Cell Motility and the Cytoskeleton 21:281-292 (1992)

Two Distinct lsoforms of Sea Urchin Egg Dynein Paula M. Grissom, Mary E. Porter, and J. Richard Mclntosh Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder Extracts of unfertilized sea urchin eggs contain at least two isofoms of cytoplasmic dynein. One exhibits a weak affinity for microtubules and is primarily soluble. The other isoform, HMr-3, binds to microtubules in an ATP-sensitive manner, but is immunologically distinct from the soluble egg dynein (Porter et al.: Journal of Biological Chemistry 263:6759-6771, 1988). We have now further distinguished these egg dynein isoforms based on differences in NTPase activity. HMr-3 copurifies with NTPase activity, but it hydrolyzes CTP at 10 times the rate of ATP. The soluble egg dynein is similar to flagellar dynein in its nucleotide specificity; its MgCTPase activity is ca. 60% of its MgATPase activity. Non-ionic detergents and salt activate the MgATPase activities of both enzymes relative to their MgCTPase activities, but this effect is more pronounced for the soluble egg dynein than for HMr-3. Sucrose gradient-purified HMr-3 promotes an ATP-sensitive microtubule bundling, as seen with darkfield optics. We have also isolated a 20 S microtubule translocating activity by sucrose gradient fractionation of egg extracts, followed by microtubule affinity and ATP release. This 20 S fraction, which contains the HMr-3 isoform, induces a microtubule gliding activity that is distinct from kinesin. Our observations suggest that soluble dynein resembles axonemal dynein, but that HMr-3 is related to the dynein-like enzymes isolated from a variety of cell types and may represent the cytoplasmic dynein of sea urchin eggs. Key words: ATPase, CTPase, minus-end-directedmicrotubule motility, cytoplasmic dynein

INTRODUCTION

Dynein was identified almost 30 years ago as a high molecular weight, microtubule-binding ATPase that drives axonemal motility in cilia and flagella [Gibbons, 19651. More recent work has established that dynein-like enzymes are also found in cells that do not form axonemes [Paschal et al., 19871 and in organisms that do not make motile cilia or flagella at any stage in their life cycle [Lye et al., 1987; Koonce and McIntosh, 19901. These “cytoplasmic dyneins” are sometimes associated with membrane bounded vesicles [Schroer et al., 19891 and are thought to contribute to the movements of such vesicles toward the minus ends of microtubules, as in retrograde axonal transport and saltatory movements toward the centrosome [reviewed in McIntosh and Porter, 1989; Vallee and Shpetner, 19901. The recent localization of cytoplasmic dynein epitopes at the kinetochores 0 1992 Wiley-Liss, Inc.

and centrosomes of mitotic spindles [Pfarr et al., 1990; Steuer et al., 19901, combined with the minus-end-directed movements of chromosomes during their attachment to microtubules both in vivo [Rieder and Alexander, 19901 and in vitro [Hyman and Mitchison, 19911, has led to speculation that dynein may also play a role in mitosis. The biochemistry of cytoplasmic dynein and a comparison of its activities with those of axonemal dy-

Received April 8, 1991; accepted October 18, 1991. Address reprint requests to Paula M. Grissom, Dept. of Molecular, Cellular, and Developmental Biology, Univ. of Colorado, Boulder, CO 80309-0347. Mary E. Porter is now at Department of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis, MN 55455.

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nein is therefore an issue of considerable biological interest. Several properties of dynein from mammalian brain have been determined and compared with those of axonemal dynein [Shpetner et al., 19881. The ATPase activities of both kinds of dynein are inhibited by vanadate ions, and the heavy chain polypeptides of both proteins are cleaved by UV light in the presence of vanadate and ADP, although the sizes of the cleavage products differ slightly [reviewed in McIntosh and Porter, 1989; Vallee and Shpetner, 19901. The MgATPase of flagellar dynein shows a higher K, for activation by microtubules [Omoto and Johnson, 19861 than does brain cytoplasmic dynein [Shpetner et al., 19881. Further, flagellar dynein is more specific for ATP than brain dynein, which hydrolyzes CTP efficiently, although with a high K, and without producing an effective mechanochemical cycle. These similarities and differences suggest that the dyneins form an enzyme family whose members are distinct. Some of the best studied cytoplasmic dyneins are the enzymes isolated from the eggs of marine invertebrates [reviewed in Pratt, 19891. A soluble dynein-like ATPase activity was first identified in sea urchin eggs by Weisenberg and Taylor [ 19681, and relevant proteins have subsequently been purified and analyzed in numerous ways [Pratt, 1980; Hisanaga and Sakai, 1980, 1983; Hollenbeck et al., 1984; Pratt et al., 1984; Scholey et al., 1984; Asai and Wilson, 1985; Penningroth et al., 1985; Foltz and Asai, 19881. Motile activities thought to depend on dynein have also been seen in these cells. The existence of a minus-end-directed translocating activity is demonstrated by the observation of bidirectional bead and organelle movements along microtubules in homogenates of sea urchin eggs [Pryer et al., 19861 and in intact embryos [Wadsworth, 19871. However, sea urchin embryos develop cilia at the late blastula stage of development [Stephens, 19721, so the egg dyneins described biochemically could be either enzymes with a cytoplasmic function or precursors for cilium formation. In a previous study, we found biochemical and immunological evidence for two distinct cytoplasmic dynein isoforms in unfertilized sea urchin eggs [Porter et al., 19881. One isoform binds weakly to microtubules and is primarily soluble. It contains 2-3 heavy chain polypeptides that are immunologically related to flagellar and embryonic ciliary dyneins and may be a ciliary precursor [Asai and Wilson, 1985; Porter et al., 19881. The second isoform, previously called HMr-3 [Scholey et al., 19841, contains a different heavy chain polypeptide and binds to microtubules in an ATP-sensitive fashion. HMr-3 exhibits vanadate-sensitive UV cleavage, but cross-reacts very weakly with antibodies to sea urchin flagellar dynein [Porter et al., 19881. Here we extend the

description of the differences between these egg dynein isoforms. We have analyzed the NTPase activity of various egg samples and find that the isoforms differ in their relative MgCTPase/MgATPase activities. We have also identified a microtubule translocating activity in 20 S fractions of ooplasm that contain the HMr-3 isoform. The microtubule motility induced by this 20 S factor is clearly distinct from kinesin. Our observations suggest that sea urchin eggs contain an axonemal dynein isoform that interacts poorly with microtubules and is set aside in a comparatively inactive form. The other dynein isoform shows many characteristics of cytoplasmic dynein and is probably the motor enzyme important for minus-enddirected motility. Thus the “egg dynein” that has been studied most thoroughly is probably a ciliary precursor and not a true cytoplasmic dynein. MATERIALS AND METHODS Materials

Sea urchins, Lytechinus pictus and Strongylocentrotus purpurutus, were obtained from Marinus Inc. (Long Beach, CA) and handled as described previously [Scholey et al., 1984; Porter et al., 1987, 19881. Taxol was a gift from Dr. M. Suffness at the National Cancer Institute. Ultrapure sucrose was purchased from Fisher Scientific (Fair Lawn, NJ), GTP from Boehringer Mannheim Biochemicals (Indianapolis, IN), [y3*P] nucleotides from ICN (Irvine, CA), Biogel A5M and antibody conjugates from Bio-Rad, Inc. (Richmond, CA), cellulose phosphate (P1 1) from Whatman Inc. (Hillsboro, OR), and all other chemicals from Sigma Chemical Co. (St. Louis, MO). Purification of Sea Urchin Cytoplasmic and Flagellar Dyneins Isolation of the two cytoplasmic dynein isoforms was carried out as described previously [Porter et al., 19881 with only minor modifications. The microtubule associated isoform, HMr-3, was purified by 10 mM MgATP extraction of taxol-assembled MTs (microtubules) followed by sucrose density centrifugation. The soluble dynein isoform was purified from MT-depleted extracts by gel filtration and phosphocellulose chromatography [Porter et al., 19881. Sea urchin flagellar axonemes were isolated by osmotic shock of live sperm and differential centrifugation [Bell et al., 19821. The native 21 S flagellar dynein was prepared by high salt extraction and purified by sucrose density gradient centrifugation in the egg extraction buffer (PMEG) [Porter et al., 19881. The ATPase activity of various dynein fractions during purification was routinely measured by the method of Waxman and Goldberg [ 19821 as modified by Pratt et al. [ 19841.

Two Isoforms of Egg Dynein

NTPase Activity

283

MgATP (1 mM), MgGTP (1 mM), or vanadate and ATP (see Table IV).

Sea urchin fractions were assayed for CTPase and ATPase activities at room temperature in PMEG buffer (unless specified in the text) by the method of Cohn et al. [1987] modified as follows. [ Y - ~ ~ P I M ~ Cand T P [y32P]MgATP(specific activity: 104-106 cprdnmol) were added to samples to give a final nucleotide concentration of 200 pM. Aliquots (100 pl) were removed at four time points ranging from 0 to 45 min. The reaction was stopped by the addition of 40 p1 of 10% Sarkosyl (Sigma Chemical Co.). Phosphate reagent (4 vol 5% w/v ammonium molybdate, 5 N H,S04:1 vol 0.1 M silicotungstic acid) was added and the phosphomolybdate complex was extracted with a xylene/isobutanol (65:35) mixture. Following centrifugation in a microfuge, 0.5 ml of the organic phase was removed, mixed with 3.5 ml of scintillation cocktail (Biofluor), and released 32Piwas measured in a Beckman scintillation counter.

MAP-free tubulin was prepared from beef brain by temperature-dependentcycles of assembly and disassembly followed by phosphocellulose chromatography [Williams and Detrich, 19791. Protein concentrations were determined by the method of Bradford [1976] and the polypeptide composition of the different fractions was assayed by SDSPAGE (SDS-polyacrylamide gel electrophoresis) as described previously [Porter and Johnson, 19831. Polypeptides were transferred to nitrocellulose [Towbin et al., 19791, probed with SUK 4,the monoclonal antibody to sea urchin kinesin [Ingold et al., 19881 or a blot affinity-purified antibody to flagellar dynein heavy chain, and visualized using the immunoperoxidase reaction.

Microtubule Bundling

RESULTS

Sucrose gradient fractions of ATP MAPs (ATP extracted microtubule-associated proteins) were assayed for microtubule bundling activity by incubation with phosphocellulose-purified microtubules (40 pg/ml) and 10 pM taxol in PMEG buffer for 1 hour at room temperature. Five microliter aliquots were removed and viewed using darkfield optics. Bundling activity was analyzed both in the absence or presence of 1 mM MgATP.

Other Biochemical Procedures

Cosedimentation of HMr-3 With NTPase Activity

The cytoplasmic dynein isoform, HMr-3, prepared by ATP extraction of microtubule pellets and sucrose density centrifugation is associated with a peak of CTPase and ATPase activities that sediments at -20 S (Fig. 1). The specific NTPase activities of the 20 S peak varied from preparation to preparation (see Table I), but the CTPase activity was always greater than the correMotility Assays sponding ATPase activity. SDS-PAGE analysis of the 20 Sucrose gradient fractions were assayed for their S peak fraction indicated that the major polypeptide that ability to induce the translocation of microtubules over a cosedimented with the CTPase activity was HMr-3 (Fig. glass surface using video-enhanced DIC microscopy as 1A). Several lower molecular weight polypeptides could described previously [Vale et al., 1985; Porter et al., also be detected in this fraction following silver staining, 19871. Samples (15 p1) were adsorbed onto coverslips but these polypeptides were present in much smaller for 20 min at room temperature in a humidified chamber. amounts and were not a consistent feature of the pattern. In some experiments, 0.2 mg/ml of protein A-purified SUK 4 [Ingold et al., 19881, a motility-blocking, mono- Comparison of Cytoplasmic and clonal antibody to sea urchin kinesin that was generously Flagellar NTPases We have analyzed the ATPase and CTPase activiprovided by Dr. J. Scholey, was added to the fraction and incubated for an additional 20 min. Taxol-stabilized ties of several fractions containing sea urchin dynein: 1) MTs to 20 pg/ml and MgATP to 2 mM were added to the the 20 S peak from a sucrose gradient fractionation of sample. The coverslip was then inverted onto a glass whole egg extract (i.e., the supernatant prepared by high speed centrifugation of homogenized eggs; this fraction slide, sealed, and assayed for MT motility. Alternatively, a perfusion chamber was used to ex- contains both the microtubule-associatedHMr-3 isoform amine the effects of GTP and vanadate on the motility of and the soluble dynein isoform); 2) the 20 S peak from a different fractions [Vale and Toyoshima, 19881. Three sucrose gradient fractionation of ATP MAPs (this mate10 pl aliquots of the same fraction were applied to a rial is prepared by MgATP extraction of taxol-stabilized perfusion chamber and then allowed to adsorb to the MTs isolated from egg extract; it contains primarily the glass for 2 min following each perfusion. The unad- HMr-3 isoform); 3) the soluble isoform of egg dynein sorbed protein was removed by perfusion with a BSA (prepared by phosphocellulose chromatography of mi(bovine serum albumin) buffer solution followed by a crotubule-depleted egg extracts); and 4) 21 S flagellar solution containing taxol MTs (20 pg/ml) and either dynein (Fig. 2). The average MgCTPase/MgATPase ra-

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A.

D SS 5 6 7 8

9 10 11 12 13 14 15 16 17 MW

CB

Ag

DY" 200

116 97

66

45

B.

9 4.0

r 2.2 s

11s

I

I

0-0

CTP

0--0

ATP x 10

Fraction Number Fig. 1. Cosedimentation of CTPase and ATPase activities with HMr3. 0.6 ml of ATP extracted MAPS (SS, starting sample) from S. purpurutus MTs was loaded onto a 5 2 0 % sucrose gradient and centrifuged. The left panel (A) is a Coomassie Blue stained 6% polyacrylamide gel of these sucrose gradient fractions. Lane D contains 21 S flagellar dynein. Lane MW contains molecular weight markers and

21 S flagellar dynein. The right panel shows the 20 S peak fraction (#9) stained with Coomassie Blue (CB) or silver nitrate (Ag). The ) ATPase (0---0) activities sedimented at peak of CTPase (Uand -20 S in fraction 9 (B). Note that the ATPase activity is plotted X 10. The gradient markers were thyroglobulin (19 S), catalase (11 S), and cytochrome C (2.2 S).

Two Isoforms of Egg Dynein

1 2 3 4

285

ATPase activity of the soluble, phosphocellulose-purified dynein was stimulated 14-fold by treatment with 0.1% Triton X-100 (specific activity, 350 nmol P,/min/ mg) as reported previously [Asai and Wilson, 1985; Porter et al., 19881, but its CTPase activity was inhibited by the treatment. Similar results were observed with flagellar dynein. On the other hand, addition of Triton X-100 to the microtubule-associated isoform, HMr-3, stimulated both its ATPase (3-fold) and CTPase (2-fold) activities. Addition of KCI mimics the effect of Triton X-100 and lowers the MgCTPase/MgATPase ratios (Table IIB). This effect was most pronounced for the soluble egg dynein. Three hundred millimolar KCI activated the ATPase activity 6-fold but inhibited the CTPase activity by 40%. On the other hand, addition of 50 mM KCI to HMr-3 resulted in a 4-fold increase in its ATPase activity with no change in the corresponding CTPase activity. These data suggest further differences in the enzyme activities of the cytoplasmic dynein isoforms and demonstrate further similarities between flagellar dynein and the soluble egg dynein isoform.

200 116

97 66

45

Fig. 2. SDS-Page analysis of sea urchin ATPases and CTPases. Fractions were run on a 6% polyacrylamide gel, assayed for NTPase activity, and the values reported in Tables I and 11. The lanes contained the following samples: 1, 20 S peak from a sucrose gradient of egg extract; 2, 20 S peak from a sucrose gradient of ATP MAPs (HMr-3 peak); 3, the peak ATPase fraction prepared by phosphocellulose chromatography of MT-depleted extract (soluble dynein); 4, 21 S flagellar dynein.

Microtubule Binding

Analysis of mechanochemical enzymes, including kinesin and both axonemal and cytoplasmic dyneins, has shown that under certain conditions their ATPase activities are sensitive to stimulation by microtubules [Omoto and Johnson, 1986; Cohn et al., 1987; Paschal et al., 19871. We examined the effect of microtubule addition on the ATPase activity of HMr-3, but under the conditio measured for the crude mixture of dyneins present tions so far tested, we have not observed any significant in the 20 S peak of an egg extract was 2.6 -+ 1.7 (mean activation. We have therefore tested other potential & standard deviation). Following purification of the mechanochemical properties of the HMr-3 polypeptide, HMr-3 isoform by microtubule affinity and sucrose gra- such as microtubule cross-bridging and motility. dient sedimentation, the 20 S fraction of ATP MAPs Fractions containing purified HMr-3 promote the gave a MgCTPase/MgATPase ratio of 10.5 8.6 (Table bundling of taxol-polymerized brain microtubules, as asI). In contrast, the MgCTPase/MgATPase ratio for the sayed by darkfield microscopy. When phosphocellulosesoluble egg dynein isoform was only 0.61 2 0.60, sim- purified, bovine brain microtubules are incubated in ilar to the ratio measured for flagellar dynein, 0.21 PMEG buffer containing 10 p M taxol and sucrose, they 0.30, both under our assay conditions and those of Gib- remain uniformly dispersed throughout the field of view. bons [ 19661. These results indicate that the microtubule- In contrast, incubation of these microtubules with differbinding cytoplasmic dynein can be distinguished from ent fractions from a sucrose gradient of S . puvpurutus the soluble cytoplasmic dynein by its greater CTPase MAPs revealed the presence of two peaks of microtubule activity relative to ATPase activity. bundling activity, one at 20 S and one at 10 S. Both peaks induced the formation of bright, dense microtubule Activation of ATPase and CTPase Activities bundles throughout the field. However, there was no We have examined the effect of detergents and evidence of aster formation as was observed in metamonovalent cations on the NTPase activity of the differ- phase extracts prepared from Xenopus eggs [Verde et al., ent egg dynein isoforms. Addition of 0.1% Triton X-100 19911. The two peaks of microtubule bundling activity to the various sea urchin dynein samples described in showed different sensitivities to the presence of ATP. Figure 2 resulted in a decrease in MgCTPase/MgATPase The addition of 1 mM MgATP to the microtubule bunratios, due, in general, to a preferential activation of dles produced by the 20 S peak resulted in their disperATPase activity over CTPase activity (Table IIA). The sion within 20 min (Table 111). Bundles formed with

*

*

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TABLE I. Specific CTPase and ATPase Activities* S. purpurutus (nmol P,/min.mg)

L. pictus (nmol P,/min.mg) CTPase ATPase

Sample Extract 20 S peak HMr-3 peak Soluble dynein Flagellar dynein

5.0 8.9 28 60

*

t 3.7 t 7.4 t 28 2

CTPase

3.2 1.5 1.4 t 0.8 32 t 6.4 300 k 35

2.1

ATPase

2.7 2 0.5 66 4.5

MgCTPase /MgATPase"

0.97

5 0.62 3.1 20

2.6 2 1.7 10.5 2 8.6 0.61 t 0.60 0.21 ? 0.30

(n = 4) (n = 4) (n = 3) (n = 2)

*All samples were assayed in PMEG buffer. The specific activities are reported as the mean t standard deviation of two or more points. "The MgCTPaseiMgATPase ratios are reported as the mean t standard deviation of all CTP/ATPase ratios from each of several enzyme preparations from both L. pictus and S. purpurutus.

TABLE 111. Characteristics of Microtubule Bundling*

TABLE 11. Effect of Triton and Salt on CTPase and ATPase Activities Triton activation L. pictus sample" A. Triton activation Extract 20 S peak HMr-3 peak Soluble dynein Flagellar dynein

+

% of CTPase

% of ATPase

PMEG

100

760 290 1,350 340

0.56 7.8 0.80 0.18

190 54 61

Microtubule bundling

MgCTPase/ MgATPase PMEG Triton 0.074 2.8 0.032 0.019

MgCTPase/MgATPase S. mmxmztus sampleb PMEG PMEG B. KC1 activation Extract 20 S peak HMr-3 peak Soluble dynein

4.3 95 0.23

+ KCl

2.0" 24" 0.015d

"Samples were assayed in PMEG in the absence or presence of 0.1% Triton X-100. The values are reported as the average of two experiments. bSamples were stored overnight on ice and assayed the following day in PMEG in the absence or presence of 50 mM KCl or 300 mM KCl. =+ 50 mM KC1. d + 300mMKC1.

material from the 10 S region of a sucrose gradient of egg MAPs did not dissociate in the presence of ATP. These data suggest that sucrose gradient-purified HMr-3 crosslinks microtubules in an ATP-sensitive manner, but that the bundling activity in the 10 S region of the gradient is induced by other microtubule-associated proteins. Similar results have been described by others for the high molecular weight MAPs from sea urchin eggs and mammalian neurons [Hollenbeck et al., 1984; Hollenbeck and Chapman, 19861 and for the cytoplasmic dynein isolated from HeLa cells [Pfarr and McIntosh, 19881. Isolation and Characterization of a 20 S Motility Factor

The HMr-3 polypeptide shows numerous similarities to motor enzymes characterized in other systems, but our standard purification protocol for this polypeptide has thus far failed to yield a microtubule translocation

Sample

PMEG

ATP MAP gradient

PMEG + 1 mMMgATP

+ +

20 S peak 10 S Deak

-

+

*Samples were prepared as described in Figure 1 and assayed for MT bundling (see "Materials and Methods").

TABLE IV. Effect of Vanadate and GTP on Motility* Treatment

Concentration

20 S motility

10 S motility

Vanadate

10 (LM 30 (LM 1 mM

t

+ +

MgGTP

-

+a

*The effect of vanadate on motility was determined in the presence of 1 mM MgATP. 'Porter et al. [1987].

activity. We have therefore developed an alternate purification scheme to seek cytoplasmic dynein-induced microtubule motility. High speed supernatants of egg extracts were fractionated by sucrose density gradient centrifugation, and the material that sedimented at 20 S was incubated with taxol-stabilized, bovine brain microtubules, centrifuged, and then extracted with MgATP as described in Figure 3. The ATP extract prepared by this method (Fig. 3 , lane 8) was composed primarily of a high molecular weight polypeptide that comigrated with the flagellar dynein heavy chain on polyacrylamide gels. This ATP extract consistently supported microtubule gliding at an average rate of 0.45 2 0.14 p,m/sec, n = 19 MTs (Fig. 4). Microtubule movement was discontinuous. Microtubules often glided for 5 to 10 sec and then slowed down, or in some cases, detached from the coverslip. This behavior contributed to the broad range in gliding rates measured for single microtubules within a preparation (0.26 to 0.75 p,m/sec). These microtubule gliding rates are considerably slower than the values reported for other cytoplasmic dyneins [Paschal et al., 1987; Lye et al., 1987; Pfarr et al., 19901 and suggest that assay conditions have not yet been optimized for

Two Isoforms of Egg Dynein

1

2

3

5

4

6

7

8

287

9

200

116

97

66

45

Fig. 3. Purification of a 20 S microtubule translocating activity from whole egg extract. 0.5 ml of L. pictus whole egg extract was loaded onto a 5-20% sucrose gradient in PMEG buffer and centrifuged. The 20 S region of the extract gradient was incubated with taxol-stabilized bovine microtubules at 1.0 mg/ml and centrifuged (40,00Og, 45 min). The pellet was washed with PMEG and repelleted (40,OOOg, 30 min). The washed pellet was extracted with 10 mM MgATP and repelleted. The fractions obtained from this preparation were analyzed by SDSPAGE on a 6% polyacrylamide gel. Lane 1: Molecular weight markers. Lane 3: The 20 S region from a sucrose gradient of extract. Lanes 4,s: Supernatant and pellet following addition of MTs and centrifugation. Lane 6: Wash supernatant. Lanes 7,s: Pellet and supernatant after ATP extraction. Lanes 2,9: 21 S flagellar dynein. The ATP extract (lane 8) supported microtubule translocation in vitro.

the ooplasmic motor. The motility induced by the 20 S ATP extract was labile and attempts to determine the polarity of microtubule translocation by assaying the movement of axoneme fragments were unsuccessful. The absence of information on polarity of the 20 S translocator prompted us to develop alternate assays to characterize this activity. These included perfusion assays with different inhibitors to test the possibility that the 20 S activity was actually due to trace levels of kinesin contaminating our preparation, even though this enzyme usually sediments at 10 S [Porter et al., 19871. Twenty and 10 S fractions were prepared from sucrose gradients of egg extracts, as described in Figure 3 , using microtubule-affinity followed by ATP extraction (see Fig. 5A). The 20 S ATP extract (Fig. 5A, lane 8) induced the gliding of single microtubules but not axonemes, while the 10 S ATP extract (Fig. 5A, lane 9), which contains kinesin, supported the movement of both microtubules and axonemes with their “plus” ends trailing, as reported previously [Porter et al., 19871. The microtubule motility induced by the 20 S material was

-

Fig. 4. Microtubule motility induced by a 20 S egg fraction. ATP extract, prepared as described in Figure 3, was assayed for in vitro microtubule translocating activity. Shown here are three frames with time indicated in seconds. The three marked microtubules glided at rates ranging from 0.26 to 0.55 p d s e c . Scale bar = 1.0 pm.

reduced upon addition of 10 pM sodium vanadate and completely inhibited at a concentration of 30 pM (Table IV). However, vanadate concentrations up to 40 pM had no detectable effect on the microtubule motility induced by the 10 S material. The microtubule translocating activity induced by the 20 S ATP extract required ATP.

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A-l 2 3 4 5 6

7 8 9 1 0

200 116

97 66

45 29 L 1’ 2’ 3’ 4’

5’

6’

7. 8’

9’10’

200 116

MT - S U K 4: MOTILITY + S U K 4:

+ + + -

Fig. 5 . Characterization of a 20 S motile fraction with a monoclonal antibody to kinesin. Twenty S and kinesin containing 10 S fractions were prepared as described in Figure 3, run on a 7% polyacrylamide gel (A), blotted to nitrocellulose, and probed with SUK 4,a monoclonal antibody to sea urchin kinesin (B). The lanes were as follows. Lane 1: Molecular weight markers. Lanes 2,3: 20 S and 10 S peaks from a sucrose gradient of egg extract. Lanes 4,6: Supernatant and pellet following addition of MTs to the 20 S peak. Lanes 5,7: Supernatant and pellet following addition of MTs to the 10 S peak. Lane 8: ATP extract of the 20 S MT pellet. Lane 9: ATP extract of the 10 S MT pellet (containing kinesin). Lane 10: 21 S flagellar dynein marker. Motility assays on the 20 S (lane 8) and 10 S (lane 9) ATP extracts were performed in the absence or presence of protein Apurified SUK 4 antibody as described in “Materials and Methods.” SUK 4 inhibited only the 10 S (kinesin)-induced microtubule motility.

Perfusion of 1 mM MgGTP into chambers containing active 20 S samples inhibited microtubule movement, but movement restarted following subsequent perfusions of buffer containing 1 mM MgATP. In contrast, sea urchin egg kinesin will induce the gliding of microtubules in the presence of 1-10 mM MgGTP [Porter et al., 1987; Cohn et al., 19891. We have also analyzed the 20 S and 10 S motility factors using SUK 4, a monoclonal antibody that blocks the motility of sea urchin kinesin [Ingold et al., 19881. Immunoblots of 20 S and 10 S samples (Fig. 5B) probed with SUK 4 have shown that kinesin heavy chain is present in fractions isolated from the 10 S region of sucrose gradients (lanes 3, 7, and 9), but that kinesin is not detectable in fractions from the 20 S region of these gradients (lanes 2, 4, 6, and 8). In vitro motility assays were used to examine the effect of SUK 4 on the microtubule gliding induced by the 20 S and 10 S ATP extracts. The 10 S-induced microtubule motility was completely inhibited in the presence of 0.2 mg/ml protein A-purified SUK 4, but the same antibody preparations had no effect on the motility induced by the 20 S ATP extract. We conclude that the microtubule translocating activity isolated from the 20 S region of sucrose gradients is dynein-like and not due to kinesin contamination in our preparations. The relationship of HMr-3 and soluble dynein to the 20 S peak containing motor activity was examined immunologically and by the use of vanadate-sensitive UV cleavage. Immunoblots of urchin fractions probed with a blot affinity-purified polyclonal antibody to sea urchin flagellar dynein show that flagellar dynein-related antigens are present in both soluble dynein and the 20 S MT translocator (Fig. 6B). The pattern of polypeptides seen by SDS-PAGE after vanadate-induced UV cleavage demonstrated that the 20 S MT translocating fraction is a mixture of both cytoplasmic dynein isoforms (Fig. 6C). DISCUSSION Sea Urchin Cytoplasmic Dynein lsoforms Exhibit Different Affinities for CTP

Two isoforms of cytoplasmic dynein have been identified in sea urchin eggs [Porter et al., 19881. The soluble isoform is enzymatically and immunologically similar to flagellar dynein; the microtubule-binding isoform HMr-3 has dynein-like characteristics but is immunologically distinct from the soluble dynein [Porter et al., 19881. Here, we have used different assays to probe the relationship between soluble egg dynein and HMr-3. Analysis of the two egg dyneins has shown that HMr-3 copurifies with NTPase activity, but that its MgCTPase activity is -10 times higher than the corresponding MgATPase activity (Fig. 1; Table I). The soluble cyto-

Two Isoforms of Egg Dynein

A 7 2 3 4

B.

7

7

1 2 3 . 4

C=

289

lV 2v 3” 4v

200

116 97 66 Fig. 6. Relationship of the 20 S MT motor to cytoplasmic dynein isoforms. Sea urchin flagellar and cytoplasmic fractions were run on a 6% polyacrylamide gel (A), transferred to nitrocellulose, and probed with a blot affinity-purified polyclonal antibody to the flagellar dynein heavy chain (B).The lanes contained the following samples. Lane 1: Flagellar dynein. Lane 2: Soluble dynein. Lane 3: ATP extract of a 20 S MT pellet (MT translocating fraction prepared by MT affinity

from a 20 S sucrose gradient peak of egg extract as described in Fig. 3). Lane 4: HMr-3 peak. These fractions were also irradiated for 60 min at 365 nm in the presence of 1 mM MgATP and 100 p M sodium vanadate and then analyzed by SDS-PAGE on a 6% polyacrylamide gel (C). The HUV and LUV cleavage fragments of each sample are designated by dots.

plasmic dynein isoform, on the other hand, hydrolyzes CTP at -60% the rate of ATP. Similarly, flagellar dynein hydrolyzes CTP at -20% the rate of ATP. Theie data support the view that the soluble egg ATPase is more closely related to flagellar dynein, whereas HMr-3 may be the sea urchin analogue of the cytoplasmic dyneins recently described in other tissues [Paschal et al., 1987; Lye et al., 1987; Euteneuer et al., 1988; Neely and Boekelheide, 1988; Collins and Vallee, 1989; Koonce and McIntosh, 1990; Pfarr et al., 19901. The preferential hydrolysis of CTP relative to ATP was observed in all samples of HMr-3, but there was a wide range in both the specific activities and the MgCTPase/MgATPase ratios from preparation to preparation, as reported in Table I. The variability may be due to several factors. First, the amount of soluble dynein that cosediments with taxol-assembled microtubules is species-dependent . We reported earlier that the soluble dynein was a minor component of taxol-assembled microtubules prepared from L . pictus compared to similar preparations of S.purpurutus microtubules [Porter et al., 19881. Second, the. relative quantities of soluble dynein and HMr-3 in a given preparation or fraction is not constant. Finally, the variability in the ratios may reflect differences in the relative activation of the soluble dynein’s “latent” ATPase activity. However, we consistently observed that HMr-3 and the soluble dynein be-

came more enzymatically distinct as they were purified away from each other. Activation of ATPase and CTPase Activities

The ATPase activities of sea urchin flagellar dynein and soluble egg dynein can be activated by treatments with non-ionic detergents and monovalent cations [Gibbons and Fronk, 1979; Asai and Wilson, 1985; Dinenberg et al., 1986; Porter et al., 19881. We found that the degree of activation of NTPase activity by such treatments demonstrated further enzymatic differences between the egg cytoplasmic dynein isoforms, HMr-3 and the soluble dynein. In the presence of Triton X-100 or KC1, the MgCTPase/MgATPase ratios decrease in all fractions due to an activation of ATPase activity relative to CTPase activity (Table 11). However, these effects were more pronounced for both the soluble cytoplasmic dynein and flagellar dynein than for the cytoplasmic isoform, HMr-3. Our data are consistent with previous observations that the soluble dynein has a “latent” ATPase activity similar to that described for flagellar dynein [Gibbons and Fronk, 1979; Porter et al., 19881. The ATPase activity of dynein is probably coupled to conformational changes that give the enzyme its mechanochemical activity [reviewed in Johnson et al., 1984; McIntosh and Porter, 19891. The rate of ATP hydrolysis by axonemal dynein is increased either by mi-

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crotubules, which speed the release of hydrolysis prod- ments described in crude egg homogenates by Pryer et ucts [Omoto and Johnson, 19861, or by Triton X-100, al. [19861 and in embryos by Wadsworth [1987]. This which increases the rate of product release independent translocator is probably analogous to the cytoplasmic dyof microtubule concentration [Gibbons and Fronk, neins described by others [Paschal et al., 1987; Lye et 19791. Indeed, Triton X- 100 changes the microtubule al., 1987; Koonce and McIntosh, 1990; Pfarr et al., binding properties of dynein [Gibbons and Gibbons, 19901. The lability of the translocator activity described 19791. Similarly, brain cytoplasmic dynein shows some by Pryer et al. [1986] is consistent with the difficulties ATPase activation by microtubules [Paschal et al., 1987; we have experienced in purifying this activity. Shpetner et al., 19881 and the dynein from C . elegans is The relative contributions of the two cytoplasmic activated by Triton [Lye et al., 19871. The data presented dynein isoforms, HMr-3 and soluble dynein, to the 20 S here indicate that the ATPase activities of both soluble MT translocating activity is still unresolved. The active egg dynein and HMr-3 are activated by Triton, while fraction contains both cytoplasmic dynein isoforms, as CTPase is only slightly activated, consistent with the shown by UV cleavage and by cross-reactivity with idea that the CTPase activity of cytoplasmic dynein is not flagellar dynein antibodies (Fig. 6). However, the flagelcoupled to a mechanochemical cycle [Shpetner et al., lar dynein antibodies that were generated in our labora19881. tory bind poorly to native soluble dynein and efforts to absorb this protein from solution were unsuccessful. Relationship of HMr-3 to Other Therefore, we were unable to separate the soluble dynein Cytoplasmic Dyneins from HMr-3 by immunoadsorption. Although the 20 S In earlier studies, Pallini and coworkers [1983] motile fraction is a mixture, neither cytoplasmic dynein identified 20 S, high molecular weight polypeptides in isoform induces MT motility when prepared by methods brain, HeLa cells and insect eggs that hydrolyze CTP at that optimize the separation of the two isoforms. Thus, it rates faster than ATP. Analogous NTPase activities have is uncertain whether microtubule translocating activity been reported for MAP lC, the cytoplasmic dynein iso- requires the presence of both dynein isoforms or simply lated from brain [Shpetner et al., 19881. Its MgCTPase/ that the new purification procedure better preserves the MgATPase ratio was 8.4, similar to the value measured activity of one form while increasing the contamination by us for HMr-3 (-10 in Table I). Likewise, we have by the other. Since the soluble dynein binds weakly to observed high MgCTPase relative to MgATPase activity microtobules, we think it likely that the HMr-3 isoform in cytoplasmic dynein preparations from Drosophilu em- is the more important for in vitro microtubule gliding, bryos (T.S. Hays and P. Grissom, unpublished observa- but a final resolution of this dilemma will require either tions) and for the cellular slime mold, Dictyostelium separation of the isoforms in an active state or the de[Koonce and McIntosh, 19901. The nucleotide specific- velopment of specific dynein heavy chain inhibitors. In conclusion, we suggest that HMr-3 is likely to ity shared by dynein-like MAPS including HMr-3 may prove useful in distinguishing true cytoplasmic dyneins be the motor molecule important for microtubule movement in vitro because it shares a number of characterisfrom axonemal dynein precursors. tics with known cytoplasmic dyneins, including an ATPIdentification of a 20 S Microtubule sensitive binding to microtubules and a high MgCTPase Translocating Activity activity relative to MgATPase activity. Analyzing the Sucrose density centrifugation of egg extracts fol- function of cytoplasmic dyneins in a sea urchin egg that lowed by microtubule affinity and ATP extraction (Fig. contains multiple dynein isoforms will be a challenging 3) yields a 20 S ATP extract that supports the in vitro problem for future study. However, our biochemical translocation of microtubules (Fig. 4). We have distin- studies in combination with the recent cloning of the guished the 20 S translocator from kinesin-induced genes for flagellar dynein heavy chains [Gibbons et movement by its sensitivity to nucleotides, inhibitors al., 1991; Ogawa, 19911 could open up opportunities for (Table IV), and a motility-blocking antibody to kinesin the detailed experiments that may unravel the functions (Fig. 5). Likewise, immunoblot analysis fails to reveal of specific dyneins in a single cell. any kinesin heavy chain in the fractions purified from the 20 S region of sucrose gradients. Thus, both pharmacol- ACKNOWLEDGMENTS ogy and immunochemistry indicate that the microtubule We are particularly grateful to Dr. Jon Scholey for motility induced by the 20 S factor is not due to kinesin providing us with protein A-purified monoclonal anticontamination; the nucleotide requirements and the senbody to sea urchin kinesin. We wish to acknowledge the sitivity to vanadate suggest that it is based on a dyneingift of taxol from Dr. Matthew Suffness of the National like enzyme. It seems probable that this 20 S translocator is responsible for the retrograde, MT-dependent move- Cancer Institute. Finally, we appreciate the photographic

Two Isoforms of Egg Dynein

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