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The identification of a dynein ATPase in unfertilized sea urchin eggs
DEVELOPMENTAL
BIOLOGY
The Identification
74,364-378 (1980)
of a Dynein ATPase in Unfertilized
Sea Urchin Eggs
M. M. PRATT* Department of Biology, Brandeis University, Waltham, Massachusetts 02154, and Marine Biological Laboratory Woods Hole, Massachusetts 02543 Received April 9, 1979; accepted July 11, 1979 A cytoplasmic dynein ATPase has been identified in three species of unfertilized sea urchin eggs, Strongylocentrotus droebachiensis, S. purpuratus, and Arbacia punctulata. The enzyme was partially purified by sucrose gradient density centrifugation, and its polypeptide chain weight and composition were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein has enzymatic characteristics in common with flagellar dynein. It is activated nearly equally by Mg2+ and Ca’+, has no activity in the presence of K’ and EDTA, shows a specificity for ATP over other nucleoside triphosphates, and is inhibited by vanadate anion. On SDS-PAGE, the enzyme shows two major bands at 320,000 and 385,000 daltons, comigrating with certain ciliary and flagellar dynein polypeptides. The enzyme, given the name “egg dynein,” constitutes 2 to 4% of the total cell protein in the unfertilized egg and maintains this high value from fertilization through the late blastula stage. It appears to be equally distributed throughout the embryo at the 16-cell stage. Possible functions of egg dynein are discussed and models for dynein-microtubule mediated movements within the cytoplasm are presented. INTRODUCTION
There has been some evidence and much speculation that microtubule-dynein ATPase interactions, analogous to those seen in cilia and flagella, might be involved in cytoplasmic motile mechanisms (cf. Stephens and Edds, 1976). Tubu!in and microtubules have been found in a wide variety of cell types, but only rarely has a dynein-like ATPase been identified (Mooseker and Tilney, 1973; Burns and Pollard, 1974; Gaskin et az., 1974). As early as 1963, Miki reported the existence of a Mg2+-ATPase in echinoderm eggs. In 1968, Weisenberg and Taylor isolated a soluble 13 S Mg”-ATPase from Arbacia eggs. They speculated that it was a ciliary precursor based on its sedimentation coefficient and nucleotide and ionic specificities. Later, Mabuchi (1973) extracted a 0.6 M KCl-soluble ATPase from isolated cortices of sea urchin eggs, which has since * Present address: Department of Anatomy, Harvard Medical School, 25 Shattuck Street, Boston, Mass. 02115.
been shown to have characteristics in common with dynein (Kobayashi et al., 1978). This report refines and extends the initial observations of Weisenberg and Taylor (1968). A 12-14 S ATPase has been isolated from three species of echinoderms and has been identified as a cytoplasmic dynein on the basis of sedimentation coefficient, comigration on SDS-polyacrylamide gels with certain axonemal dynein polypeptides, and characteristic enzymatic properties. Further, it has been shown to constitute a significant portion of the total cell protein throughout early development. Since it was first isolated from unfertilized eggs, it will be referred to here as “egg dynein.” A preliminary account of some of this work has appeared in abstract form (Pratt and Stephens, 1977). MATERIALS
Organisms: Obtaining dling Embryos
Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
Gametes and Han-
Strongylocentrotus droebachiensis and Arbacia pun&data were obtained from the supply department of the Marine Bio364
OOlZ-1606/80/020364-15$0.200/O
AND METHODS
M. M. PRATT
Dynein ATPase
logical Laboratory, Woods Hole, Massachusetts, and held in tanks of running seawater at 10 and 20X’, respectively. S. purpuratus was obtained from Pacific Bio-Marine, Venice, California, and held in running seawater tanks at 10°C. Gametes were obtained by injection of 25 ml of 0.5 M KC1 into the urchin coelom. Sperm were shed into a dry Syracuse dish. Eggs were shed into Millipore-filtered seawater (MPFSW)’ and washed twice by settling. Eggs suspended in MPFSW were fertilized by addition to an equal volume of MPFSW containing 5-10 drops of diluted sperm (1 drop of dry sperm to 10 ml MPFSW). The embryos were grown in MPFSW in culture dishes in an incubator set at 8°C for S. droebachiensis, 14°C for S. purpuratus, and 23°C for Arbacia punctulata. For experiments of greater than 24 hr duration, sulfadiazine was added to S. droebachiensis cultures to a final concentration of 0.01%. Preparation
of Egg Dynein
Egg dynein was prepared according to the method of Weisenberg and Taylor (1968) with slight modifications. Typically, 3-5 ml of packed dejellied eggs were suspended in 2-3 vol of an isotonic buffer containing 1 M sucrose, 5 mM MgC12, 50 n&I Tris-HCl, pH 8.0, 1 m&I EDTA (SMTE). Unless otherwise stated, all preparations were carried out at 4°C. The eggs were broken by 8-10 passes of a ground glass homogenizer. The homogenate was spun at 100,OOOg(35,000 rpm, Type 40 rotor) for 2 hr. The pellet was retained on ice. The supernatant was dialyzed against 50 mJ4 Tris-HCl, pH 8.0, 1 mM EDTA for 510 hr. Samples of the whole homogenate, supernatant, and pellet were assayed for protein content and ATPase activity. An ’ Abbreviations used: EGTA, ethylene glycol bis (/3-aminoethyl ether) N,N’-tetraacetic acid; MA, mitotic apparatus; MPFSW, Millipore-filtered seawater; OD-cm, optical density x centimeters; SMTE, 1 M sucrose, 5 mI14 MgCI, 50 mh4 Tris-HCl, pH 8.0, 1 mM EDTA; V(V). vanadate anion in the pentavalent state.
365
in Sea Urchin Eggs
appropriate volume of the dialyzed supernatant was layered onto a 12- or 56-ml 520% (w/v) sucrose gradient, prepared in 50 mJ4 Tris-HCl, pH 8.0, 50 n&I NaCl, and spun for 12 or 16 hr at 200,OOOg(35,000 rpm, SW 40 rotor) or 24 hr at 100,OOOg(25,000 rpm, SW 25.2 rotor). Fractions were collected and the absorbance at 280 nm (A& was monitored. Appropriate fractions were assayed for protein content and ATPase activity. Preparation of Cilia, nemal Dynein
Flagella,
and Axo-
Cilia were prepared from embryos cultured as described above by suspending the washed blastulas in hypertonic seawater (Stephens, 1977a). Deciliated embryos were sedimented in a hand centrifuge and resuspended in MPFSW if regeneration of cilia was desired. The cilia were pelleted from the hypertonic supernatant at 10,OOOgfor 10 min in a Sorvall HS4 rotor, and washed once in 10 mJ4 Tris-HCl, pH 8.0. Whole cilia were demembranated by incubation in 0.25% Nonidet P-40, 30 miV Tris-HCl, pH 8.0, 3 mJ4 MgCle, 0.1 mM EDTA (NTME) , and collected by centrifugation at 10000 g for 15 min (9000 rpm, Sorvall HS4 rotor). Sperm flagella were prepared according to the method of Stephens (1970). Tails were sheared from heads by 20 rapid passes of a ground glass homogenizer. Flagella were isolated by differential centrifugation and demembranated in 10 vol of NTME without an incubation period. Axonemes were collected as described above. Dynein was prepared by dialysis of flagellar axonemes against 1 miI4 Tris-HCl, pH 8.0, 0.1 mM EDTA, 0.05% mercaptoethanol according to the method of Gibbons and Fronk (1972). Separation
of Blastomeres
Preparation of blastomeres was modified from the procedure of Hynes and Gross (1970). Approximately 0.5 ml of S. purpuratus eggs were fertilized as above. Fertili-
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DEVELOPMENTAL BIOLOGY
zation membrane formation was prevented by the presence of 0.2% papain and 0.04% cysteine (Tyler and Speigel, 1956). Embryos were cultured as above until the 16cell stage. They were spun down and washed twice in a 19:l mixture of 0.5 M NaCl and 0.5 M KC1 containing 16 r&f Tris-HCl, pH 8.0 (19:l Tris), using a hand centrifuge. The embryos were suspended in 5-10 ml fresh 19:l Tris and gently pipetted and swirled until the cells separated from one another (approximately 10 mm). Five milliliters of the cell suspension was layered onto a .30-ml gradient of 5-15% Ficoll (w/v) in 19:l Tris. The gradients were spun for 1 min at 1250rpm (Sorvall HS4 rotor). Crude fractions were collected from the top using a Pasteur pipet. Each was monitored for cell content and pure meso-, macro-, and micromeres were recovered by centrifugation. The pellets of cells were solubilized for gel electrophoresis as outlined below.
VOLUME 74,198O
flagellar dynein and 45 min with the egg enzyme. Assays were always run for less than these maxima, the absolute times being adjusted to the amount of enzyme used so that the ATzOremained in an easily quantitated range. For studies of ATPase inhibitors, oligomycin was stored at -20°C as a 25-mg/ml solution in 100%ethanol. Ouabain was prepared as a 10e3 M solution in deionized water, and stored at -20°C. Sodium vanadate (NaV03) was prepared as a 10 r&l4 stock solution in 10 n-&f Tris-HCl, pH 8.0, heated in boiling water for 1 min to dissolve, and stored at -20°C. Gel Electrophoresis and Densitometry
All samples for sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) were prepared in a sample buffer containing 2.5 mM Tris-glycine, pH 8.3, 1% SDS, 1% mercaptoethanol, 5% glycerol, 0.05% bromphenol blue (Bryan, 1974; SteDetermination of Protein Content and phens, 1975), and 50 pg/ml phenyhnethylATPase Activity sulfonyl fluoride (PMSF). Samples preProtein content was determined by the pared from whole unhatched embryos were method of Lowry et al. (1951), standardized spun at 10,000 rpm (Sorvall HS4 rotor) to pellet the fertilization membranes which with bovine serum albumin (BSA). Adenosine triphosphatase activity was did not solubilize in sample buffer. SDSassayed at 20°C in 50 mM Tris-HCl, pH PAGE was carried out either with a phos8.0, 0.1 M KCl, 5 mM cation, and 1 mM phate buffer system (Shapiro et al., 1967) nucleotide. Deionized water was used and a 3% polyacrylamide gel, or with a throughout. When Mg’+-ATPase was Tris-glycine system (Bryan, 1974; Stephens, 1975) using a 5% gel. Gels were quanmeasured, the mix also contained 1 r&f titatively stained by the method of Weber EGTA. For detection of myosin-like ATPase, 5 mM EDTA was included in lieu of and Osborn (1969) in 5 vol of 0.1% Coomasany cation. Typically, the reaction was sie brilliant blue (R-250) or 0.25%fast green started by the addition of enzyme to one of dissolved in 50% methanol, 10% acetic acid for 2 hr at 45°C. Staining was continued in the mixtures described above. The reaction was stopped and inorganic phosphate gen- fresh stain for 2 hr at 45°C. The gels were erated was measured by the method of destained in 5% ethanol, 7.5% acetic acid. Pollard and Korn (1973), a modification of Stained gels were scanned either in a Joyce-Loebl MK III microdensitometer usthe original method of Martin and Doty (1949). For both flagellar and egg enzymes, ing white light or in an Ortec 4310 densiphosphate release was linear for the tometer using a green filter for Coomassie blue or a red filter for fast green. The area amount of enzyme added. At concentrations below 10 fig/ml for flagellar and 100 under the curve (in OD-cm) was measured pg/ml for egg dynein, the amount of phos- by planimetry and was compared with BSA phate generated was linear for 20 min with standards to determine the amount of pro-
M. M. PRATT
Dynein ATPase
367
in Sea Urchin Eggs
tein in various bands. For purposes of calculation, all overlapping peaks were assumed to be Gaussian. RESULTS
Purification
of Egg Dynein
Approximately 30-40s of the total Mg*+ATPase activity of unfertilized eggs from the sea urchin Strongylocentrotus droebachiensis is readily soluble and can be found in the high speed supernatant of eggs broken in SMTE. The remainder of the activity stays in the pellet and is not purified further. The soluble activity can be partially purified by sucrose density gradient centrifugation. To allow visualization of highly aggregated material and for comparison with standard gradients used in the purification of axonemal dynein (Gibbons, 1966), a relatively steep 5-20s gradient was used. When resolved on such a 5-20s sucrose gradient, the dialyzed egg supernatant separates into two major protein peaks-a small peak of aggregated material near the bottom of the gradient and a large, broad peak at the top. Flagellar dynein, extracted from axonemes by low ionic strength dialysis, was run in a parallel gradient as a 14 S marker (Gibbons and Fronk, 1972). The sea urchin egg ATPase duplicated the distribution of flagellar dynein ATPase. The ATPase activity did not coincide with either major protein peak but, instead, formed a nearly symmetrical peak centered at 14 S (Fig. la). Identical results were obtained with the sea urchin S. purpuratus. When analyzed on SDS-PAGE, the 14 S peak appears highly enriched in two high molecular weight polypeptides, and contains lesser amounts of lower molecular weight proteins (Fig. lb). The two high molecular weight bands have been named egg dynein. Significant ATPase activity first appears along with these two heavy bands in fractions 14 and 16. In addition, these high molecular weight bands repre-
2
18
28
b FIG. 1. Sucrose gradient analysis of S. droebachiensis egg extract. (a) 5-20s sucrose (w/v) in 10 m44 Tris-HCl, pH 8.0, 10 m&f NaCl, 1 rmI4 EDTA. There are two protein peaks and one peak of ATPase activity at fraction 18. Sixteen-hour gradient run. (b) 3% polyacrylamide gels in phosphate buffer of evennumbered gradient fractions, 2-28. Note the enrichment of the two highest molecular weight proteins and some lower molecular weight species in fractions 14 20, corresponding to the ATPase peak.
sent the major portion of the protein in fractions 16-20. Upon centrifugation of homogenized eggs and analysis of the fractions on polyacrylamide gels, the protein in these two bands represents a larger percentage of the total protein in the supernatant than in the pellet. In contrast, the total ATPase is greater in the pellet, and thus there seems to be a distinct enrichment of egg dynein in the soluble extract. It can be noted in Fig. 1 that the absolute
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DEVELOPMENTALBIOLOGY
amounts of protein in these two high molecular weight bands do not correspond directly with the ATPase activity peak. Fraction 20, especially, contains a large amount of egg dynein protein but lacks a correspondingly high ATPase activity. This apparent discrepancy, strictly reproducible in all egg dynein gradient preparations and often seen in gradient purifications of flagellar dynein also, is best explained by the fact that dynein is a multisubunit enzyme (Gibbons, 1966; Gibbons et al., 1976; Gibbons and Fronk, 1979). At S values lower than 14 S (found closer to the top of the gradient) a portion of the enzyme may be partially dissociated, rather than existing as an intact 14 S complex. Thus, gel electrophoresis would indicate large amounts of the enzyme’s constituent proteins, but the ATPase activity would be low, since the proteins are not in the form of an active complex. Gibbons and Fronk (1979) have found that the most active form of the enzyme is the 14 S molecule. Low ATPase activities toward the top of the gradient may also be due to the fact that the two high molecular weight bands comprise a lower percentage of the total protein in these fractions. Another possibility is that the lower molecular weight proteins appearing in later fractions may inhibit the enzyme activity. The two high molecular weight bands are undoubtedly the major components of egg dynein, but sucrose gradient preparation provides only minimal separation of egg dynein from some other proteins. Further purification of egg dynein away from these lower molecular weight components has proved rather difficult. Ammonium sulfate fractionation was unsuccessful, precipitating the bulk of the protein, egg dynein, and contaminants, between 50 and 60% saturation. Gel chromatography on Sephadex G-200 or Bio-Gel Al5 (Burns, 1976) also failed to purify the high molecular weight egg dynein from certain low molecular weight proteins, suggesting
VOLUME 74.1980
either that these proteins exist as aggregates approximating the size of native egg dynein, or that they are tightly associated with the enzyme, perhaps as subunits of the ATPase. Purification of the enzyme on DEAE-Sephadex was attempted with only moderate success. It should be pointed out that axonemal dynein has not yet been purified to homogeneity (Gibbons et al., 1976; Ogawa and Gibbons, 1976). It has recently been hypothesized that certain low molecular weight proteins, which were earlier thought to be contaminants, may be part of the enzyme complex (Gibbons and Fronk, 1979).
Enzymatic Properties of Egg Dynein When the major fractions of the sucrose gradient within the 14 S ATPase peak are combined and assayed under a variety of conditions, the enzymatic activity shows a marked similarity to that of axonemal dynein. Table 1 illustrates a comparison of axonemal and egg dynein under conditions which examine the unique character of dynein ATPases. The data reported are from one preparation of egg and flagellar dynein but similar data were obtained from other experiments. Absolute specific activities measured never varied more than 25% from the published values and the ratios between the specific activities under different conditions had a variance of 10%. Egg dynein exhibits the low ratio of Ca”’ to Mg*‘-ATPase (defined as the Ca*+/Mg” ratio) characteristic of ciliary dynein (Gibbons, 1966) as well as the absence of activity in K+ and EDTA. However, the Ca’+/Mg*+ ratio is even lower than that of flagellar dynein. Egg dynein is also less discriminating between ATP and other nucleotides (GTP and ITP). In addition, the specific activity of egg dynein is approximately lo-fold lower than that of axonemal dynein. This difference is not surprising when the energy requirements of a flagellum are compared to those of a sea urchin egg. Estimates have been
M. M. PRATT
Dynein ATPase TABLE
369
in Sea Urchin Egg.9
1
SPECIFIC ACTIVITIES OF FLACELLAR DYNEIN AND EGG DYNEIN UNDER VARYING ASSAY CONDITIONS” Mg’+-ATPase (‘i inMg’+-GTPase K+-EDTA Mg”‘-ATPase Ca”‘-ATPase hibiin2pM activity activity activity ATPase actionl NaVO j tivity L9
purpuratus flagellar dynein S. purpuratus egg dynein S. droebachiensis flagellar dynein S. droebachiensis egg dynein
1.50 f 0.08
0
0.019 f 0.001
1.13 f 0.06
(461
0.257 f 0.013
0.170 + 0.008
0
0.010 +- 0.001
0.232 f 0.01 I
(IO)
1.40 + 0.01
1.31 -t 0.07
0.012 f 0.001
0.983 + 0.049
IX))
0.399 + 0.020
0.209 + 0.010
0.248 (0.120 0.020 (0.090
0.296 k 0.015
(26)
--2.10-t
0.11
0.007 f 0
f + + +
0.012 0.006)” 0.001 0.005)”
” 14 S flagellar dynein and egg dynein were assayed under conditions shown. The assay medium was 50 mM Tris-HCl, pH 8.0, 0.1 M KCl, 5 mM cation or EDTA, 1 mM nucleotide. Specific activities are expressed as micromoles inorganic phosphate released/milligram dynein x minutes f 5% instrumental error. Details of the assay method can be found in the text. The actual amount of dynein heavy chain present was determined by gel densitometry as described under Results for egg dynein quantitation. ” These values are for Mg”‘-ITPase activity.
made that sperm flagella expend 3-12 x lo-’ erg/set/cell when beating at lo-40 Hz (cf. Hiramoto, 1974). Sea urchin embryos do not generate such obvious motility, but chromosome movement during cell division can be used as an example of an energyrequiring event in these cells. Amoore (1963) has calculated that all of anaphase in pea endosperm requires only 0.8 x lo-l3 erg/set/cell, a value six to seven orders of magnitude lower than that for flagella. Furthermore, the specific activity for egg dynein reported here is lo-fold higher than the original value reported by Weisenberg and Taylor (1968)) and 5- to lo-fold higher than the values reported for a dynein-like enzyme found in the brain (Burns and Pollard, 1974; Gaskin et al., 1974). Gibbons et al. (1978) have shown recently that vanadium in the pentavalent state, V(V), is a potent inhibitor of flagellar dynein ATPase. At 100 @4 NaV03, the Mg’+-ATPase activity of S. droebachiensis flagellar dynein is nearly 75% inhibited, while nearly 1 n&I NaV03 is required for 75% inhibition of egg dynein ATPase. A better comparison of the effect of vanadate on the two enzymes can be made at 2 fl NaVO:j, where both are inhibited less than 50%. At 2 fl NaVO:s, the inhibition of S. purpuratus egg dynein is only lo%, but for
S. droebachiensis it is 26%, compared to 46 and 30% for the respective flagellar enzymes. These and other differences seen in the egg dynein are similar to those recently found for a dynein associated with ciliary membranes (Dentler et al., 1978). To rule out contamination by Na’-K’ or mitochondrial ATPases, the Mg”‘-ATPase activity of egg dynein was assayed in the presence of two different inhibitors-l, 5, and 10 pg/ml oligomycin, or lo-” and lo-” M ouabain. These agents are known to inhibit H’- or Na’-K’-ATPases, respectively, at these concentrations (Gelfand et al., 1978). The Mg”‘-ATPase activities of both egg and flagellar dynein were unaffected or even stimulated, rather than inhibited by these agents. When an effect was seen, it was considerably less in the case of egg dynein (Table 2). SDS-Polyacrylamide
Gel Electrophoresis
Axonemal dynein is characterized by an extremely large minimal polypeptide chain weight and a specific band profile on SDSpolyacrylamide gels. Figure 2 illustrates the typical pattern obtained for ciliary versus flagellar dyneins on 5% Tris-glycine and 3% phosphate gels. The four bands appearing on 3% gels of flagellar axonemes have been designated C, A, D, and B as shown (Gib-
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DEVELOPMENTALBIOLOGY
bons et al., 1976). A similar nomenclature has been adopted here for ciliary dyneins, although the banding pattern is clearly different (see figure). By comparison with molecular weight standards, including myosin heavy-chain dimer from scallop myofibrils, the axonemal dyneins have molecular weights ranging from 335,000-385,000. Using a variety of other gel buffer systems, the same values are obtained, basically confuming those published by Burns and Pollard (1974) rather than higher values reported previously (Kincaid et al., 1973; Linck, 1973). Egg dynein contains two of these characteristically high molecular weight polypeptide chains. Figure lb shows 3% gel patterns of fractions from the corresponding gradient. Fractions within the peak of ATPase activity of both S. purpuratus and S. droebachiensis show a distinct enrichment of electrophoretic bands with molecular weights of 320,000 and 385,000. The upper band comigrates with flagellar dynein C and the lower band runs just below dynein B of both flagellar and ciliary dynein of the same species (Fig. 3). (Preparation of egg dynein from Arbacia punctulata yields the same two bands in different proportions, along with a higher molecular weight band.) TABLE 2 ACTIVITIES OF FLAGELLARDYNEIN AND EGG DYNEIN IN THE PRESENCEOF INHIBITORSOF MITOCHONDRIALAND Na+-K+-ATPases” Percentage of Mg’+ATPase activity
Mg’+-ATPase +l pg/ml oligomycin +5 pg/ml oligomycin +lO pg/ml oligomycin + W5 M ouabain + 10m4M ouabain
Flagellar dynein
Egg dynein
100 140 146 154 163 168
100 100 141 100 100 147
a S. droebachiensti flagellar dynein and egg dynein were assayed for Mg’+-ATPase activity in the absence and presence of oligomycin or ouabain. Assay conditions were 50 mM Tris-HCl, pH 8.0,O.l M KCl, 5 mM MgC12,1 mM ATP, and the designated inhibitor.
VOLUME 74.1980
Dy=
LXP-
FC
FC
FIG. 2. Electrophoretic profiles of S. purpuratus cilia (C) and flagella (F). The gels on the left are 5% polyacrylamide in Tris-glycine buffer. The dynein doublet (Dy) and tubulin (Y-and P-chains are labeled. The gels on the right are 3% polyacrylamide in phosphate buffer. The four dynein polypeptides are labelled (C, A, D, and B), as is the tubulin band (T). Note that the tubulin subunits are not separated on this gel system.
Each egg dynein peak fraction also contains lower molecular weight polypeptides which may represent the medium weight dynein components recently identified by Gibbons and Fronk (1979). Stoichiometry Once identified, the two polypeptides characteristic of the egg dynein ATPase region of the gradients are easily visualized on SDS-polyacrylamide gels of whole eggs and embryos. The amount of egg dynein in whole eggs could therefore be quantitated using gel electrophoresis and densitometry. A similar method was used to estimate the amount of actin in Acanthamoeba castelZani (Pollard and Kern, 1972).
M. M. PRATT
C
C+P
P
Dynein ATPase
F+P
F
a
c
371
in Sea Urchin Eggs
c+w
P
F+P
F b
FIG. 3. Comigrations of egg dynein with ciliary and flagellar dynein. (a) The species is S. purpuratus. From left to right, the 3% polyacrylamide gels in phosphate buffer are cilia (C), cilia + the sucrose gradient ATPase peak fraction from an egg dynein preparation (C+P), the same ATPase peak fraction (P), flagella + the same ATPase peak fraction (F+P), and flagella (F). (b) The species is S. droebachiensis. From left to right the 39 polyacrylamide gels in phosphate buffer are cilia (C), cilia + a whole embryo preparation (C+W), the sucrose gradient ATPase peak fraction from an egg dynein preparation (P), flagella + the same ATPase peak fraction (F+P), and flagella (F).
Varying amounts of bovine serum albumin were run on gels identical to those used for quantitation and were stained with either Coomassie blue or fast green as described under Materials and Methods. The stained gels were scanned in a densitometer and the peak areas were determined for the known amounts of BSA. Figure 4a shows a plot of peak areas obtained for l-10 pg of BSA loaded. The function is linear within this range. For quantitation of egg proteins, whole embryos were solubilized at various intervals from fertilization through hatching. Samples of the whole embryos were run on 5% polyacrylamide gels and stained as previously described. Figure 4b shows a densitometer trace of such a gel. The amount
of protein in the egg dynein bands was determined by comparison of the area under the idealized Gaussian curve with the peak areas obtained for BSA. A similar calculation was made for total egg protein by integrating the area under the entire curve. The percentage of total egg or embryo protein represented by egg dynein was calculated by dividing the micrograms of protein in the two egg dynein bands by the micrograms of total cell protein and multiplying by 100. Often, the amount of protein in the egg dynein bands was measured on 3% gels loaded with an equal volume of sample. These allowed better separation and therefore quantitation of the bands. Using this method, it can be established that the two high molecular weight egg
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DEVELOPMENTAL
BIOLOGY
VOLUME 74,198O
FIG. 4. Stoichiometric analysis of egg dynein by gel electrophoresis. (a) Graph of peak areas (in OD-cm), obtained by densitometry and measured by planimetry, versus amounts of BSA run on polyacrylamide gels and stained with Coomassie blue or fast green. (b) Densitometer tracing of a 5% polyacrylamide gel pattern of a whole egg homogenate from S. droebachiensis. The egg dynein bands are shaded. The percentage of total protein in these bands was determined as described in the text.
dynein polypeptides represent 3.7 f 0.3% (n = 9) of the total Coomassie blue staining protein in embryos of S. purpuratus and 1.9 & 0.1% (n = 8) in S. droebachiensis. These are both surprisingly high figures and they remain nearly constant throughout early development, decreasing only slightly from fertilization through the swimming blastula stage. It is important to note that a large portion of the high molecular weight egg dynein found in the soluble egg supernatant, and isolated on sucrose gradients, sediments at 14 S. The total protein collected from a gradient preparation of S. droebachiensis egg dynein was estimated from 5% gels of the fractions, while the protein in the two high molecular weight bands was estimated from 3% gels of the fractions within the 14 S peak. The egg dynein polypeptides within the 14 S peak represent 33.5% of the total protein on the gradient.
This value is even higher than that calculated for the percentage of egg dynein in whole eggs and reflects the enrichment of this protein in the egg supernatant. The distribution of egg dynein within the embryo was examined at the 16-cell stage, when three different cell types are easily separated on the basis of size (Hynes and Gross, 1970). These cells, mesomeres, macromeres, and micromeres, establish three different embryonic regions and have different fates in development (Okazaki, 1975). Each separated cell type, shown in Fig. 5, contains approximately the same amount of egg dynein (determined as a percentage of total protein), and the values fall well within those for whole eggs and embryos (Fig. 6; Table 3). DISCUSSION
A 14 S Mg”-ATPase has been found in echinoderm eggs. It has enzymatic proper-
M. M. PRATT
Dynein ATPase
373
in Sea Urchin Eggs
leted ATPase. The apparent Mg”‘-ATPase specific activity, based on actual amounts of egg dynein, is much greater in the pellet than the supernatant, possibly signifying a higher activity of the bound form. However, the considerable activity in the pellet is more likely due to other ATPases, Nat-K+, H’, Ca’+, or others, which undoubtedly exist in the egg (Petzelt, 1972: Petzelt and von Ledebun-Vilhger, 1973)) since the amount of egg dynein in the pellet is a lower percentage of the total protein than that in either the whole egg or the supernatant. The supernatant, on the other hand, seems to contain primarily egg dynein ATPase. Only one peak of Mg”‘-ATPase activity is seen on sucrose gradients, at 14 S, and the enzyme in this peak shows a
Fro. 5. Isolated mesomeres (a), macromeres (bl, and micromeres (cl of S. purpuratus. The cells were examined using phase contrast microscopy and were photographed immediately after removal from Ficoll gradients. The magnification is 364~. Scale bar = 10 km.
ties in common with axonemal dynein, comigrates with certain dynein bands on SDS-polyacrylamide gels, and is a prominent and constant cell component prior to fertilization and throughout early development. The enzyme, which is referred to as egg dynein, can be partially purified from a soluble extract of sea urchin eggs. However, a large portion of the Mg’+-ATPase activity remains in the high-speed pellet. This may be due to a tightly bound form of egg dynein since the pellet also contains substantial amounts of the two high molecular weight polypeptides identified as egg dynein. Weisenberg and Taylor (1968) showed that further extraction in various KC1 and MgCL concentrations did not solubilize the pel-
-ED -
Me
Ma
Mi
FIG. 6. Egg dynein in three types of blastomeres of S. purpuratus. The gels are 3% polyacrylamide in phosphate buffer. The samples shown are whole isolated mesomeres (Me), macromeres (Ma), and micromeres (Mi). The egg dynein bands are indicated.
374
DEVELOPMENTAL BIOLOGY
marked overall similarity to axonemal dynein. Egg dynein exhibits enzymatic specificities much like those of flagellar dynein, but in some cases they differ in degree. These differences between egg and flagellar dyneins may reflect the fact that the assay conditions chosen were those known to be optimal for axonemal dynein, but these may not be ideal for egg dynein. A detailed analysis of the unique ionic, nucleotide, and pH requirements of the enzyme must await further purification. The discrepancies may also be due to the association of lipids with the enzyme (see below). Another possibility is that the enzyme requires a specific activation, analogous to the phosphorylation of nonmuscle myosins (Korn, 1978), which cannot take place in the partially purified preparation. In addition to possessing the appropriate enzymatic characteristics, egg dynein also resembles axonemal dynein in terms of native molecular weight as judged from sedimentation coefficient. The 14 S enzyme seems to be primarily composed of two high molecular weight subunits. It also, however, may contain some lower molecular weight components. This seems more plausible in view of the report by Gibbons and Frank (1979) that native flagellar dynein may include three protein components of less than 150,000 daltons. A direct association of egg dynein and tubulin has not been shown but there is experimental evidence which suggests an association. Tubulin can be crystallized from unfertilized sea urchin eggs by incubating them with 0.01% vinblastine sulfate. A small amount of a high molecular weight band, corn&rating with axonemal dynein C, consistently appears on gels of the vinblastine-tubulin crystals (cf. Stephens, 1978). In accordance with this observation, Nagayama and Dales (1970) have reported ATPase activity associated with vinblastine crystals from mammalian brain. In addition, the isolated mitotic apparatus (MA)
VOLUME 74.1980 TABLE
3
PERCENTAGEOFTOTALCELLPROTEIN REPRESENTEDBYEGGDYNEIN IN S.purpuratus BLASTOMERES~ Cell type Whole embryo Mesomeres Macromeres Micromeres
Percentage of total protein in egg dynein 3.7 3.3 3.4 3.5
f f k f
0.3 0.4 0.8 0.5
“The three cell types were isolated as described under Materials and Methods. The percentage of total protein in the egg dynein bands was determined by gel densitometry, as described under Results. The whole embryo value is an average of 10 experimental points. The blastomere values are averages of three measurements for each cell type. The variance is expressed as standard error.
of S. droebachiensis, a major tubulin-containing organelle, shows Mg2+-ATPase activity and protein species which corn&ate with certain ciliary dynein bands (Salmon and Jenkins, 1977; Pratt and Otter, in preparation). An evaluation of the capacity of egg dynein for tubulin interaction has been reserved for future studies. Cosedimentation of egg dynein and brain tubules is being examined along with possible copolymerization of the two proteins. The amount of egg dynein found in embryos is considerably higher than the amount of dynein which the embryo incorporates into cilia. Rough calculations show that ciliary protein accounts for about 0.25% of total embryonic protein and dynein accounts for about one-fifth of this. Thus, ciliary dynein can be estimated to represent no more than 0.25% of total embryonic protein, even after allowing for pools of ciliary dynein five times as large as needed for one crop of cilia. This is a maximum amount which might be present, as deduced from protein synthesis studies (Auclair and Seigel, 1966; Pratt, manuscript in preparation). Egg dynein pools must, then, contain a minimum of 7- to 15-fold more protein than would be necessary for ciliary incorporation. The ratio of egg dynein to egg tubulin is
M. M.
PRATT
Dynein ATPase
also significant as compared to that of cilia. Estimates by vinblastine precipitation (Burnside et al., 1973) and colchicine binding (Raff et al., 1971) of the amount of tubulin present in echinoderm and other marine eggs range from 3 to 10% of the total cell protein. Even using the largest of these values, the dynein:tubulin ratio in the whole egg is no more than 1:3 in S. purpuratus and 1:5 in S. droebachiensis. These values are equal to or higher than the average 1:5 ratio seen in cilia. This is in sharp contrast to cytoplasmic actomyosin systems where the myosin:actin ratios are considerably lower than those found in striated muscle cells (Korn, 1978). It seems unlikely that egg dynein serves only as a precursor to ciliary dynein as was first supposed (Weisenberg and Taylor, 1968; Stephens, 1972). First, it does not exhibit as varied an electrophoretic pattern as ciliary dynein even in hatched embryos. Second, the amount of egg dynein in the unfertilized egg and in the ciliated blastula is far greater than the amount of dynein ever found in cilia, even if one considers multiple regenerations. Finally, pulse-labeling studies of dynein synthesis using [3H]leucine have shown clearly that embryos can and do synthesize all of the ciliary dynein polypeptide chains and incorporate them into cilia, but that very little synthesis of egg dynein occurs after fertilization (Pratt and Stephens, 1978). The mitotic apparatus (MA) may provide one possible location and function for cytoplasmic dynein. Mohri et al. (1976) have shown that a fluorescent antibody to a fragment of flagellar dynein stains the mitotic apparatus during cleavage of sea urchin embryos, The same antibody has been reported to stop chromosome movement in isolated sea urchin MAs (Sakai et al., 1976) and to inhibit the dynein-like ATPase activity associated with the egg cortex (Kobayashi et al., 1978). It has also been shown that isolated spindles contain ATPase activity characteristic of dynein though it is
in Sea Urchin Eggs
375
not clear whether this activity is due to cortical contamination (Salmon and Jenkins, 1977). A role for egg dynein in mitosis is consistent with reports that very little new protein is synthesized for incorporation into the mitotic apparatus (Wilt et al., 1967), since it exists in large quantities in unfertilized eggs. Further studies of the nature of the MA dynein-like ATPase are in preparation. It seems likely that if dynein does play a role in mitosis, it may be in late anaphase during pole to pole elongation, as cross bridges have been seen, most often, in thin sections of the interzone region (Hepler et al., 1970; McIntosh et al., 1976). Another reasonable possibility is that egg dynein is active in microtubule-associated transport of cytoplasmic constituents, most likely of membrane-bound organelles along microtubule “railways.” Such a mechanism has been proposed for axonal transport, clearly a microtubule-related phenomenon (Heslop, 1975). There has been some evidence for dynein associated with neuronal systems (Burns and Pollard, 1974; Gaskin et al., 1974). In one case (Gaskin et al., 1974), the enzyme isolated from brain seemed to be membrane associated. In addition, Dentler et al. (1978) have recently reported a “membrane dynein” associated with ciliary membranes, which also contain large amounts of tubulin (Stephens, 1977b; Dentler, 1978). Egg dynein has certain enzymatic characteristics in common with this enzyme, namely, nucleotide specificity and sensitivity to vanadate inhibition. There also seems to be some evidence that egg dynein may be a hydrophobic protein or may be associated with hydrophobic moieties such as membrane domains. When prepared in the presence of detergent, egg dynein shows a marked tendency to shift its sedimentation coefficient to lower values and concomitantly to lose its ATPase properties. This may represent a conformational change due to detergent association. Alternatively,
376
DEVELOPMENTAL BIOLOGY
there may be a dissociation of the two different-sized polypeptide chains which seem to comprise the enzyme, resulting in destruction of the active site. It is tempting to speculate that within the egg there exists a membrane-dyneinmicrotubule interaction, analogous to that found in the ciliary membrane and suspected in nerve cell axons. Figure 7 shows three possible ways that such a system could support the transport of membranated organelles along microtubule pathways.
VOLUME 74,198O
This study was supported by USPHS Grants GM 20,644 and GM 21,040 to Dr. R. E. Stephens. The study was submitted to the Graduate School of Arts and Sciences of Brandeis University in partial fulfiiment of the requirements of the degree Doctor of Philosophy. The author is grateful to Dr. R. E. Stephens for his helpful suggestions and discussions during the course of this work, and for comment on the manuscript. The author also thanks E. A. Morales for the artistic representation in Fig. 7. Note added in proof. After the submission of this manuscript, it was determined that the two high molecular weight egg dynein polypeptides are carbohydrate containing, by the periodic acid-Schiff reagent (PAS) staining method for polyacrylamide gels, and therefore may be membrane associated. While this may raise some doubt as to the molecular weight of these polypeptides, as determined by SDS-PAGE, the data support the model shown in Fig. 7b, where the dynein ATPase is associated with vesicle membranes. REFERENCES
b
FIG. 7. Three models for cytoplasmic vesicle transport. (a) Cytoplasmic dynein is associated with microtubules and moves vesicles along in “conveyer belt” fashion, by cyclic interaction with tubulin in the vesicle membranes. (b) Cytoplasmic dynein is associated with vesicle membranes. The dynein interacts cyclically with microtubules, and vesicles roll along stationary microtubules via the dynein bridges. (c) Dynein bridges are associated with both vesicles and microtubules. Vesicles move actively as in (b) and passively as in (a).
AMOORE, J. E. (1963). Non-identical mechanisms of mitotic arrest by respiratory inhibitors in pea root and sea urchin eggs. J. Cell Biol. l&555-567. AUCLAIR, W., and SEIGEL, B. W. (1966). Cilia regeneration in the sea urchin embryo: Evidence for a pool of ciliary proteins. Science 154, 913-915. BRYAN, J. (1974). Biochemical properties of microtubules. Fed. Proc. 33, 152-157. BURNS, R. G. (1976). Some properties of the high molecular weight proteins of sea urchin sperm tail flagella and of re-assembled brain microtubules. In “International Symposium on Contractile Systems in Non-Muscle Tissues” (S. V. Perry, A. Margreth, and R. S. Adelstein, ed.), pp. 215-227. Elsevier, Amsterdam. BURNS, R. G., and POLLARD, T. D. (1974). A dyneinlike protein from brain. Fed. Eur. Biochem. Sot. Lett. 40, 274-280. BURNSIDE, B., KOZAK, C., and KAFATOS, F. C. (1973). Tubulin determination by an isotope dilution-vinblastine precipitation method. The tubulin content of Spisula eggs and embryos. J. Cell Biol. 59,755769. DENTLER, W. L. (1978). Isolation and characterization of Tetrahymena ciliary membrane. J. Cell Biol. 79, 284a. DENTLER, W. L., PRATT, M. M., and STEPHENS, R. E. (1978). Microtubule-membrane interactions in cilia. J. Cell Biol. 79, 290a. EPEL, D. (1975). The program of and mechanisms of fertilization in the echinoderm egg. Amer. Zool. 15, 507-522. GASKIN, F., KRAMF.R, S. B., CANTOH, C. R., ADEI~STF,IN, R., and QH~ANSKI, M. I,. (1974). A dynein-
M. M. PRATT
Dynein ATPase
like protein associated with neurotubules. Fed. Eur. Biochem. Sot. Lett. 40, 281-286. GELFAND, V. I., GYOEVA, F. R., ROSENBLAT, V. A., and SHANINA, N. A. (1978). A new ATPase in cytoplasmic microtubule preparations. Fed. Eur. Biothem. Sot. Lett. 88,197-200. GIBBONS, I. R. (1966). Studies on the adenosine triphosphatase activity of 14 S and 30 S dynein from cilia of Tetrahymena. J. Biol. Chem. 241, 55905596. GIBBONS, I. R., COSSON, M. P., EVANS, J. A., GIBBONS, B. H., HOUCK, B., MARTINSON, K. H., SALE, W. S., and TANG, W.-J. Y. (1978). Potent inhibition of dynein adenosine triphosphatase and of the motility of cilia and sperm flagella by vanadate. Proc. Nat. Acad. Sci. USA 75,2220-2224. GIBBONS, I. R., and FRONK, E. (1972). Some properties of bound and soluble dynein from sea urchin sperm flagella. J. Cell Biol. 54, 365-381. GIBBONS, I. R., and FRONK, E. (1979). A latent adenosine triphosphatase form of dynein 1 from sea urchin sperm flagella. J. Biol. Chem. 254, 187-196. GIBBONS, I. R., FRONK, E., GIBBONS, B. H., and OGAWA, K. (1976). Multiple forms of dynein in sea urchin sperm flagella. In “Cell Motility” (R. D. Goldman, Pollard, and Rosenbaum, eds.), Vol. C, pp. 915-932. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. HEPLER, P. K., MCINTOSH, J. R., and CLELAND, S. (1970). Intermicrotubule bridges in mitotic spindle apparatus. J. Cell Biol. 45, 438-444. HF,SLOP, J. (1975). Axonal flow and fast transport in nerves. Advan. Comp. Physiol. Biochem. 6, 75-163. HIRAMOTO, Y. (1974). Mechanisms of ciliary movements. In “Cilia and Flagella” (M. A. Sleigh, ed.), pp. 177-196. Academic Press, New York/London. HYNIXS, R. O., and GROSS, P. R. (1970). A method for separating cells from early sea urchin embryos. Derselop. Biol. 21, 383-402. KINCAI~, H. L., JR., GIBBONS, B. H., and GIBBONS, I. K. (1973). The salt-extractable fraction of dynein from sea urchin sperm flagella: An analysis by gel electrophoresis and by adenosine triphosphatase activity. J. Supramol. Struct. 1, 461-470. KOHAYASHI, Y., OGAWA, K., and MOHRI, H. (1978). Evidence that the Mg”-ATPase in the cortical layer of sea urchin eggs is dynein. Exp. Cell Res. 114, 285-292. KoRP*‘, E. D. (1978). Biochemistry of actomyosin-dependent cell motility (a review). Proc. Nat. Acad. Sci. USA 75, 588-599. LINCK, It. W. (1973). Chemical and structural differences between cilia and flagella of the lamellibranch mollusc, Aequipecten L-radians. J. Cell Sci. 12, 951-981. LOWHY, 0. H., ROSEBROUGH, N., FARR, A. L., and RANDALL, H. J. (1951). Protein measurements with
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the folin phenol reagent. J. Biol. Chem. 193, 265275. MABUCHI, I. (1973). ATPase in the cortical layer of sea urchin eggs. Its properties and interaction with cortical protein. Biochem. Biophys. Acta 297, 317332. MCINTOSH, J. R., CANDE, W. Z., LAZARIDES, E., MCDONALD, K., and SNYDER, J. (1976). Fibrous elements of the mitotic spindle. In “Cell Motility” (R. D. Goldman, Pollard, and Rosenbaum, eds.), Vol. C, pp. 1261-1272. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. MARTIN, J. B., and DOTY, D. M. (1949). Determination of inorganic phosphate. Modification of isobutyl alcohol procedure. Anal. Chem. 21, 965-967. MIKI, T. (1963). ATPase activity of the egg cortex during the first cleavage of sea urchin eggs. Exp. Cell Res. 33, 575-578. MOHRI, H., MOHRI, T., MABUCHI, I., YAZAKI, I., SAKAI, A., and OGAWA, K. (1976). Localization of dynein in sea urchin eggs during cleavage. Develop. Growth Differ. 18, 391-298. MOOSEKER, M. S., and TILNEY, L. G. (1973). Isolation and reactivation of the axostyle. Evidence for a dynein-like ATPase in the axostyle. J. Cell Biol. 56, 13-26. NAGAYAMA, A., and DALES, S. (1970). Rapid purification and the immunological specificity of mammalian microtubular paracrystals possessing an ATPase activity. Proc. Nat. Acad. Sci. USA 60,464-471. OGAWA, K. and GIBBONS, I. R. (1976). Dynein 2: A new adenosine triphosphatase from sea urchin sperm flagella. J. Biol. Chem. 251,5793-5801. OKAZAKI, K. (1975). Spicule formation by isolated micromeres of the sea urchin embryo. Amer. Zool. 15, 567-581. PETZEI,T, C. (1972). Ca++ activated ATPase during the cell cycle of the sea urchin S. purpuratus. Exp. Cell Res. 70, 333-339. PETZELT, C., and VON LEDEBUR-VILLIGER, M. (1973). Cati stimulated ATPase during the early development of parthenogenetically activated eggs of the sea urchin Paracentr0tu.s lividus. Exp. Cell Res. 81, 87-94. POLLARD, T. D., and KORN, E. D. (1972). The “contractile” proteins of Acanthamoeba castellanl. Cold Spring Harbor Symp. Quant. Biol. 37, 573-583. POLLARD, T. D., and KORN, E. D. (1973). Acanthamoeba myosin. I. Isolation from Acanthamoeba castellani of an enzyme similar to muscle myosin. cJ. Biol. Chem. 248,4682-4690. PRATT, M. M. and STEPHENS, R. E. (1978). Dynein synthesis in sea urchin embryos. ,J. Cell Biol 79, 300a. PRATT, M. M., and STEPHENS, R. E. (1977). Evidence for a cytoplasmic dynein. Biophys. J. 17, Z68a. RAFF, R. A., GREENHOUSE. G., GROSS, K., and GROSS,
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P. R. (1971). Synthesis and storage of microtubule proteins by sea urchin embryos. J. Cell Biol. 50, 516-527. SAKAI, H., MABUCHI, I., SHIMODA, S., KURIYAMA, R., OGAWA, K., and MOHRI, H. (1976). Induction of chromosome motion in the glycerol isolated mitotic apparatus. Nucleotide specificity and effects of antidynein and myosin sera on the motion. Develop. Growth Differ. l&211-219. SALMON, E. D., and JENKINS, R. (1977). Isolated mitotic spindles are depolymerized by fl calcium and show evidence of dynein. J. Cell Biol. 75,295a. SHAPRIO, A. L., VINUELA, E., and MAISEL, J. V., JR. (1967). Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide
gels. Biochem. Biophys. Res. Commun. 28.815-820. STEPHENS, R. E. (1970). Thermal fractionation of outer fiber doublet microtubules into A- and Bsubfiber components: A- and B-tubulin. J. Mol.
Biol. 47,353-363. STEPHENS, R. E. (1972). Studies on the development of the sea urchin Strongylocentrotus droebachiensis. I. Ecology and normal development. Biol. Bull. 142,489-504. STEPHENS, R. E. (1975). High-resolution preparative SDS-polyacrylamide gel electrophoresis: Fluorescent visualization and electrophoretic elution-concentration of protein bands. Anal. B&hem. 65,
369-379.
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STEPHENS, R. E. (1977a). Differential protein synthesis and utilization during cilia formation in sea urchin embryos. Develop. Biol. 61, 311-329. STEPHENS, R. E. (197713). Major membrane protein differences in cilia and flagella: Evidence for a membrane associated tubulin. Biochemistry 16, 20472058. STEPHENS, R. E. (1978). Primary structural differences among tubulin subunits from flagella, cilia, and the cytoplasm. Biochemistry 17,2882-2891. STEPHENS, R. E., and EDDS, K. T. (1976). Microtubules: Structure, chemistry and function. Physiol.
Rev. 56,709-777. TYLER, A., and SPEIGEL, M. (1956). Elevation and retraction of the fertilization membrane of echinoderm eggs fertilized in papain solutions. Biol. Bull. 110,196-200. WEBER, K., and OSBORN, M. (1969). The reliability of molecular weight determinations by dodecyl-sulfate polyacrylamide gel electrophoresis. J. Biol. Chem. 244,4406-4442. WEISENBERG, R., and TAYLOR, E. W. (1968). Studies on ATPase activity of sea urchin eggs and the isolated mitotic apparatus. Exp. Cell Res. 53, 372384. WILT, F. H., SAKAI, H., and MAZIA, D. (1967). Old and new protein in the formation of the mitotic apparatus in cleaving sea urchin eggs. J. Mol. Biol. 27, 1-7.
BIOLOGY
The Identification
74,364-378 (1980)
of a Dynein ATPase in Unfertilized
Sea Urchin Eggs
M. M. PRATT* Department of Biology, Brandeis University, Waltham, Massachusetts 02154, and Marine Biological Laboratory Woods Hole, Massachusetts 02543 Received April 9, 1979; accepted July 11, 1979 A cytoplasmic dynein ATPase has been identified in three species of unfertilized sea urchin eggs, Strongylocentrotus droebachiensis, S. purpuratus, and Arbacia punctulata. The enzyme was partially purified by sucrose gradient density centrifugation, and its polypeptide chain weight and composition were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein has enzymatic characteristics in common with flagellar dynein. It is activated nearly equally by Mg2+ and Ca’+, has no activity in the presence of K’ and EDTA, shows a specificity for ATP over other nucleoside triphosphates, and is inhibited by vanadate anion. On SDS-PAGE, the enzyme shows two major bands at 320,000 and 385,000 daltons, comigrating with certain ciliary and flagellar dynein polypeptides. The enzyme, given the name “egg dynein,” constitutes 2 to 4% of the total cell protein in the unfertilized egg and maintains this high value from fertilization through the late blastula stage. It appears to be equally distributed throughout the embryo at the 16-cell stage. Possible functions of egg dynein are discussed and models for dynein-microtubule mediated movements within the cytoplasm are presented. INTRODUCTION
There has been some evidence and much speculation that microtubule-dynein ATPase interactions, analogous to those seen in cilia and flagella, might be involved in cytoplasmic motile mechanisms (cf. Stephens and Edds, 1976). Tubu!in and microtubules have been found in a wide variety of cell types, but only rarely has a dynein-like ATPase been identified (Mooseker and Tilney, 1973; Burns and Pollard, 1974; Gaskin et az., 1974). As early as 1963, Miki reported the existence of a Mg2+-ATPase in echinoderm eggs. In 1968, Weisenberg and Taylor isolated a soluble 13 S Mg”-ATPase from Arbacia eggs. They speculated that it was a ciliary precursor based on its sedimentation coefficient and nucleotide and ionic specificities. Later, Mabuchi (1973) extracted a 0.6 M KCl-soluble ATPase from isolated cortices of sea urchin eggs, which has since * Present address: Department of Anatomy, Harvard Medical School, 25 Shattuck Street, Boston, Mass. 02115.
been shown to have characteristics in common with dynein (Kobayashi et al., 1978). This report refines and extends the initial observations of Weisenberg and Taylor (1968). A 12-14 S ATPase has been isolated from three species of echinoderms and has been identified as a cytoplasmic dynein on the basis of sedimentation coefficient, comigration on SDS-polyacrylamide gels with certain axonemal dynein polypeptides, and characteristic enzymatic properties. Further, it has been shown to constitute a significant portion of the total cell protein throughout early development. Since it was first isolated from unfertilized eggs, it will be referred to here as “egg dynein.” A preliminary account of some of this work has appeared in abstract form (Pratt and Stephens, 1977). MATERIALS
Organisms: Obtaining dling Embryos
Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
Gametes and Han-
Strongylocentrotus droebachiensis and Arbacia pun&data were obtained from the supply department of the Marine Bio364
OOlZ-1606/80/020364-15$0.200/O
AND METHODS
M. M. PRATT
Dynein ATPase
logical Laboratory, Woods Hole, Massachusetts, and held in tanks of running seawater at 10 and 20X’, respectively. S. purpuratus was obtained from Pacific Bio-Marine, Venice, California, and held in running seawater tanks at 10°C. Gametes were obtained by injection of 25 ml of 0.5 M KC1 into the urchin coelom. Sperm were shed into a dry Syracuse dish. Eggs were shed into Millipore-filtered seawater (MPFSW)’ and washed twice by settling. Eggs suspended in MPFSW were fertilized by addition to an equal volume of MPFSW containing 5-10 drops of diluted sperm (1 drop of dry sperm to 10 ml MPFSW). The embryos were grown in MPFSW in culture dishes in an incubator set at 8°C for S. droebachiensis, 14°C for S. purpuratus, and 23°C for Arbacia punctulata. For experiments of greater than 24 hr duration, sulfadiazine was added to S. droebachiensis cultures to a final concentration of 0.01%. Preparation
of Egg Dynein
Egg dynein was prepared according to the method of Weisenberg and Taylor (1968) with slight modifications. Typically, 3-5 ml of packed dejellied eggs were suspended in 2-3 vol of an isotonic buffer containing 1 M sucrose, 5 mM MgC12, 50 n&I Tris-HCl, pH 8.0, 1 m&I EDTA (SMTE). Unless otherwise stated, all preparations were carried out at 4°C. The eggs were broken by 8-10 passes of a ground glass homogenizer. The homogenate was spun at 100,OOOg(35,000 rpm, Type 40 rotor) for 2 hr. The pellet was retained on ice. The supernatant was dialyzed against 50 mJ4 Tris-HCl, pH 8.0, 1 mM EDTA for 510 hr. Samples of the whole homogenate, supernatant, and pellet were assayed for protein content and ATPase activity. An ’ Abbreviations used: EGTA, ethylene glycol bis (/3-aminoethyl ether) N,N’-tetraacetic acid; MA, mitotic apparatus; MPFSW, Millipore-filtered seawater; OD-cm, optical density x centimeters; SMTE, 1 M sucrose, 5 mI14 MgCI, 50 mh4 Tris-HCl, pH 8.0, 1 mM EDTA; V(V). vanadate anion in the pentavalent state.
365
in Sea Urchin Eggs
appropriate volume of the dialyzed supernatant was layered onto a 12- or 56-ml 520% (w/v) sucrose gradient, prepared in 50 mJ4 Tris-HCl, pH 8.0, 50 n&I NaCl, and spun for 12 or 16 hr at 200,OOOg(35,000 rpm, SW 40 rotor) or 24 hr at 100,OOOg(25,000 rpm, SW 25.2 rotor). Fractions were collected and the absorbance at 280 nm (A& was monitored. Appropriate fractions were assayed for protein content and ATPase activity. Preparation of Cilia, nemal Dynein
Flagella,
and Axo-
Cilia were prepared from embryos cultured as described above by suspending the washed blastulas in hypertonic seawater (Stephens, 1977a). Deciliated embryos were sedimented in a hand centrifuge and resuspended in MPFSW if regeneration of cilia was desired. The cilia were pelleted from the hypertonic supernatant at 10,OOOgfor 10 min in a Sorvall HS4 rotor, and washed once in 10 mJ4 Tris-HCl, pH 8.0. Whole cilia were demembranated by incubation in 0.25% Nonidet P-40, 30 miV Tris-HCl, pH 8.0, 3 mJ4 MgCle, 0.1 mM EDTA (NTME) , and collected by centrifugation at 10000 g for 15 min (9000 rpm, Sorvall HS4 rotor). Sperm flagella were prepared according to the method of Stephens (1970). Tails were sheared from heads by 20 rapid passes of a ground glass homogenizer. Flagella were isolated by differential centrifugation and demembranated in 10 vol of NTME without an incubation period. Axonemes were collected as described above. Dynein was prepared by dialysis of flagellar axonemes against 1 miI4 Tris-HCl, pH 8.0, 0.1 mM EDTA, 0.05% mercaptoethanol according to the method of Gibbons and Fronk (1972). Separation
of Blastomeres
Preparation of blastomeres was modified from the procedure of Hynes and Gross (1970). Approximately 0.5 ml of S. purpuratus eggs were fertilized as above. Fertili-
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DEVELOPMENTAL BIOLOGY
zation membrane formation was prevented by the presence of 0.2% papain and 0.04% cysteine (Tyler and Speigel, 1956). Embryos were cultured as above until the 16cell stage. They were spun down and washed twice in a 19:l mixture of 0.5 M NaCl and 0.5 M KC1 containing 16 r&f Tris-HCl, pH 8.0 (19:l Tris), using a hand centrifuge. The embryos were suspended in 5-10 ml fresh 19:l Tris and gently pipetted and swirled until the cells separated from one another (approximately 10 mm). Five milliliters of the cell suspension was layered onto a .30-ml gradient of 5-15% Ficoll (w/v) in 19:l Tris. The gradients were spun for 1 min at 1250rpm (Sorvall HS4 rotor). Crude fractions were collected from the top using a Pasteur pipet. Each was monitored for cell content and pure meso-, macro-, and micromeres were recovered by centrifugation. The pellets of cells were solubilized for gel electrophoresis as outlined below.
VOLUME 74,198O
flagellar dynein and 45 min with the egg enzyme. Assays were always run for less than these maxima, the absolute times being adjusted to the amount of enzyme used so that the ATzOremained in an easily quantitated range. For studies of ATPase inhibitors, oligomycin was stored at -20°C as a 25-mg/ml solution in 100%ethanol. Ouabain was prepared as a 10e3 M solution in deionized water, and stored at -20°C. Sodium vanadate (NaV03) was prepared as a 10 r&l4 stock solution in 10 n-&f Tris-HCl, pH 8.0, heated in boiling water for 1 min to dissolve, and stored at -20°C. Gel Electrophoresis and Densitometry
All samples for sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) were prepared in a sample buffer containing 2.5 mM Tris-glycine, pH 8.3, 1% SDS, 1% mercaptoethanol, 5% glycerol, 0.05% bromphenol blue (Bryan, 1974; SteDetermination of Protein Content and phens, 1975), and 50 pg/ml phenyhnethylATPase Activity sulfonyl fluoride (PMSF). Samples preProtein content was determined by the pared from whole unhatched embryos were method of Lowry et al. (1951), standardized spun at 10,000 rpm (Sorvall HS4 rotor) to pellet the fertilization membranes which with bovine serum albumin (BSA). Adenosine triphosphatase activity was did not solubilize in sample buffer. SDSassayed at 20°C in 50 mM Tris-HCl, pH PAGE was carried out either with a phos8.0, 0.1 M KCl, 5 mM cation, and 1 mM phate buffer system (Shapiro et al., 1967) nucleotide. Deionized water was used and a 3% polyacrylamide gel, or with a throughout. When Mg’+-ATPase was Tris-glycine system (Bryan, 1974; Stephens, 1975) using a 5% gel. Gels were quanmeasured, the mix also contained 1 r&f titatively stained by the method of Weber EGTA. For detection of myosin-like ATPase, 5 mM EDTA was included in lieu of and Osborn (1969) in 5 vol of 0.1% Coomasany cation. Typically, the reaction was sie brilliant blue (R-250) or 0.25%fast green started by the addition of enzyme to one of dissolved in 50% methanol, 10% acetic acid for 2 hr at 45°C. Staining was continued in the mixtures described above. The reaction was stopped and inorganic phosphate gen- fresh stain for 2 hr at 45°C. The gels were erated was measured by the method of destained in 5% ethanol, 7.5% acetic acid. Pollard and Korn (1973), a modification of Stained gels were scanned either in a Joyce-Loebl MK III microdensitometer usthe original method of Martin and Doty (1949). For both flagellar and egg enzymes, ing white light or in an Ortec 4310 densiphosphate release was linear for the tometer using a green filter for Coomassie blue or a red filter for fast green. The area amount of enzyme added. At concentrations below 10 fig/ml for flagellar and 100 under the curve (in OD-cm) was measured pg/ml for egg dynein, the amount of phos- by planimetry and was compared with BSA phate generated was linear for 20 min with standards to determine the amount of pro-
M. M. PRATT
Dynein ATPase
367
in Sea Urchin Eggs
tein in various bands. For purposes of calculation, all overlapping peaks were assumed to be Gaussian. RESULTS
Purification
of Egg Dynein
Approximately 30-40s of the total Mg*+ATPase activity of unfertilized eggs from the sea urchin Strongylocentrotus droebachiensis is readily soluble and can be found in the high speed supernatant of eggs broken in SMTE. The remainder of the activity stays in the pellet and is not purified further. The soluble activity can be partially purified by sucrose density gradient centrifugation. To allow visualization of highly aggregated material and for comparison with standard gradients used in the purification of axonemal dynein (Gibbons, 1966), a relatively steep 5-20s gradient was used. When resolved on such a 5-20s sucrose gradient, the dialyzed egg supernatant separates into two major protein peaks-a small peak of aggregated material near the bottom of the gradient and a large, broad peak at the top. Flagellar dynein, extracted from axonemes by low ionic strength dialysis, was run in a parallel gradient as a 14 S marker (Gibbons and Fronk, 1972). The sea urchin egg ATPase duplicated the distribution of flagellar dynein ATPase. The ATPase activity did not coincide with either major protein peak but, instead, formed a nearly symmetrical peak centered at 14 S (Fig. la). Identical results were obtained with the sea urchin S. purpuratus. When analyzed on SDS-PAGE, the 14 S peak appears highly enriched in two high molecular weight polypeptides, and contains lesser amounts of lower molecular weight proteins (Fig. lb). The two high molecular weight bands have been named egg dynein. Significant ATPase activity first appears along with these two heavy bands in fractions 14 and 16. In addition, these high molecular weight bands repre-
2
18
28
b FIG. 1. Sucrose gradient analysis of S. droebachiensis egg extract. (a) 5-20s sucrose (w/v) in 10 m44 Tris-HCl, pH 8.0, 10 m&f NaCl, 1 rmI4 EDTA. There are two protein peaks and one peak of ATPase activity at fraction 18. Sixteen-hour gradient run. (b) 3% polyacrylamide gels in phosphate buffer of evennumbered gradient fractions, 2-28. Note the enrichment of the two highest molecular weight proteins and some lower molecular weight species in fractions 14 20, corresponding to the ATPase peak.
sent the major portion of the protein in fractions 16-20. Upon centrifugation of homogenized eggs and analysis of the fractions on polyacrylamide gels, the protein in these two bands represents a larger percentage of the total protein in the supernatant than in the pellet. In contrast, the total ATPase is greater in the pellet, and thus there seems to be a distinct enrichment of egg dynein in the soluble extract. It can be noted in Fig. 1 that the absolute
368
DEVELOPMENTALBIOLOGY
amounts of protein in these two high molecular weight bands do not correspond directly with the ATPase activity peak. Fraction 20, especially, contains a large amount of egg dynein protein but lacks a correspondingly high ATPase activity. This apparent discrepancy, strictly reproducible in all egg dynein gradient preparations and often seen in gradient purifications of flagellar dynein also, is best explained by the fact that dynein is a multisubunit enzyme (Gibbons, 1966; Gibbons et al., 1976; Gibbons and Fronk, 1979). At S values lower than 14 S (found closer to the top of the gradient) a portion of the enzyme may be partially dissociated, rather than existing as an intact 14 S complex. Thus, gel electrophoresis would indicate large amounts of the enzyme’s constituent proteins, but the ATPase activity would be low, since the proteins are not in the form of an active complex. Gibbons and Fronk (1979) have found that the most active form of the enzyme is the 14 S molecule. Low ATPase activities toward the top of the gradient may also be due to the fact that the two high molecular weight bands comprise a lower percentage of the total protein in these fractions. Another possibility is that the lower molecular weight proteins appearing in later fractions may inhibit the enzyme activity. The two high molecular weight bands are undoubtedly the major components of egg dynein, but sucrose gradient preparation provides only minimal separation of egg dynein from some other proteins. Further purification of egg dynein away from these lower molecular weight components has proved rather difficult. Ammonium sulfate fractionation was unsuccessful, precipitating the bulk of the protein, egg dynein, and contaminants, between 50 and 60% saturation. Gel chromatography on Sephadex G-200 or Bio-Gel Al5 (Burns, 1976) also failed to purify the high molecular weight egg dynein from certain low molecular weight proteins, suggesting
VOLUME 74.1980
either that these proteins exist as aggregates approximating the size of native egg dynein, or that they are tightly associated with the enzyme, perhaps as subunits of the ATPase. Purification of the enzyme on DEAE-Sephadex was attempted with only moderate success. It should be pointed out that axonemal dynein has not yet been purified to homogeneity (Gibbons et al., 1976; Ogawa and Gibbons, 1976). It has recently been hypothesized that certain low molecular weight proteins, which were earlier thought to be contaminants, may be part of the enzyme complex (Gibbons and Fronk, 1979).
Enzymatic Properties of Egg Dynein When the major fractions of the sucrose gradient within the 14 S ATPase peak are combined and assayed under a variety of conditions, the enzymatic activity shows a marked similarity to that of axonemal dynein. Table 1 illustrates a comparison of axonemal and egg dynein under conditions which examine the unique character of dynein ATPases. The data reported are from one preparation of egg and flagellar dynein but similar data were obtained from other experiments. Absolute specific activities measured never varied more than 25% from the published values and the ratios between the specific activities under different conditions had a variance of 10%. Egg dynein exhibits the low ratio of Ca”’ to Mg*‘-ATPase (defined as the Ca*+/Mg” ratio) characteristic of ciliary dynein (Gibbons, 1966) as well as the absence of activity in K+ and EDTA. However, the Ca’+/Mg*+ ratio is even lower than that of flagellar dynein. Egg dynein is also less discriminating between ATP and other nucleotides (GTP and ITP). In addition, the specific activity of egg dynein is approximately lo-fold lower than that of axonemal dynein. This difference is not surprising when the energy requirements of a flagellum are compared to those of a sea urchin egg. Estimates have been
M. M. PRATT
Dynein ATPase TABLE
369
in Sea Urchin Egg.9
1
SPECIFIC ACTIVITIES OF FLACELLAR DYNEIN AND EGG DYNEIN UNDER VARYING ASSAY CONDITIONS” Mg’+-ATPase (‘i inMg’+-GTPase K+-EDTA Mg”‘-ATPase Ca”‘-ATPase hibiin2pM activity activity activity ATPase actionl NaVO j tivity L9
purpuratus flagellar dynein S. purpuratus egg dynein S. droebachiensis flagellar dynein S. droebachiensis egg dynein
1.50 f 0.08
0
0.019 f 0.001
1.13 f 0.06
(461
0.257 f 0.013
0.170 + 0.008
0
0.010 +- 0.001
0.232 f 0.01 I
(IO)
1.40 + 0.01
1.31 -t 0.07
0.012 f 0.001
0.983 + 0.049
IX))
0.399 + 0.020
0.209 + 0.010
0.248 (0.120 0.020 (0.090
0.296 k 0.015
(26)
--2.10-t
0.11
0.007 f 0
f + + +
0.012 0.006)” 0.001 0.005)”
” 14 S flagellar dynein and egg dynein were assayed under conditions shown. The assay medium was 50 mM Tris-HCl, pH 8.0, 0.1 M KCl, 5 mM cation or EDTA, 1 mM nucleotide. Specific activities are expressed as micromoles inorganic phosphate released/milligram dynein x minutes f 5% instrumental error. Details of the assay method can be found in the text. The actual amount of dynein heavy chain present was determined by gel densitometry as described under Results for egg dynein quantitation. ” These values are for Mg”‘-ITPase activity.
made that sperm flagella expend 3-12 x lo-’ erg/set/cell when beating at lo-40 Hz (cf. Hiramoto, 1974). Sea urchin embryos do not generate such obvious motility, but chromosome movement during cell division can be used as an example of an energyrequiring event in these cells. Amoore (1963) has calculated that all of anaphase in pea endosperm requires only 0.8 x lo-l3 erg/set/cell, a value six to seven orders of magnitude lower than that for flagella. Furthermore, the specific activity for egg dynein reported here is lo-fold higher than the original value reported by Weisenberg and Taylor (1968)) and 5- to lo-fold higher than the values reported for a dynein-like enzyme found in the brain (Burns and Pollard, 1974; Gaskin et al., 1974). Gibbons et al. (1978) have shown recently that vanadium in the pentavalent state, V(V), is a potent inhibitor of flagellar dynein ATPase. At 100 @4 NaV03, the Mg’+-ATPase activity of S. droebachiensis flagellar dynein is nearly 75% inhibited, while nearly 1 n&I NaV03 is required for 75% inhibition of egg dynein ATPase. A better comparison of the effect of vanadate on the two enzymes can be made at 2 fl NaVO:j, where both are inhibited less than 50%. At 2 fl NaVO:s, the inhibition of S. purpuratus egg dynein is only lo%, but for
S. droebachiensis it is 26%, compared to 46 and 30% for the respective flagellar enzymes. These and other differences seen in the egg dynein are similar to those recently found for a dynein associated with ciliary membranes (Dentler et al., 1978). To rule out contamination by Na’-K’ or mitochondrial ATPases, the Mg”‘-ATPase activity of egg dynein was assayed in the presence of two different inhibitors-l, 5, and 10 pg/ml oligomycin, or lo-” and lo-” M ouabain. These agents are known to inhibit H’- or Na’-K’-ATPases, respectively, at these concentrations (Gelfand et al., 1978). The Mg”‘-ATPase activities of both egg and flagellar dynein were unaffected or even stimulated, rather than inhibited by these agents. When an effect was seen, it was considerably less in the case of egg dynein (Table 2). SDS-Polyacrylamide
Gel Electrophoresis
Axonemal dynein is characterized by an extremely large minimal polypeptide chain weight and a specific band profile on SDSpolyacrylamide gels. Figure 2 illustrates the typical pattern obtained for ciliary versus flagellar dyneins on 5% Tris-glycine and 3% phosphate gels. The four bands appearing on 3% gels of flagellar axonemes have been designated C, A, D, and B as shown (Gib-
370
DEVELOPMENTALBIOLOGY
bons et al., 1976). A similar nomenclature has been adopted here for ciliary dyneins, although the banding pattern is clearly different (see figure). By comparison with molecular weight standards, including myosin heavy-chain dimer from scallop myofibrils, the axonemal dyneins have molecular weights ranging from 335,000-385,000. Using a variety of other gel buffer systems, the same values are obtained, basically confuming those published by Burns and Pollard (1974) rather than higher values reported previously (Kincaid et al., 1973; Linck, 1973). Egg dynein contains two of these characteristically high molecular weight polypeptide chains. Figure lb shows 3% gel patterns of fractions from the corresponding gradient. Fractions within the peak of ATPase activity of both S. purpuratus and S. droebachiensis show a distinct enrichment of electrophoretic bands with molecular weights of 320,000 and 385,000. The upper band comigrates with flagellar dynein C and the lower band runs just below dynein B of both flagellar and ciliary dynein of the same species (Fig. 3). (Preparation of egg dynein from Arbacia punctulata yields the same two bands in different proportions, along with a higher molecular weight band.) TABLE 2 ACTIVITIES OF FLAGELLARDYNEIN AND EGG DYNEIN IN THE PRESENCEOF INHIBITORSOF MITOCHONDRIALAND Na+-K+-ATPases” Percentage of Mg’+ATPase activity
Mg’+-ATPase +l pg/ml oligomycin +5 pg/ml oligomycin +lO pg/ml oligomycin + W5 M ouabain + 10m4M ouabain
Flagellar dynein
Egg dynein
100 140 146 154 163 168
100 100 141 100 100 147
a S. droebachiensti flagellar dynein and egg dynein were assayed for Mg’+-ATPase activity in the absence and presence of oligomycin or ouabain. Assay conditions were 50 mM Tris-HCl, pH 8.0,O.l M KCl, 5 mM MgC12,1 mM ATP, and the designated inhibitor.
VOLUME 74.1980
Dy=
LXP-
FC
FC
FIG. 2. Electrophoretic profiles of S. purpuratus cilia (C) and flagella (F). The gels on the left are 5% polyacrylamide in Tris-glycine buffer. The dynein doublet (Dy) and tubulin (Y-and P-chains are labeled. The gels on the right are 3% polyacrylamide in phosphate buffer. The four dynein polypeptides are labelled (C, A, D, and B), as is the tubulin band (T). Note that the tubulin subunits are not separated on this gel system.
Each egg dynein peak fraction also contains lower molecular weight polypeptides which may represent the medium weight dynein components recently identified by Gibbons and Fronk (1979). Stoichiometry Once identified, the two polypeptides characteristic of the egg dynein ATPase region of the gradients are easily visualized on SDS-polyacrylamide gels of whole eggs and embryos. The amount of egg dynein in whole eggs could therefore be quantitated using gel electrophoresis and densitometry. A similar method was used to estimate the amount of actin in Acanthamoeba castelZani (Pollard and Kern, 1972).
M. M. PRATT
C
C+P
P
Dynein ATPase
F+P
F
a
c
371
in Sea Urchin Eggs
c+w
P
F+P
F b
FIG. 3. Comigrations of egg dynein with ciliary and flagellar dynein. (a) The species is S. purpuratus. From left to right, the 3% polyacrylamide gels in phosphate buffer are cilia (C), cilia + the sucrose gradient ATPase peak fraction from an egg dynein preparation (C+P), the same ATPase peak fraction (P), flagella + the same ATPase peak fraction (F+P), and flagella (F). (b) The species is S. droebachiensis. From left to right the 39 polyacrylamide gels in phosphate buffer are cilia (C), cilia + a whole embryo preparation (C+W), the sucrose gradient ATPase peak fraction from an egg dynein preparation (P), flagella + the same ATPase peak fraction (F+P), and flagella (F).
Varying amounts of bovine serum albumin were run on gels identical to those used for quantitation and were stained with either Coomassie blue or fast green as described under Materials and Methods. The stained gels were scanned in a densitometer and the peak areas were determined for the known amounts of BSA. Figure 4a shows a plot of peak areas obtained for l-10 pg of BSA loaded. The function is linear within this range. For quantitation of egg proteins, whole embryos were solubilized at various intervals from fertilization through hatching. Samples of the whole embryos were run on 5% polyacrylamide gels and stained as previously described. Figure 4b shows a densitometer trace of such a gel. The amount
of protein in the egg dynein bands was determined by comparison of the area under the idealized Gaussian curve with the peak areas obtained for BSA. A similar calculation was made for total egg protein by integrating the area under the entire curve. The percentage of total egg or embryo protein represented by egg dynein was calculated by dividing the micrograms of protein in the two egg dynein bands by the micrograms of total cell protein and multiplying by 100. Often, the amount of protein in the egg dynein bands was measured on 3% gels loaded with an equal volume of sample. These allowed better separation and therefore quantitation of the bands. Using this method, it can be established that the two high molecular weight egg
372
DEVELOPMENTAL
BIOLOGY
VOLUME 74,198O
FIG. 4. Stoichiometric analysis of egg dynein by gel electrophoresis. (a) Graph of peak areas (in OD-cm), obtained by densitometry and measured by planimetry, versus amounts of BSA run on polyacrylamide gels and stained with Coomassie blue or fast green. (b) Densitometer tracing of a 5% polyacrylamide gel pattern of a whole egg homogenate from S. droebachiensis. The egg dynein bands are shaded. The percentage of total protein in these bands was determined as described in the text.
dynein polypeptides represent 3.7 f 0.3% (n = 9) of the total Coomassie blue staining protein in embryos of S. purpuratus and 1.9 & 0.1% (n = 8) in S. droebachiensis. These are both surprisingly high figures and they remain nearly constant throughout early development, decreasing only slightly from fertilization through the swimming blastula stage. It is important to note that a large portion of the high molecular weight egg dynein found in the soluble egg supernatant, and isolated on sucrose gradients, sediments at 14 S. The total protein collected from a gradient preparation of S. droebachiensis egg dynein was estimated from 5% gels of the fractions, while the protein in the two high molecular weight bands was estimated from 3% gels of the fractions within the 14 S peak. The egg dynein polypeptides within the 14 S peak represent 33.5% of the total protein on the gradient.
This value is even higher than that calculated for the percentage of egg dynein in whole eggs and reflects the enrichment of this protein in the egg supernatant. The distribution of egg dynein within the embryo was examined at the 16-cell stage, when three different cell types are easily separated on the basis of size (Hynes and Gross, 1970). These cells, mesomeres, macromeres, and micromeres, establish three different embryonic regions and have different fates in development (Okazaki, 1975). Each separated cell type, shown in Fig. 5, contains approximately the same amount of egg dynein (determined as a percentage of total protein), and the values fall well within those for whole eggs and embryos (Fig. 6; Table 3). DISCUSSION
A 14 S Mg”-ATPase has been found in echinoderm eggs. It has enzymatic proper-
M. M. PRATT
Dynein ATPase
373
in Sea Urchin Eggs
leted ATPase. The apparent Mg”‘-ATPase specific activity, based on actual amounts of egg dynein, is much greater in the pellet than the supernatant, possibly signifying a higher activity of the bound form. However, the considerable activity in the pellet is more likely due to other ATPases, Nat-K+, H’, Ca’+, or others, which undoubtedly exist in the egg (Petzelt, 1972: Petzelt and von Ledebun-Vilhger, 1973)) since the amount of egg dynein in the pellet is a lower percentage of the total protein than that in either the whole egg or the supernatant. The supernatant, on the other hand, seems to contain primarily egg dynein ATPase. Only one peak of Mg”‘-ATPase activity is seen on sucrose gradients, at 14 S, and the enzyme in this peak shows a
Fro. 5. Isolated mesomeres (a), macromeres (bl, and micromeres (cl of S. purpuratus. The cells were examined using phase contrast microscopy and were photographed immediately after removal from Ficoll gradients. The magnification is 364~. Scale bar = 10 km.
ties in common with axonemal dynein, comigrates with certain dynein bands on SDS-polyacrylamide gels, and is a prominent and constant cell component prior to fertilization and throughout early development. The enzyme, which is referred to as egg dynein, can be partially purified from a soluble extract of sea urchin eggs. However, a large portion of the Mg’+-ATPase activity remains in the high-speed pellet. This may be due to a tightly bound form of egg dynein since the pellet also contains substantial amounts of the two high molecular weight polypeptides identified as egg dynein. Weisenberg and Taylor (1968) showed that further extraction in various KC1 and MgCL concentrations did not solubilize the pel-
-ED -
Me
Ma
Mi
FIG. 6. Egg dynein in three types of blastomeres of S. purpuratus. The gels are 3% polyacrylamide in phosphate buffer. The samples shown are whole isolated mesomeres (Me), macromeres (Ma), and micromeres (Mi). The egg dynein bands are indicated.
374
DEVELOPMENTAL BIOLOGY
marked overall similarity to axonemal dynein. Egg dynein exhibits enzymatic specificities much like those of flagellar dynein, but in some cases they differ in degree. These differences between egg and flagellar dyneins may reflect the fact that the assay conditions chosen were those known to be optimal for axonemal dynein, but these may not be ideal for egg dynein. A detailed analysis of the unique ionic, nucleotide, and pH requirements of the enzyme must await further purification. The discrepancies may also be due to the association of lipids with the enzyme (see below). Another possibility is that the enzyme requires a specific activation, analogous to the phosphorylation of nonmuscle myosins (Korn, 1978), which cannot take place in the partially purified preparation. In addition to possessing the appropriate enzymatic characteristics, egg dynein also resembles axonemal dynein in terms of native molecular weight as judged from sedimentation coefficient. The 14 S enzyme seems to be primarily composed of two high molecular weight subunits. It also, however, may contain some lower molecular weight components. This seems more plausible in view of the report by Gibbons and Frank (1979) that native flagellar dynein may include three protein components of less than 150,000 daltons. A direct association of egg dynein and tubulin has not been shown but there is experimental evidence which suggests an association. Tubulin can be crystallized from unfertilized sea urchin eggs by incubating them with 0.01% vinblastine sulfate. A small amount of a high molecular weight band, corn&rating with axonemal dynein C, consistently appears on gels of the vinblastine-tubulin crystals (cf. Stephens, 1978). In accordance with this observation, Nagayama and Dales (1970) have reported ATPase activity associated with vinblastine crystals from mammalian brain. In addition, the isolated mitotic apparatus (MA)
VOLUME 74.1980 TABLE
3
PERCENTAGEOFTOTALCELLPROTEIN REPRESENTEDBYEGGDYNEIN IN S.purpuratus BLASTOMERES~ Cell type Whole embryo Mesomeres Macromeres Micromeres
Percentage of total protein in egg dynein 3.7 3.3 3.4 3.5
f f k f
0.3 0.4 0.8 0.5
“The three cell types were isolated as described under Materials and Methods. The percentage of total protein in the egg dynein bands was determined by gel densitometry, as described under Results. The whole embryo value is an average of 10 experimental points. The blastomere values are averages of three measurements for each cell type. The variance is expressed as standard error.
of S. droebachiensis, a major tubulin-containing organelle, shows Mg2+-ATPase activity and protein species which corn&ate with certain ciliary dynein bands (Salmon and Jenkins, 1977; Pratt and Otter, in preparation). An evaluation of the capacity of egg dynein for tubulin interaction has been reserved for future studies. Cosedimentation of egg dynein and brain tubules is being examined along with possible copolymerization of the two proteins. The amount of egg dynein found in embryos is considerably higher than the amount of dynein which the embryo incorporates into cilia. Rough calculations show that ciliary protein accounts for about 0.25% of total embryonic protein and dynein accounts for about one-fifth of this. Thus, ciliary dynein can be estimated to represent no more than 0.25% of total embryonic protein, even after allowing for pools of ciliary dynein five times as large as needed for one crop of cilia. This is a maximum amount which might be present, as deduced from protein synthesis studies (Auclair and Seigel, 1966; Pratt, manuscript in preparation). Egg dynein pools must, then, contain a minimum of 7- to 15-fold more protein than would be necessary for ciliary incorporation. The ratio of egg dynein to egg tubulin is
M. M.
PRATT
Dynein ATPase
also significant as compared to that of cilia. Estimates by vinblastine precipitation (Burnside et al., 1973) and colchicine binding (Raff et al., 1971) of the amount of tubulin present in echinoderm and other marine eggs range from 3 to 10% of the total cell protein. Even using the largest of these values, the dynein:tubulin ratio in the whole egg is no more than 1:3 in S. purpuratus and 1:5 in S. droebachiensis. These values are equal to or higher than the average 1:5 ratio seen in cilia. This is in sharp contrast to cytoplasmic actomyosin systems where the myosin:actin ratios are considerably lower than those found in striated muscle cells (Korn, 1978). It seems unlikely that egg dynein serves only as a precursor to ciliary dynein as was first supposed (Weisenberg and Taylor, 1968; Stephens, 1972). First, it does not exhibit as varied an electrophoretic pattern as ciliary dynein even in hatched embryos. Second, the amount of egg dynein in the unfertilized egg and in the ciliated blastula is far greater than the amount of dynein ever found in cilia, even if one considers multiple regenerations. Finally, pulse-labeling studies of dynein synthesis using [3H]leucine have shown clearly that embryos can and do synthesize all of the ciliary dynein polypeptide chains and incorporate them into cilia, but that very little synthesis of egg dynein occurs after fertilization (Pratt and Stephens, 1978). The mitotic apparatus (MA) may provide one possible location and function for cytoplasmic dynein. Mohri et al. (1976) have shown that a fluorescent antibody to a fragment of flagellar dynein stains the mitotic apparatus during cleavage of sea urchin embryos, The same antibody has been reported to stop chromosome movement in isolated sea urchin MAs (Sakai et al., 1976) and to inhibit the dynein-like ATPase activity associated with the egg cortex (Kobayashi et al., 1978). It has also been shown that isolated spindles contain ATPase activity characteristic of dynein though it is
in Sea Urchin Eggs
375
not clear whether this activity is due to cortical contamination (Salmon and Jenkins, 1977). A role for egg dynein in mitosis is consistent with reports that very little new protein is synthesized for incorporation into the mitotic apparatus (Wilt et al., 1967), since it exists in large quantities in unfertilized eggs. Further studies of the nature of the MA dynein-like ATPase are in preparation. It seems likely that if dynein does play a role in mitosis, it may be in late anaphase during pole to pole elongation, as cross bridges have been seen, most often, in thin sections of the interzone region (Hepler et al., 1970; McIntosh et al., 1976). Another reasonable possibility is that egg dynein is active in microtubule-associated transport of cytoplasmic constituents, most likely of membrane-bound organelles along microtubule “railways.” Such a mechanism has been proposed for axonal transport, clearly a microtubule-related phenomenon (Heslop, 1975). There has been some evidence for dynein associated with neuronal systems (Burns and Pollard, 1974; Gaskin et al., 1974). In one case (Gaskin et al., 1974), the enzyme isolated from brain seemed to be membrane associated. In addition, Dentler et al. (1978) have recently reported a “membrane dynein” associated with ciliary membranes, which also contain large amounts of tubulin (Stephens, 1977b; Dentler, 1978). Egg dynein has certain enzymatic characteristics in common with this enzyme, namely, nucleotide specificity and sensitivity to vanadate inhibition. There also seems to be some evidence that egg dynein may be a hydrophobic protein or may be associated with hydrophobic moieties such as membrane domains. When prepared in the presence of detergent, egg dynein shows a marked tendency to shift its sedimentation coefficient to lower values and concomitantly to lose its ATPase properties. This may represent a conformational change due to detergent association. Alternatively,
376
DEVELOPMENTAL BIOLOGY
there may be a dissociation of the two different-sized polypeptide chains which seem to comprise the enzyme, resulting in destruction of the active site. It is tempting to speculate that within the egg there exists a membrane-dyneinmicrotubule interaction, analogous to that found in the ciliary membrane and suspected in nerve cell axons. Figure 7 shows three possible ways that such a system could support the transport of membranated organelles along microtubule pathways.
VOLUME 74,198O
This study was supported by USPHS Grants GM 20,644 and GM 21,040 to Dr. R. E. Stephens. The study was submitted to the Graduate School of Arts and Sciences of Brandeis University in partial fulfiiment of the requirements of the degree Doctor of Philosophy. The author is grateful to Dr. R. E. Stephens for his helpful suggestions and discussions during the course of this work, and for comment on the manuscript. The author also thanks E. A. Morales for the artistic representation in Fig. 7. Note added in proof. After the submission of this manuscript, it was determined that the two high molecular weight egg dynein polypeptides are carbohydrate containing, by the periodic acid-Schiff reagent (PAS) staining method for polyacrylamide gels, and therefore may be membrane associated. While this may raise some doubt as to the molecular weight of these polypeptides, as determined by SDS-PAGE, the data support the model shown in Fig. 7b, where the dynein ATPase is associated with vesicle membranes. REFERENCES
b
FIG. 7. Three models for cytoplasmic vesicle transport. (a) Cytoplasmic dynein is associated with microtubules and moves vesicles along in “conveyer belt” fashion, by cyclic interaction with tubulin in the vesicle membranes. (b) Cytoplasmic dynein is associated with vesicle membranes. The dynein interacts cyclically with microtubules, and vesicles roll along stationary microtubules via the dynein bridges. (c) Dynein bridges are associated with both vesicles and microtubules. Vesicles move actively as in (b) and passively as in (a).
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