View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by PubMed Central

Cytoplasmic -like ATPase Cross-links in an ATP-sensitive Manner

PETER J. HOLLENBECK, FRANK SUPRYNOWICZ, and W. ZACHEUS CANDE Department of Botany, University of California at Berkeley, Berkeley, California 94720. Dr. Suprynowicz's present address is Hopkins Marine Station, Pacific Grove, California 93950.

ABSTRACT We have prepared dynein-like ATPase from the eggs of the sea urchin StrongyIo- centrotus purpuratus using differential centrifugation and column chromatography. This ATPase preparation is inhibited by vanadate and erythro-9-(3-[2-hydroxynonyl]) adenine (EHNA) at concentrations similar to those that inhibit reactivated flagellar beating and spindle elongation in lysed cell models. Using affinity and ATP-induced release, we can purify this ATPase activity to a composition on SDS PAGE of four peptides ranging in molec~ular weight from 180,000-300,000. When viewed in darkfield optics, this affinity-purified ATPase caused extensive parallel bundling of microtubule-associated protein-free microtubules. These bun- dles were dispersed by 1 mM ATP but not by ATP~,S or AMP-5'-adenylimidodiphosphate. The reformation of microtubule bundles after dispersal by ATP required ATP hydrolysis; bundles did not reform in the presence of 10 #M vanadate. Negative stain electron microscopy of these bundled microtubules revealed that they are arranged in parallel networks with extensive close lateral association.

Microtubule systems are often organized into orderly arrays projecting from most of the microtubules in the midzone (27, by lateral interactions between tubules. In many cases, these 28). lateral interactions are mediated by cross-bridges that can be A second line of evidence for dynein involvement in ana- seen in the electron microscope or purified and characterized phase comes from physiological studies using permeabilized biochemically. Such systems include the ciliary or flagellar PtKI cell spindle models. These models, which permit the axoneme (12, 42, 48, 52, 53, 56), the protozoan axostyle (2, study of anaphase under controlled conditions, have shown 3, 17), the pharyngeal basket of certain ciliates (51), the that separation of the spindle poles requires ATP and is heliozoan axopod (50), and the mitotic apparatus (20, 27, 28). inhibited by vanadate and erythro-9-(3-[2-hydroxynonyl])ad- While some cross-bridges may merely be static links others enine (EHNA) ~ (6, 7) which are both potent inhibitors of are likely to generate force for microtubule-based movements. dynein ATPase activity (23, 34). In addition, it has been Of these systems, only one, the axoneme, has had its cross- reported that isolated sea urchin embryo mitotic spindles bridging elements characterized both morphologically and move chromosomes and that this movement is blocked by biochemically. They consist of both static bridges such as the vanadate and antibodies to flagellar dynein (40). nexin links and the mechanochemical protein, dynein. Ifa dynein ATPase is a component of the mitotic apparatus, While the mitotic apparatus lacks the order of the axoneme, then it should be identifiable in preparations of mitotic ap- it has been suggested that a dynein-like cross-bridge plays a paratuses or cytoplasm. A MgATPase activity was originally role in its formation and function, and particularly in aria- reported in isolated spindles of the sea urchin egg by Mazia phase chromosome movement. Two kinds of studies have et al. (26). Weisenberg and Taylor (55) suggested that this provided evidence for this view. First, morphological studies ATPase was a precursor to embryonic ciliary dynein bound of diatoms reveal a highly ordered spindle midzone in which nonspecifically to spindle preparations, but subsequent work microtubules from opposite half-spindles interdigitate in a ~Abbreviations used in this paper. AMPPNP, 5'-adenylimidodi- regular array. The distance between nearest neighbor micro- phosphate; EHNA, erythro-9-(3-[2-hydroxynonyl]);MAP, microtu- tubules in this midzone is appropriate for dynein cross-bridg- bule-associated protein; PME, 100 mM PIPES, I mM MgSO4, I mM ing, and in some preparations arms or bridges can be seen EGTA.

THE JOURNAL OF CELL BIOLOGY . VOLUME 99 OCTOBER 1984 1251-1258 © The Rockefeller University Press . 0021-9525/84[10/1251108 $1.00 1 25 1 by Pratt (36, 37, 39) made this seem unlikely, as did the claim Material was assayed for ATPase activity by incubation at 25"C in 100 mM of Pallini et al. (32) of having isolated similar dynein-like Tris HC1, pH 8.0, 100 mM KC1, 5 mM MgSO4 or CaC12, 1 mM EGTA, and 2 mM ATP for 18 min. (When assaying in the presence of EHNA, ATP was from cells that do not give rise to cilia (32). adjusted to 10% of the EHNA concentration.) Samples were withdrawn from A dynein-like ATPase has now been prepared and charac- the incubation mixture every 3 rain from 6 through 18 min and assayed for terized from the mitotic apparatuses and cytoplasm of sea inorganic phosphate after the method of Fiske and Subbarow (9). Vanadate urchin embryos by several investigators (21, 33, 38). It has was prepared for inhibition studies by making a l-mM stock solution of sodium the high sedimentation coefficient, high molecular weight gel orthovanadate (Na3VO~) in distilled H20 and placing it briefly in a boiling water bath. This stock was prepared fresh each day. bands, and Mg +÷ and Ca ++ activation expected for dynein, The Km for ATP was determined by assaying the sizing column peak fraction and it shows inhibition by vanadate and EHNA. But these at ATP concentrations ranging from 0. I to 4 mM. The data was analyzed using are not the only properties of dynein critical to a role in Eadie-Hofstee plots and gave straight lines with correlation coefficients of mitosis and other microtubule-based movements. To be a <-0.996. Samples were assayed for protein by the method of Bradford (4), using BSA as a standard. likely candidate for involvement in these processes, this AT- Microtubule Pelleting Binding Assay: Microtubules were pre- Pase must bind to and cross-link microtubules. Recent work pared by polymerizing DEAE-purified with taxol. Column-enriched by Hisanaga and Sakai (22) addressed this question using an ATPase was combined with microtubules and incubated in the presence of no ATPase preparation purified to a single high molecular weight Mg+÷, 5 mM MgSO4, or 5 mM MgSO4 + 0.5 mM ATP for 30 min. Incubation polypeptide. As judged by gels, this polypeptide pelleted with mixtures were then layered over a pad of 3 vol of 20% sucrose in HE buffer containing the same cation and/or nucleotide concentration as the incubation microtubules in an ATP-sensitive manner. Binding of the mixture and 0.2% protease inhibitor stock. They were centrifuged in an SWS0. l ATPase activity to microtubules was not measured directly, rotor (Beckman Instruments, Inc., Pain Alto, CA) at 125,000 g for 60 min at but activity disappeared from the supernatant when micro- 4"C and pellets were assayed for ATPase activity. As controls, either microtu- tubules were pelleted. bules or ATPase alone was layered over pads and centrifuged. In this study, we prepared cytoplasmic dynein-like ATPase Darkfield Microscopy: Microtubule-associated protein (MAP)-free from sea urchin eggs by size filtration chromatography fol- taxol-stabilized microtubules were diluted to 0.2 mg/ml with PME or micro- tubule affinity-purified ATPase. In the latter case, this resulted in a ratio of 0.4 lowed by microtubule affinity and ATP-induced release. We mg nontubulin peptides/1 mg microtubules. These mixtures were incubated used dark-field optics and negative stain electron microscopy under various ionic and nucleotide conditions and viewed after 5 and 30 rain. to demonstrate the microtubule cross-linking properties of Glass slides were thoroughly rinsed with Millipore-filtered (Millipore Corp., this preparation and to correlate its cross-linking activity with Bedford, MA) distilled water and dried by air jet. Samples of 1-2/~I were placed on these slides and covered with air dusted coverslips. Slides were viewed using its ATPase activity. a Zeiss dark-field condensor and a Zeiss 100x plan objective with an iris. Photographs of typical fields were taken with Kodak Tri-X film at ASA 1600. MATERIALS AND METHODS Electron Microscopy: Samples of microtubules or microtubules plus Cytoplasmic Dynein-like ATPase Preparation: sea urchinsof ATPase were removed from the dark-field assay mixtures and applied to the species Strongylocentrotuspurpuratus were spawned by filling the coelom Formvar-coated grids. After 10 s, the samples were drawn off and the grids with 0.5 M KCI. Eggs were collected in filtered natural sea water, dejeUied by were prepared by floating them upside down on a series of solutions after the passage through 190-~m nitex mesh, and washed twice with filtered natural sea method of Langford (25): 5 s each on two changes of PME, 30-60 s on PME water. After one washing with gluconate buffer (10 mM HEPES, 0.3 M plus 0.1% glutaraldehyde, 5 s each on two changes of PME, 5 s each on two potassium gluconate, 0.33 M glycine, 10 mM NaCI, 5 mM MgSO4, 1 mM changes of distilled HzO, 5 s each on four changes of bacitracin (40 ug/ml in EGTA, pH 7.4), eggs were resuspended in 3 vol of the same buffer at 4"C distilled H20), excess liquid was drawn off, then 5 s each on three changes of containing 0.5% protease inhibitor stock (0.2 mg/ml leupeptin, 2 mg/ml aqueous 1% uranyl acetate. Excess stain was drawn offand grids were allowed soybean trypsin inhibitor, 0.2 mg/ml pepstatin A, 2 mg/ml N-a-benzoyl-L- to air dry. Grids were viewed with a Zeiss EM 109. arginine methyl ester, 2 mg/ml p-tosyl-L-arginine methyl ester, 2 mg/ml L-I Gel Electrophoresis: Samples were denatured and run on 4-8% tosylamide-2-phenylethylchloromethyl ketone) and disrupted by 15 strokes polyacrylamide gradient gels made after the method of Laemmli (24). These with a glass Dounce hand homogenizer. The cell homogenate was centrifuged were stained with Coomassie Blue and stored in 10% acetic acid. Gels were at 40,000 g for 30 min at 4"C and the supernatant was carefully removed and photographed with Kodak Pan-X film. further centrifuged at 125,000 g for 75 min at 4"C. The resulting high speed Molecular weight determinations were made using standard proteins of supernatant was concentrated by ammonium sulfate precipitation at 40% molecular weights 45,000, 66,200, 92,500, 116,250, and 200,000. In this gel saturation. Precipitated protein was resuspended in HE buffer (10 mM HEPES, system, the molecular weights and Rf values of these standards have a nearly 0.1 mM EDTA, pH 7.4) containing 0.5 mM a-mercaptoethanol and 0.2% perfect linear relationship, with a correlation coefficient of <-0.999. protease inhibitor stock and dialyzed three times at 4"C against 150 vol of the Stained gels were scanned with a densitometer (E-C Apparatus Corp., St. same buffer, three h per change. The dialysate was clarified by centrifugation Petersburg, FL) with a 570-nm filter. The percent of total protein in each band at 125,000 g for 60 rain at 4"C. The resulting supernatant was loaded onto a was assumed to be proportional to the percent of the total area under its curve. 2.6 x 80 cm Bin-Gel AlSm sizing column equilibrated in HE buffer plus 0.6 M KCI and 0.2% protease inhibitor stock. This column was run at 2--4"C at a linear flow rate of 6 cm/h and 7.5-ml fractions were collected. MgATPase R ES U LTS activity eluted in a peak with Ktv of 0.3-0.5 (Fig. 1). The peak fractions were pooled and dialyzed twice at 4"C against 100 vol of 50 mM PIPES, 1 mM Purification and Enzymatic Properties of MgSO4, 1 mM EGTA (PME), and 0.2% protease inhibitor stock, pH 6.94. It Cytoplasmic Dynein-like A TPase was then clarified by spinning at 125,000 g for 60 min. This pooled, dialyzed, clarified peak was then used to dilute DEAE-purified tubulin to a tubulin We prepared dynein-like ATPase from sea urchin eggs using concentration of 1.6-2.0 mg/ml. The tubulin was polymerized into microtu- a protocol that exploits its high molecular weight and micro- bules by addition of taxol equimolar with tubulin dimers and the microtubules tubule-binding properties. Differential centrifugation, am- were pelleted at 125,000 g for 45 rain at 4*(2. The resulting microtubule pellet monium sulfate precipitation, and size filtration (Figs. 1 and was resuspended to a concentration of 10 mg/ml in PME plus 2 mM MgATP and pelleted again at 125,000 g for 45 min. This process was repeated with the 2) provided a rapid 40-fold enrichment of MgATPase activity second pellet and the supernatants were combined. These supernatants con- with a 15 % recovery (Table I). When assayed under various tained MgATPase activity. conditions (Table II), this column-enriched ATPase showed Tubulin Preparation: Twice-cycled microtubule protein was pre- cationic requirements and oligomycin and ouabain insensitiv- pared by the method of Shelanski et al. (45) and stored at -80"C. Tubulin was ity similar to dynein-like ATPase prepared in different ways purified from this preparation by ion exchange chromatography on DEAE- Sephadex after the method of Murphy and Borisy (31) and dialyzed against by other investigators (21, 33, 37). In addition, it was inhibited 200 vol of PME plus 0.1 mM GTP for 60 min. by EHNA in a concentration range very similar to that found A TPase Activity Assays and Protein Determinations: for dynein-like ATPase from urchin eggs by Penningroth and

1252 THE JOURNAL OF CELL BIOLOGY • VOLUME 99, 1984 I ! ! 07 I 06 / -',,,/\ o 3 ..-~. O5 ; A\ 04 02 0 / co E & cJ 0,3 / / <

O2 OI --

OI 0

, / ...... \ 0 m 14 18 22 26 30 34 38 42 46 50 0 cl

Fraction number < FIGURE 1 Elution profile of cytoplasmic dynein MgATPase on a sizing column. The clarified dialyzed ammonium sulfate fraction was subjected to gel filtration on a 2.6 x 80-cm column of Bio-Gel A15m equilibrated and eluted with 0.6 M KCI, 10 mM HEPES/KOH, 0.1 mM EDTA, 0.5 mM ~x-mercaptoethanol, and 0.2% protease inhibitor stock, pH 7.4. It was run at 2-4°C at a linear flow rate of 6 cm/h and 7.5-ml fractions were collected and assayed for absorb- ance at 280 nm and MgATPase activity as described in Materials and Methods. The activity eluted in a peak with Kavof 0.3-0.5.

Cheung (33) and from brain by Pallini et al. (32). However, our ATPase preparation was more sensitive to vanadate in- hibition than those reported by other workers, with our half- inhibitory concentration (1 vM) as much as 10-fold lower than reported values. This may be due to the relative hetero- geneity of their preparations, or to the presence in their stocks of vanadate not in the +5 oxidation state. Assays with other nucleotides (Table II) showed that the Mg-stimulated activity was relatively ATP specific. The Km of the column-enriched FIGURE 2 SDS PAGE of fractions from the purification procedure ATPase for ATP was 66 vM under our assay conditions. using a 4-8% gradient gel. Lanes A-D contain 20 p.g each of (A) The MgATPase was further purified from the column- high speed cytoplasmic supernatant; (B) clarified dialyzed 40% enriched fraction by a microtubule affinity and ATP-induced ammonium sulfate fraction; (C) Bio-Gel A15m column peak fraction; release procedure. When taxol-polymerized brain microtu- (D) fraction released from microtubule binding by ATP. Lane E bules were incubated with this column-enriched ATPase and contains molecular weight standards: (200,000);/~-galacto- then pelleted, 75% of the MgATPase activity pelleted with sidase (116,300); phosphorylase b (92,500); BSA (66,200); and oval- bumin (45,000). them. When these pellets were extracted with 2 mM MgATP and repelleted, they released 25-35% of the activity to the supernatant. When examined on 4-8% gradient SDS poly- acrylamide gels, this ATP-released ATPase preparation re- vealed only four major peptides along with tubulin extracted TABLE I from the microtubules (Fig. 2, lane D). This extraction of Purification of Dynein-like A TPase tubulin was due to the slight lability of taxol-polymerized microtubules in the cold. The nontubulin peptides had M, Total Enrich- Total pro- MgATPase Specific ment for values of 295,000, 246,000, 243,000, and 180,000. The pres- Fraction tein activity activity each step ence of soluble tubulin in the supernatant necessitated the use mg of densitometry of gel lanes to determine the percentage of Homogenate 1,500 7.5 0.005 -- nontubulin peptides there. Densitometry indicated that the $125 300 5.2 0.017 3.4x four nontubulin peptides composed 3.6% of the protein in cdAMS 107 4.3 0.040 2.3x the ATP-released supernatant while tubulin composed the Column peak 5.5 1.1 0.200 5.0x rest. This allowed the calculation of an estimated specific Mt affinity 0.35 .30 0.850 4.5x ATPase activity for these peptides of 0.850 #moles Pi/min 5125, high speed supernatant of cytoplasmic homogenate; cdAMS, clarified per mg protein. dialyzed 40% ammonium sulfate fraction of $125; column peak, MgATPase peak from Bio-Gel A15m sizing column; Mt affinity, fraction released from binding to microtubules by ATP. All fractions were assayed for ATPase in 100 Pelleting A TPase Assay mM Tris/HCl, pH 8.0, 100 mM KCI, 5 mM MgSO4, and 2 mM ATP. Homog- enate was assayed in the presence of 10/~g/ml of oligomycin. Total MgATPase In the presence of 5 mM Mg +÷, 32% of the ATPase activity activity is expressed in micromoles inorganic phosphate per minute and incubated with microtubules pelleted with them through the specific activity is expressed in micromoles inorganic phosphate per (minute x milligram protein). Total protein and specific activity of the microtubule sucrose pad. In the absence of Mg ÷÷, this was reduced to affinity fraction are adjusted for nontubulin peptides only as determined by 9.5%, while in the presence of 5 mM Mg ÷÷ and 0.5 mM ATP, densitometric measurements of this fraction on SDS gels.

HOLLENBECK ET AL. Cytoplasmic Dynein-like ATPase Cross-linking ] 25 3 14% of the activity pelleted. In the absence of microtubules, (Fig. 4A) resulted in their complete dissociation within 5 min no detectable activity pelleted. into scattered individual microtubules (Fig. 4B). After an interval sufficient to allow complete hydrolysis of ATP by the Characterization of A TPase/Microtubule ATPase (30 min), microtubules again appeared in bundles Interactions by Darkfield Optics (Fig. 4 C). However, when 1 mM ATP + 10 #M vanadate were added, the bundles (Fig. 4D) dispersed but failed to form Microtubules polymerized from purified tubulin and di- again over a period of 60 min (Fig. 4, E and F). The addition luted to 0.2 mg/ml with PME were visible in dark-field of I mM ATP'vS or 1 mM AMP-5'-adenylimidodiphosphate microscopy as bright individual filaments distributed evenly (PNP) to microtubules bundled by the ATPase (Fig. 4, G and across the field (Fig. 3A). When these microtubules were J) resulted in very little dispersal of the bundles (Fig. 4, H, I, diluted to 0.2 mg/ml with affinity-purified ATPase in the K, and L). presence of 5 mM Mg÷÷, the dark-field image was dramati- In the absence of Mg+÷, incubation of microtubules with cally different (Fig. 3 B). Essentially all of the microtubules in ATPase did not result in any visible bundling of microtubules. the field were seen to be gathered into bright dense bundles, Likewise, the addition of Mg÷÷ to microtubules without AT- with individual microtubules apparent at the edges. Pase gave no bundling. When the dark-field assays described The addition of 1 mM MgATP to microtubules so bundled above were performed with sizing column-enriched ATPase rather than affinity-purified material, the results were essen- TABLE 11 tially the same (data not shown). Nucleoside Triphosphatase Activities Negative Stain Electron Microscopy Control ATPase ac- Assay conditions tivity To observe the fine structure of microtubules bundled by 0,6 the ATPase, we prepared samples of the dark-field incubation mixtures for electron microscopy. Microtubules alone showed 2 mM ATP, 5 mM MgSO4 100 fairly typical negative stain morphology, appearing long and +10 #g/ml oligomycin 103 straight with excellent resolution of protofilaments and indi- +0.1 mM ouabain 102 vidual dimers (Fig. 5, A and B). The microtubules were free and unassociated and were distributed evenly across the grid. +0.5/,,M vanadate 76 But microtubules incubated with dynein-like ATPase in the +1.0 #M vanadate 54 presence of Mg ÷÷ were gathered into parallel bundles of +2.0 ~M vanadate 0 various size. Within these bundles, microtubules parallel to one another and were frequently separated by a gap of 5- +1 mM EHNA 46 20 nm. Also, globular structures were seen along some micro- +2 mM EHNA 0 tubules, but no stretches of periodic decoration were observed 2 mM GTP 6 (Fig. 5, C and D). 2 mM ITP 6 DISCUSSION 2 mM CTP 15 2 mM ATP, 5 mM CaCI2 83 In the search for a dynein-like ATPase that might be involved All assayswere performed using sizing column peak fractions as describedin in anaphase chromosome movement, we must ask ourselves Materials and Methods. two questions: first, is there a candidate ATPase in the cyto-

FIGURE 3 Darkfield microscopy shows the bundling of microtubules produced by incubation with cytoplasmic dynein-like ATPase. MAP-free microtubules diluted to 0.2 mgJml with PME are shown in A. In B, the microtubules have been diluted to the same concentration with cytoplasmic dynein-like ATPase prepared by microtubule affinity and ATP-induced release. Nearly all of the microtubules are gathered into large bright bundles, x 1,000.

1254 THE JOURNAL OF CELL BIOLOGY • VOLUME 99, 1984 FiGUrE 4 Darkfield microscopy reveals the effects of four nucleotide treatments on microtubule bundling induced by cytoplasmic dynein-like ATPase. Bundled rnicrotubules (,4) are dispersed at 5 min after ATP addition (B); after 30 rain they are again bundled together (C). Bundles (D) are also dispersed by ATP plus vanadate after 5 rain (E), but fail to reassociate after 60 rain (F). Bundtes (G) are not appreciably dispersed by ATP'yS after 5 rain (H) or 30 rain (I). Bundles (J) are also not noticeably dispersed by addition of AMP-PNP after 5 rain (K) or 30 min (L). x 800.

plasm? and second, does it interact with microtubules in the molecular weight and gel bands in the 300,000-mol-wt range. manner expected for dynein? In this study, we have addressed In in vitro assays, the sizing column-enriched ATPase fraction these questions using several different techniques. showed a ratio of Mg- to Ca-stimulation (1.2) and an ATP We have developed an enrichment scheme for dynein-like specificity similar to axonemal . It was unlike mito- ATPase from the sea urchin egg that exploits its size and chondrial or Na/K ATPases in showing no inhibition by microtubule-binding properties. We recovered an ATPase oligomycin or ouabain. In addition, we found the cytoplasmic fraction that was dynein-like by the criteria outlined by Gib- ATPase to be dynein-like and not myosin-like by virtue of bons et al. (14)--it contained a MgATPase with a high complete lack of K/EDTA-stimulated ATPase activity. Fur-

HOLLENBECK ET AL. Cytoplasmic Dynein-like A TPase Cross-linking 1255 FIGURE 5 Negative stain electron microscopy of dark-field incubation mixtures. Microtubules alone: typical views are shown in A and B. Microtubles plus cytoplasmic dynein-like ATPase are shown in C and D. x 20,000 (A and C); x 160,000 (B and D).

thermore, this MgATPase activity was inhibited by EHNA in absence of Mg+÷, indicated that the ATPase had a Mg- the same range (1-2 mM) as axonemal dyneins, and, like dependent interaction with microtubules. The pelleting assay those dyneins, was highly sensitive to vanadate (1-2 #M). also showed that the binding was ATP sensitive; inclusion of Although the inhibitor data and Eadie-Hofstee plots suggested 0.5 mM ATP in the assay reduced the pelleting ATPase that there was only one ATPase in this preparation, we cannot activity by >50%. This was similar to the findings of Mitchell completely rule out the possibility that it contained more than and Warner (29) for dynein-B subfiber interactions in Tetra- one. hymena ciliary axonemes. Furthermore, it suggested that the Another characteristic we expect ofdynein, and an essential ATPase could be further purified from the enriched column one if it is to play a role in force generation, is the ability to peak by microtubule affinity and ATP-induced release. bind to and cross-link microtubules. We assessed this capacity This proved to be possible. When the peak MgATPase in three ways, the first of which was the pelleting binding fractions from the sizing column were dialyzed, clarified, and assay. Here we found that sizing column-enriched MgATPase incubated with taxol-polymerized microtubules, and the mi- activity pelleted with MAP-free microtubules through a su- crotubules were pelleted, 75 % of the MgATPase activity pel- crose pad in the presence of Mg++. This, considered along leted with them. When these pellets were extracted with 2 with the 75% reduction in pelleting ATPase activity in the mM MgATP, they released 25-35% of the activity to the

1256 THE JOUrNaL OF CELL BIOLOGY • VOLUME 99, 1984 supernatant. Viewed on 4-8 % gradient SDS polyacrylamide since vanadate apparently does not inhibit initial hydrolysis, gels, this supernatant showed four major peptides in addition but rather binds to an product complex, preventing to tubulin extracted from the microtubules (Fig. 1D). As product release (13, 15, 23, 41, 46, 47). In fact, Penningroth described in Results, these polypeptides had Mr values of et al. (35) have obtained evidence that ATP hydrolysis is 295,000, 246,000, 243,000, and 180,000. This was consistent necessary for relaxation of rigor axonemes. with the complex polypeptide composition of axonemal dy- Recent evidence suggests that the binding of MAP2 to neins such as the 21S urchin flagellar dynein 1, which has microtubules is inhibited by phosphorylation (5). Our result several peptides that fall into three size classes (53). Although with ATP-rS makes it unlikely that phosphorylation is respon- the Mr of 295,000 is less than reported values for the high sible for the release seen here. Although ATP'rS is not hydro- molecular weight bands of axonemal dyneins, in our gel lyzed by ATPases it serves as a good substrate for kinases, system axonemal peptides from S. purpuratus sperm tails and producing irreversible thiophosphorylation (16). Tetrahymena cilia also migrate in the 295,000-300,000-mol- To further compare cytoplasmic dynein-like ATPase with wt region (data not shown). The estimated specific activity of axonemal dynein, we prepared samples from the dark-field the nontubulin peptides of the affinity-purified ATPase prep- assay for negative stain electron microscopy. In samples of aration of 0.850 #mol Pi/min per protein was similar to the microtubules bundled by the ATPase, we observed extensive values obtained for dynein-like ATPase prepared by Hisanaga close association of microtubules (Fig. 5). However, we ob- and Sakai from a different species (21, 22). We will refer to served no stretches of obvious periodic bridges, and there may our microtubule affinity/ATP-released ATPase fraction as have been two reasons for this. First, because of material cytoplasmic dynein-like ATPase. limitations, the ratio of ATPase to tubulin was only around By its design, our purification procedure provided us with one-third of that used to saturate microtubules with axonemal a protein that was dynein-like, having the appropriate ATPase dynein decoration (18, 19, 43). This may have precluded activity, size, and ATP-sensitive microtubule binding char- seeing periodic decoration. A second possibility is that sea acteristics. Using this microtubule affinity-purified ATPase, urchin cytoplasmic ATPase is difficult to preserve for electron we wanted to assess its capacity to cross-link microtubules microscopy. This would be consistent with our experience and to relate its cross-linking properties to its ATPase activity. with sea urchin flagellar dynein, which gave poor decoration To this end we employed dark-field optics, which allowed us even when ATPase assays indicated that the microtubules to observe a very large population of microtubules under were saturated with dynein (unpublished data). By scaling up various conditions while retaining the ability to resolve indi- our purification procedure and trying different methods of vidual microtubules. sample preparation, we hope to overcome these difficulties. When the ATPase was added to MAP-free taxol-polymer- To conclude, we return to the two questions posed at the ized brain microtubules, virtually all of the microtubules were beginning of this discussion. First, concerning whether there gathered into bundles (Fig. 3). This suggested that the ATPase is an ATPase in the cytoplasm that resembles dynein, we have did in fact have the capacity to cross-link microtubules. To shown that there is an ATPase in sea urchin eggs that shares relate this cross-linking capacity to the ATPase activity, we a number of characteristics with axonemal dynein. Its size, added ATP, ATP + vanadate, ATP.,/S, or AMP-PNP to these complex polypeptide composition, cationic requirements, and bundled microtubules. Addition of ATP or ATP plus vana- sensitivity to inhibitors are strikingly similar to the axonemal date caused complete dispersal of the bundles to individual ATPase. As for the question of its microtubule interactions, microtubules, indicating detachment of the cross-linking ele- we have shown that the cytoplasmic ATPase binds to micro- ment (Fig. 4, B and E). This was consistent with results tubules in a Mg-dependent, ATP-sensitive manner. Further- obtained with axonemal dynein in intact axonemes, decorated more, as judged by the dark-field assay, it mediates an inter- outer doublets and brain microtubule, and trypsin-digested action between microtubules that is related to ATP binding axonemes, where ATP addition results in the detachment of and hydrolysis in precisely the manner demonstrated for rigor bound dynein arms from microtubules and this detach- axonemal dynein. ment is not prevented by vanadate (41, 43, 49). If, after We suggest that cytoplasmic dynein-like ATPase is a good dispersing the bundles with ATP, we allowed sufficient time candidate for the force-generating element in microtubule- for the ATPase to hydrolyze the nucleotide, the microtubules based intracellular movements. It not only possesses the nec- formed bundles again (Fig. 4C). However, in samples dis- essary microtubule-binding characteristics, but also shows persed by ATP plus sufficient vanadate to inhibit ATPase inhibition by EHNA and vanadate which closely matches activity, microtubules failed to form bundles again (Fig. 4F). their inhibition of anaphase B in permeabilized cell models This too is consistent with results obtained for axonemal (6, 7), of pronucleus migration in fertilized sea urchin eggs dynein decoration of doublet and brain microtubules (43, 49) (44), and of particle movements in axons (11), erythrophores where rebinding after ATP-induced release was blocked by (1), and permeabilized fibroblasts (10) and melanophores (8). vanadate. In the axonemal system, ATP-induced release and To relate the observed properties of cytoplasmic dynein-like the coupling of hydrolysis and product release to dynein ATPase to its potential function in vivo, we would like to reattachment are presumed to be part of the mechanochemi- know if it can generate force between microtubules. To this cal cross-bridge cycle; we do not know what role they may end, we are using dark-field microscopy to observe whether play for cytoplasmic dynein-like ATPase. microtubules slide over each other during the formation and When we added the nonhydrolyzable ATP analogues ATP-induced release of microtubules bundled by the ATPase. ATP-rS or AMP-PNP to bundled microtubules, we observed little if any release of bundling. This suggested that either We wish to thank Drs. David Asai, Kent McDonald, Stephen Pen- these analogues bind to the ATPase very poorly or that ningroth, and Richard Vallee for helpful discussions. We are also hydrolysis is necessary for release. It should be noted that our grateful to Susan Eiliger for technical assistance. ATP-vanadate result does not rule out the latter possibility, This work was supported by National Institute of Health grant

HOLLENBECK ETAL. Cytoplasmic Dyneinqike A TPase Cross-linking 1257 GM23238 to W. Z. Cande. P. J. Hollenbeck was supported by a mitotic apparatus. Pro¢. Natl. Acad. Sci. USA. 47:788-790. 27. McDonald, K. L., M. K. Edwards, and J. R. Mclntosh. 1979. Cross-sectional structure National Science Foundation predoctoral fellowship. of the central mitotic spindle of Diatoma vulgare: evidence for specific interactions between antiparallel microtubnles. J. Cell Biol. 83:443-461. 28. McDonald, K. L., J. R. Mclntosh, and D. H. Tippit. 1977. On the mechanism of Received for publication 19 September 1983, and in revised form 12 anaphase spindle elongation in Diatoma vulgare. J. Cell Biol. 74:377-388. June 1984. 29. Mitchell, D. R., and F. D. Warner. 1980. Interactions of dynein arms with B subfibers of Tetrahymena cilia: quantitation of the effects of magnesium and adenosine triphos- phate. J. Cell Biol. 87:84-97. 30. Murofushi, H., Y. Minami, G. Matsumoto, and H. Sakai. 1983. Bundling of microtu- bules in vitro by a high molecular weight protein prepared from the squid axon. J. REFERENCES Biochem. (Tokyo). 93:639-650. 31. Murphy, D. B., and G. G. Borisy. 1975. Association of high-molecular-weight proteins 1. Beckerle, M. C., and K. R. Porter. 1982. lnhibitors ofdynein block intracellular transport with microtubules and their role in microtubule assembly in vitro. Proc. NatL Acad Sci. in erythropholes. Nature (Loud). 295:701-703. USA. 72:2696-2700. 2. Bloodgood, R. A. 1975. Biochemical analysis of axostyle motility. Cytobios. 14:101- 32. Pallini, V., C. Mencarelli, L. Bracci, M. Contorui, P. Ruggiero, A. Tiezzi, and R. 120. Manetti. 1983. Cytoplasmic nucleoside-triphosphatases similar to axonemal dynein can 3. Bloadgood, R. A., and K. R. Miller. 1974. Freeze fracture of microtubules and bridges occur widely in different cell types. J. Submicrosc. Cytol. 15:229-235. in motile axostyles. J. Cell Biol. 62:660--671. 33. Penningroth, S. M., and A. Chenng. 1983. Erythro-9-[3-(2-hydroxynonyl)] adenine 4. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram (EHNA): a new link between cytoplasmic and axonemal dyneins. J. Submicrosc. Cytol. quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 15:223-227. 72:248-254. 34. Penningroth, S. M., A. Cheung, P. Bouchard, C. Gagnon, and C. W. Bardin. 1982. 5. Bums, R. G., K. Islam, and R. Chapman. 1983. The graduated phosphorylation of Dynein ATPase is inhibited selectively in vitro by erythro-9-[3-(2-hydruxynonyl)] ade- MAP2 regulates the MAP2:microtubule interaction. Z Cell Biol. 97(5, pt. 2):200a. nine. Biochem. Biophys. Res. Commun. 104:234-240. (Abstr.) 35. Penningroth, S. M., A. Cheung, K. Olehnik, and R. Kosiosky. 1982. Mechanoehemical 6. Cantle, W. Z. 1982. Inhibition of spindle elongation in permcabilized cells by erythro- coupling in the relaxation of rigor wave sea urchin sperm flagella. J. Cell Biol. 92:733- 9[3-(2-hydroxynonyl)] adenine. Nature (Loud.). 295:700-701. 41. 7. Cande, W. Z. 1982. Nucleotide requirements for anaphase chromosome movements in 36. Pratt, M. M. 1979. Comparison of flagellar and egg dynein. J. Cell Biol. 83(2, Pt. 2): permeabilized mitotic cells: anaphase B but not anaphase A requirm ATP. Cell. 28:15- 350a. (Abstr.) 22. 37. Pratt, M. M. 1980. The identification of a dynein ATPase in unfertilized sea urchin 8. Clark, T. G., and J. L. Rosenhaum. 1982. Pigment particle translocation in detergent eggs. Dev. Biol. 74:364-378. permeabilized melanophores of Fundulns heteroclitus. Proc. Natl. Acad. ScL USA. 38. Pratt, M. M., Otter, T., and E. D. Salmon. 1980. Dynein-like Mg+ ATPas¢ in mitotic 79:4655-4659. spindles isolated from sea urchin embryos (Strongylocentrotus droebachiensis). ,L Cell 9. Fiske, C. H., and Y. Subbarow. 1925. The colorimctric determination of phosphorus. Biol. 86:738-745. J. Biol. Chem. 66:375-400. 39. Pratt, M. M., and R. Stepbeus. 1978. Dynein synthesis in sea urchin embryos. Z Cell 10. Forman, D. S. 1982. Vanadate inhibits saltatory organelle movement in a permeabilized BioL 79(2, Pt. 2):300a. (Abstr.) cell model. Exp. Cell Res. 141:139-147. 40. Sakai, H. 1978. The isolated mitotic apparatus and chromosome motion. Int. Bey. 11. Forman, D. S., K. J. Brown, and D. R. Livengood. 1983. Fast axonal transport in CytoL 55:23-48. permeabilized lobster giant axons is inhibited by vanadate. J. Neurosci. 3:1279-1288. 41. Sale, W. S., and I. R. Gibbons. 1979. Study of the mechanism of vanadate inhibition of 12. Gibbons, B. H., and I. R. Gibbons. 1976. Functional recombination ofdynein 1 with the dynein cross-budge cycle in sea urchin sperm flagella. Z Cell Biol. 82:291-298. demembranated sea urchin sperm partially extracted with KCl. Biochem. Biophys. Res. 42. Satir, P. 1979. Basis of flagellar motility in spermatozoa: current status. In The Sper- Commun. 73:1-6. matozoon: Maturation, Motility, and Surface Properties. D. W. Fawcett and J. M. 13. Gibbons, I. R., M. P. Cosson, L A. Evans, B. H. Gibbons, B. Houck, K. H. Martinson, Bedford, editors. Urban and Scharzenberg, Baltimore. 8L-90. W. S. Sale, and W. Y. Tang. 1978. Potent inhibition ofdynein adenosinetriphosphatase 43. Satir, P., J. Wais-Steider, S. Lebduska, A. Nasr, and J. Avolio. 1981. The mechanochem- and of the motility of cilia and sperm flagella by vanadate. Proc. Natl. Acad Sci. USA. ical cycle of the dynein arm. CellMotility. 1:303-327. 75:2220-2224. 44. Schatten, G., R. Balczon, C. Clioc, and H. Schatten. 1982. EHNA, a dynein inhibitor, 14. Gibbons, I. R., E. Fronk, B. H. Gibbons, and K. Ogawa. 1976. Multiple forms of dynein blocks the nuclear movements during sea urchin fertilization. J. Cell Biol. 95(2, Pt. in sea urchin sperm flagella. CoM Spring Harbor Conf. Cell Prolif 3(Book A):915-932. 2): 166 a. (Abstr.) 15. Goodno, C. C. 1979. Inhibition of myosin ATPase by vanadate ion. Proc. NatL Acad 45. Shelanski, M. L., F. Gaskin, and R. Cantor. 1973. Microtnbule assembly in the absence Sei. USA 76:2620-2624. of added nucleotides. Proc. Natl. Acad. Sci. USA. 70:765-768. 16. Gratecos, D., and E. H. Fischer. 1974. Adenosine 5'-0(3-thiotriphosphate)in the control 46. Shimizu, T. 1981. Steady-state kinetic study of vanadate-induced inhibition of dynein of phosphorylase activity. Biochem. Biophys. Res. Commun. 58:960-7. adenosine triphosphataso activity from Tetrahymena. Biochemistry. 20:4347-4354. 17. Grimstone, A. V., and L. R. Cleveland. 1965. The fine structure and function of the 47. Shimizu, T., and K. A. Johnson. 1983. Presteady state kinetic analysis of vanadate- contractile axostyles of certain flagellates. J. Cell Biol. 24:387-400. induced inhibition of the dynein ATPase. Z Biol. Chem. 258:13833-13840. 18. Haimo, L. T., and B. R. Telzer. 1981. Dynein-microtubule interactions: ATP-sensitive 48. Summers, K. E., and I. R. Gibbons. 1973. Effects of trypsin digestion of flagellar dynein binding and the structural polarity of mitotic microtubules. Cold Spring Harbor structures and their relationship to motility. J. Cell BioL 58:618-629. Syrup. Quant. Biol. 46:207-2t7. 49. Takahashi, M., and Y. Tonomura. 1978. Binding of the 30S dynein with the B-tubule 19. Haimo, L. T., B. R. Telzer, and J. L. Rosenhaum. 1979. Dynein binds to and cross- of the outer doublet of axonemes from Tetrahymena pyriformis and adenosine triphos- bridges cytoplasmic microtubules. Proc. Natl. Acad. Sei. USA. 76:5759-5763. phate-indnced dissociation of the complex. J. Biochem. (Tokyo). 84:1339-1355. 20. Hepler, P. K., J. R. Mclntosh, and S. Cleveland. 1970. Intermicrotubule bridges in 50. Tilncy, L. G. 1971. How microtubule patterns are generated. The relative importance mitotic spindle apparatus. J. Cell Biol. 45:438--444. of nucleation and bridging of microtubules in the formation of the axoneme of Radi- 21. Hisanaga, S. I., and H. Sakai. 1980. Cytoplasmic dynein of the sea urchin egg: a partial ophrys. £ Cell Biol. 51:837-854. purification and characterization. Dev. Growth Differ. 22:373-384. 51. Tucker, J. B. 1968. Fine structure and function of the cytopharyngeal basket in the 22. Hisanaga, S. 1., and H. Sakai. 1983. Cytoplasmic dynein of the sea urchin egg. 11. ciliate Nassula. £ Cell Sci. 3:493-514. Purification, characterization, and interaction with mierotubules and Ca-calmodulin. J. 52. Warner, F. D. 1978. Cation-induced attachment of ciliary dynein cross-bridges. J. Cell Biochem. (Tokyo). 93:87-98. Biol. 77:RI9-R26. 23. Kohayashi, T., T. Martensen, J. Nath, and M. Flavin. 1978. Inhibition ofdynein ATPase 53. Warner, F. D. 1980. Dynein: the mechanochemical coupling adenosine triphosphatase by vanadate, and its possible use as a probe for the role of dynein in cytoplasmic of microtubule-hased sliding filament mechanisms. Int. Rev. Cytol. 66:1-43. motility. Biochem. Biophys. Res. Commun. 81:1313-1318. 54. Warner, F. D., and D. R. Mitchell. 1981. Polarity of dynein-microtubule interactions 24. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head in vitro: cross-bridging between parallel and antiparallel mierotubules. J. Cell Biol. of bacteriophage 1"4. Nature (Loud). 227-680-685. 89:35-44. 25. Langford, G. M. 1984. Length and appearance of projections on neuronal microtubules 55. Weisenberg, R. C., and E. W. Taylor. 1968. Studies on ATPase activity of sea urchin in vitro after negative staining: evidence against a crosslinking function for MAPs. £ eggs and the isolated mitotic apparatus. Exp. CellRes. 53:372-384. Ultrastruct. Res. 85:1-10. 56. Zanetti, N. C., D. R. Mitchell, and F. D. Warner. 1979. Effects of divalent cations on 26. Mazia, D., R. R. Chaffee, and R. M. Iverson. 1961. Adenosine triphosphatasc in the dynein cross-bridging and ciliary dynein sliding J. Cell Biol. 80:573-588.

1258 THE JOURNAL OF CELL BIOLOGY • VOLUME 99, 1984