and AMP-PNP. Lane 1: Cytosolicextract. Lanes 2 and 3: Supernatant (S1) and pellet (P1) after taxol-assisted microtubule assemblywithout nucleotide. Dynein (D), kinesin (K), and the 100-kDasubunit of dynamin are prominent components of P1. Lanes 4 and 5: Supernatant ($2) and pellet (P2) after first buffer wash of microtubules. Lanes 6 and 7: Supernatant ($3) and pellet (P3) after second buffer wash of microtubules. Lanes 8 and 9: Supernatant (E') and pellet (P4) after extraction of microtubules with GTP and AMP-PNP. In addition to the 100-kDa protein, a 37-kDa polypeptide is specificallyextracted from the microtubules. Fractions were mixed with an equal volume of gel sample buffer and analyzed on an 8% polyacrylamidegel stained with Coomassie Blue. T, Tubulin. 18,000 rpm for 30 min at 30 °. The supernatant ($1'), which should be quantitatively depleted o f the 100-kDa polypeptide, is removed with a Pasteur pipette and saved for use in subsequent assays o f ATPase activity and force production. The pellet (PI') is resuspended in 1.3 ml P / G buffer plus 0.25 ml 0.1 M MgATP (pH 7.0) with a Pasteur pipette until homogeneous ( - 2 0 times). One milliliter o f a 0.5 M solution o f sodium phosphate (pH 7.0) is slowly added to the resuspended microtubules, which are further resuspended 1 0 - 2 0 times. The microtubules are centrifuged as in the preceding step, and the supernatant ($2'; volume = 2.5 ml) is desalted on a PD-10 column as described above. The $2' supernatant should contain the 100-kDa protein as its major component, in addition to tubulin, variable
[ 17]
PURIFICATION AND CHARACTERIZATION OF DYNAMIN
E' SI'
197
PI' $2' P2'
-100
m-T
-37 FIG. 3. Fractionation of the GTP/AMP-PNP extract (E') by microtubule affinity. Taxolstabilized microtubules composed of DEAE-purified tubulin were added to the E' extract and centrifuged. The pellet (PI') contained all of the 100-kDa protein and some of the 37-kDa species, while the supernatant (SI') contained the dynamin activating factor. The P I ' pellet was extracted with MgATP (10 raM) and sodium phosphate (0.2 M) and centrifuged; the 100-kDa protein and other nontubulin species were found in the supernatant ($2'), leaving only tubulin in the pellet (P2'). Fractions were mixed with an equal volume of gel sample buffer and analyzed on an 8% polyacrylamide gel stained with Coomassie Blue.
amounts of the 37-kDa species, and trace contaminants in the 150- to 300-kDa range. The desalted $2' supernatant is applied to a DEAE-Sepharose CL-6B column (volume--0.3 ml) preequilibrated in P/G buffer. P/G buffer (1.5 ml) containing 2 m M MgATP is applied to the column, which is then eluted with a 12-ml gradient of sodium phosphate (0-0.35 M; pH 7.0) in P/G buffer containing 2 m M MgATP, at a rate of 2.5 ml/hr, in 0.5-ml aliquots. The 100-kDa protein elutes from the column at the position of 125 m M sodium phosphate. One milliliter of the purified protein is applied to a prepacked NAP-10 column (Pharmacia-LKB). The column is washed with 0.35 ml P/G buffer and the DEAE-purified 100-kDa protein (total yield -- 100-200 ag) is collected by eluting with 1.0 ml P/G buffer.
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$2' 5 7
MICROTUBULES, MAPs, ANI3 MICROTUBULEMOTORS
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[17]
19 30
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-T
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FI~. 4. Purification of the 100-kDa protein by anion-exchange chromatography. Numbers above the lanes indicate the column fractions. The $2' supernatant was applied to a DEAESepharose column and eluted with a gradient of 0-0.6 M sodium glutamate in P/G buffer (pH 7). The 100-kDa protein eluted in fractions 10-13. A trace amount of tubulin eluted slightly afterward. Lane 5 shows part of the flow through from the column, which contained the 37-kDa polypeptide at low concentration. Fraction 30 contained a 1M NaCI wash. Only those fractions that contained detectable protein are shown. Fractions were prepared as described in the text, except that the column was eluted with sodium glutamate rather than sodium phosphate. Fractions were mixed with an equal volume of gel sample buffer and analyzed on an 8% polyacrylamide gel stained with Coomassie Blue. W h e n stored on ice, the microtubule-activated ATPase activity of dyna m i n generally declines by 1 0 - 2 0 % / d a y , although in a few preparations full activity was seen after 1 week. The extent o f cross-bridge formation appears to decline somewhat m o r e rapidly, possibly due to denaturation a n d / o r aggregation o f the protein, and both bundling and motility assays should be performed within 48 hr after purification. F u n c t i o n a l A s s a y s for D y n a m i n As noted earlier, d y n a m i n forms axially periodic cross-bridges between microtubules that exhibit distinctive structural and enzymatic properties. Cross-bridges between microtubules are formed by the DEAE-purified 100-kDa protein alone, and are assayed by mixing the DEAE-purified 100-kDA protein (final concentration = 5 0 - 1 0 0 / t g / m l ) with taxol-stabi-
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199
lized microtubules composed of DEAE-purified tubulin (final concentration = 0.2#g/ml). Microtubule bundles exhibiting numerous periodic striations (axial repeat = 13.0 __. 1.1 nm) should be readily detectable by negative stain electron microscopy. Bundling activity should be apparent from the E' stage of the preparation onward, and is dramatically reduced when ATP is added prior to reconstitution of the protein with microtubules. Microtubule bundling is also readily detectable by video-enhanced direct interference contrast (DIC) microscopy. However, the formation of cross-bridges must be confirmed by negative stain electron microscopy. Both microtubule-activated ATPase activity and bundle elongation require, in addition to the purified 100-kDa protein, an additional activating factor present in the S 1' supernatant. ATPase assay mixtures contain 20pl each of the purified 100-kDa protein (final concentration = 1530 pg/ml), the S 1' supernatant, and either microtubules in P/G buffer or P/G buffer alone, plus 15/tl of [y-32p]ATP (final concentration -- 1 raM). Microtubules are prepared from frozen stocks of DEAE-purified tubulin ( - 5 mg/ml)s stored in PEM buffer (100 m M PIPES, pH 6.6, 1.0 m M MgSO4, 1 m M EGTA) plus 0.1 m M GTP. The tubulin is polymerized by addition of taxol (100 pM) and centrifuged at 18,000 rpm in a Sorvall SS-34 rotor at 37 ° for 30 min. The microtubules are resuspended in P/G buffer containing an additional 100 m M sodium glutamate to minimize solubilization oftubulin. The microtubules are recentrifuged as above, and the pellet resuspended in P/G buffer to a final concentration o f - 1.5 mg/ml. Saturation of the microtubule-activated ATPase in P/G buffer has been seen at microtubule concentrations as low as 0.18 mg/ml, and we routinely assay our preparations at concentrations o f - 0.4 mg/ml microtubules. ATPase assays are performed for 20-25 min at 37 °, using the method described in the preceding chapter? When visualized by video-enhanced DIC microscopy, dynamin-induced bundles are seen to fragment and elongate upon perfusion with ATP. Bundles are prepared by mixing 20 ~1 each of the purified 100-kDa protein and the S 1' supernatant with 4/A of microtubules ( - 1.5 mg/ml) in P/G buffer, prepared as described above. Twenty to 25 ~1 of the mixture is placed on a glass slide, and two parallel 4-mm strips of Parafilm "M" (American National Can, Greenwich, CT) are placed on either side of the mixture. A glass coverslip is placed on the Parafilm strips so as to slightly overhang the front edge of the slide. The edges of the perfusion chamber are sealed with Valap B (a 1:1:1 mixture of petrolatum, lanolin, and beeswax), leaving small openings along the front and back edges through which solutions can be introduced and withdrawn. The perfusion chamber is mounted on a Zeiss (Thornwood, NY) IM 35 inverted microscope and 9 B. M. Paschal, H. S. Shpetner, and R. B. Vallee, this volume [16].
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visualized by video-enhanced DIC microscopy, as described previously.4 A number of bundles will be seen to be loosely attached to the slide. When perfused with a solution of 4 m M MgATP in the S 1' supernatant, bundles will be seen to partially detach from the slide and fragment along their longitudinal axis, in some cases elongating as microtubules are extruded from the ends of the bundle. Remarks The yield and quality ofdynamin preparations depend on a number of factors. As noted for dynein in the preceding chapter, optimal preparations of dynamin are obtained when the tissue is chilled immediately after slaughtering. Delay at this stage reduces the amount of dynamin in the initial microtubule pellet. Between slaughtering and homogenization, insofar as is possible, the tissue is kept in constant contact with an ice-cold solution of 1 m M sodium phosphate (pH 7), 1 m M MgSO4, and 0.1 m M EGTA. All procedures are done as rapidly as possible, and purification is usually completed 9 - 10 hr after homogenization. The relatively low ionic strength of the P/G extraction buffer is intended to prevent dissociation of the high-molecular-weight MAPs from the crude microtubule pellet (P1-P4). However, microtubules tend to pack somewhat loosely in this buffer, and, therefore, in the early stages of the preparation microtubules are centrifuged at higher g forces than are customary. If it is necessary to perform these steps at lower than the recommended g forces, considerable care should be taken when decanting the supernatants not to disturb the pellets. Dynamin is extremely sensitive to changes in both buffer and ionic strength. Phosphate/glutamate buffer has been chosen because it appears to optimally stabilize the microtubule-activated ATPase of dynamin (H. S. Shpetner and R. B. Vallee, unpublished observations). However, we have also obtained enzymatically active preparations using the HEPES/PIPES extraction buffer described in the preceding chapter,9 through the nucleotide extraction step. Incubation of dynamin at high ionic strength can result in irreversible loss of ATPase activity. ATP appears to mitigate this effect, and it has therefore been included at those stages of the preparation at which high concentrations of salt are required. We have found sodium phosphate, at high concentrations, to be less deleterious than sodium glutamate, and have used the former to elute dynamin from both the P 1' pellet and DEAE-Sepharose in the preparation described here (cf. Ref. 1). Potentially, significant amounts of protein can be lost by nonspecific adsorption to Sephadex G-25 during the desalting steps. We have found it useful to pretreat both the PD-10 and the NAP-5 desalting columns with a
[18]
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201
half-volume of bovine serum albumin (5 mg/ml) in P/G buffer, followed by four column volumes of P/G buffer. Pretreatment of the DEAE-Sepharose column does not seem necessary. We have occasionally noted proteolysis of the 100-kDa protein after the ion exchange step. Preliminary observations suggest that degradation is due to trace levels of a protease, possibly salt activated, in the $2' supernatant. While we do not routinely include protease inhibitors during the latter part of the preparation, such precautions could prove useful. It is recommended that the DEAE-Sepharose column not be reused, since it could contain residual protease. NOTE ADDED IN PROOF. Recently, primary sequence analysis of a cDNA encoding the 100-kDa dynamin polypeptide has indicated the presence of GTP-binding consensus elements20 Enzymological studies suggest that GTP is, in fact, the preferred substrate of dynamin, microtubule-activated hydrolysis requiring only the 100-kDa protein.~' 10R. Obar, C. A. Collins, J. A. Hammarback, H. S. Shpetner, and R. B. Vallee, Nature (London) 347, 256 (1990). " H. S. Shpetner and R. B. Vallee, J. Cell Biol. abstract, in press (1990).
[ 18] P u r i f i c a t i o n a n d C h a r a c t e r i z a t i o n o f Salmo gairdneri Outer Arm Dynein
By ANTHONY G. MOSS, JEAN-LUC GATTI, STEPHENM. KING, and GEORGE B. W I T M A N Introduction Dyneins are multimeric ATPases which make up the inner and outer arms that bridge the outer doublet microtubules of eukaryotic cilia and flagella. The dyneins are responsible for the generation of sliding between outer doublets, which in turn is the basis for the formation and propagation of bending waves in both cilia and flagella? The general biochemical and pharmacological properties of axonemal dynein are inferred from the characteristics of a limited phylogeny? the 22S outer arm dynein from Tetrahymena cilia, 3 sea urchin outer arm dynein from Tripneustes,4 and ~G. B. Witman, in "Structure and Function of Ciliary and Flageilar Surfaces" (R. A. Bloodgood, ed.), p. 1. Plenum, New York, 1990. 2 G. B. Witman, in "The Dynein ATPases" (F. D. Warner, P. Satir, and I. R. Gibbons, eds.), Vol. l, p. 25. Alan R. Liss, New York, 1989. 3 K. A. Johnson, Annu. Rev. Biophys. Biophys. Chem. 14, 161 (1985). 4 I. R. Gibbons and E. Frnnk, J. Biol. Chem. 254, 187 (1979).
METHODS IN ENZYMOLOGY, VOL. 196
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
