tubulin characterization during embryogenesis of ascaris suum

DEVELOPMENTAL BIOLOGY T&47-57 (1980)

Tubulin Characterization during Embryogenesis of Ascaris suum’

PAUL A. FRIEDMAN,*‘~ EDWARD G. PLATZER,* AND EDWARD J. CARROLL, JR.t

* Department of Nematology and t Department of Biology, University of California, Riverside, California 92521

Received April 16, 1979; accepted in revised form September 27, 1979

Tubulin in cytosolic fractions of Ascaris sum embryos was characterized on the basis of its specific colchicine binding and known properties of the tubulin-colchicine complex. Cytosolic fractions of early (eight-cell) and late (gastrula) embryos maintained at 37°C exhibited significant colchicine binding reaching pseudosaturation at 6 hr. No binding was detected in samples incubated for 8 hr at 0°C. Colchicine binding activity of late embryo cytosolic fractions in the absence of guanosine 5’-triphosphate or vinblastine sulfate decayed with first-order kinetics and had a h/2 of 377 min and a k of 1.84 x 10e3 min-‘. In the presence of 1 n&f guanosine 5’-triphosphate (tl,P = 563 min, k = 1.23 X 10e3 min-‘) or 0.5 n&f vinblastine sulfate (tl12 = 877 min, k = 0.79 X 10m3 mix-‘), the tubulin-colchicine interaction was stabilized. Colchicine binding to late embryo tubulin was competitively inhibited by podophyllotoxin with a K; of 1.1 x 10e6 M. The association constants of early and late soluble embryo tubulin for colchicine were 4.35 x lo4 M-’ and 1.86 x

ld M-l, respectively. Although the affinity of early tubulin for colchicine was less than late embryo tubulin, the titratable soluble tubulin pools were equal in these stages. The tubulin pool was estimated to be 0.3% of the soluble embryo protein. The change in affinity of tubulin for colchicine during embryogenesis appeared to be unique to this organism. The importance of this change and the mechanisms involved in the regulation of tubulin affinities are discussed.

INTRODUCTION

Cleavage and embryogenesis are depend- ent on functional tubulin, a structural protein of microtubules and the mitotic apparatus (Mazia, 1961; Inoue and Sato, 1967). The presence of a preformed pool of tubulin in the unfertilized egg and its maintenance at a constant level during development has been demonstrated in Drosophila melanogaster (Green et al., 1975), sea urchin (Raff et al., 1971; Raff and Kaumeyer, 1973; Raff, 1975), the clam Spisula solidissima (Burn- side et al., 1973), and the Mexican axolotl Ambystoma mexicanum (Raff, 1977). The importance of this tubulin pool and its utilization during embryogenesis has been reviewed (Raff, 1975; Raff et al., 1975; Raff and Raff, 1978). However, the mechanisms

’ This project was supported in part by BRSG Grant RR07010-11, Biomedical Research Support Grant Program Division of Research Resources.

’ Present address: Department of Biology, Univer- sity of Notre Dame, Notre Dame, Ind. 46556.

involved in regulating tubulin function, e.g., microtubule assembly or formation of the mitotic apparatus are not completely un- derstood.

To provide additional information on the importance of tubulin and the regulation of tubulin function during embryogenesis, we have characterized tubulin from cytosolic fractions of developing embryos of the in- testinal nematode of pigs, Ascaris suum. The availability of large numbers of eggs and their highly synchronous development makes A. suum a suitable organism for developmental and biochemical studies. We have described embryo tubulin on the basis of its specific binding to colchicine and the properties of the tubulin-colchicine interaction known for other eukaryotic orga- nisms (Wilson and Bryan, 1974). The soluble colchicine binding in early (eight-cell) and late (gastrula) embryos of A. suum shared the general properties exhibited by tubulin in other animal species. However,

47

0012-1606/80/050947-11$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

48 DEVELOPMENTAL BIOLOGY VOLUME 76,198O

the change in affinity of tubulin for colchi- tine during embryogenesis appeared to be unique to this organism. The function of this change and the mechanisms involved in the regulation of tubulin affinities are discussed.

MATERIALS AND METHODS

Preparation of eggs. Adult Ascaris suum were collected in 0.15 M sodium chloride and maintained at 37°C upon return to the laboratory. Mature females were dissected and the uteri collected and placed in ice cold 0.5 N sodium hydroxide. Fertilized eggs are unaffected by dilute base and acid (see under Results) (Fairbairn, 1957). Eggs were prepared by a modification of the method of Kaulenas and Fairbairn (1968). Uteri were mechanically stirred in ice cold 0.5 N sodium hydroxide for 4 hr to digest the uterine walls and any permeable eggs (see under Results). The egg suspension was then placed at 4°C and allowed to settle. The resulting supernatant solution containing uterine debris and empty egg shells was removed by aspiration. This procedure was repeated several times with fresh ice cold 0.5 N sodium hydroxide until contamina- tion by uterine debris and egg shells was minimal. The egg preparation was then centrifuged at 900g for 2 min at 4’C, the supernatant solution was removed by aspiration, and the egg pellet was resuspended in distilled water. This procedure was repeated several times to obtain a clean egg preparation. The final egg preparation was resuspended in 0.1 N sulfuric acid and stored at 4°C. The concentration of eggs in this suspension was 3.3 x lo6 eggs/ml. Fur- ther development of A. suum eggs was initiated by adding 5 ml of the egg suspension to 1.4 ml of 0.1 N sulfuric acid in a 50-ml screw cap Erlenmeyer flask and incubating at 30°C in an Eberbach shaker bath.

Preparation of egg and embryo cytosolic extracts. Suspensions of eggs or embryos were washed and neutralized by repeated

centrifugations at 9OOg for 2 min at 4°C in PMgG buffer (0.01 M sodium phosphate, 0.02 M magnesium acetate, and 0.1 M sodium glutamate, pH 7.0). The final egg or embryo pellet was resuspended in 2 vol of cold PMgG buffer and passed twice through an Aminco French pressure cell at 16,000 psi. Two passes were required to ensure complete disruption of eggs or embryos. The homogenate was collected and centrifuged at 50,OOOg for 30 min at 4’C in a Beckman J21C preparative centrifuge. The resulting supernatant solution was drawn off and filtered through Pyrex wool filtering fiber to remove contaminating lipids. The clarified 50,OOOg supernatant was used for subsequent colchicine binding assays. Pro- tein concentration was determined by the method of Lowry et al. (1951) with bovine serum albumin as the standard.

Colchicine binding assay. The colchicine binding activity of 50,OOOg A. suum egg or embryo supernatants was determined by a modification of the method of Sherline et al. (1974). Aliquots of supernatants (280 pg soluble egg or embryo protein/100 ~1 assay volume) were incubated with [3H]colchi- tine for 90 min at 37°C. Blanks containing aliquots of supernatant (280 pg soluble egg or embryo protein/100 ~1 assay volume), [3H]colchicine, and 1 x 10m3 M unlabeled colchicine were incubated in the same manner. The reaction was stopped by adding 1 ml of a 6 mg/ml activated charcoal suspension. The suspension was mixed, allowed to settle for 10 min on ice, and centrifuged at 8OOOg for 3 min in a Beckman Microfuge B. A 500~~1 aliquot of the supernatant solution was added to 10 ml of Aquasol II and counted in a Beckman 100 C Liquid Scin- tillation Counter at 45% efficiency.

Time-decay colchicine binding assay. Loss of colchicine binding activity of 50,OOOg A. suum embryo supernatants was determined in the absence and presence of guanosine 5’-triphosphate (GTP) or vinblastine sulfate as described by Bamburg et al. (1973). Tubes containing aliquots of su-

FRIEDMAN, PLATZER, AND CARROLL

pernatant (280 pg soluble embryo protein/ 100 ~1 assay volume) were preincubated at 37°C for 0,2,4, and 6 hr in the absence and presence of 1 x 10e3 M GTP or 5 x 10d4 M vinblastine sulfate. After the preincubation period bound colchicine was determined by a modification (supra vide) of the method of Sherline et al. (1974) with 4 x low6 M [3H]colchicine. Results were expressed as disintegrations per minute per microgram soluble embryo protein (dpm/pg protein).

Determination of binding constants. Al- iquots of 50,OOOg A. suum embryo supernatants (280 pg soluble embryo protein/100 ~1 assay volume) were incubated with various concentrations (2 to 40 x lo-” M) of [3H]colchicine for 6 hr at 37°C. Colchicine binding activity was previously determined to be maximal at 6 hr. Blanks containing aliquots of supernatant (280 pg soluble embryo protein/100 ~1 assay volume), [3H]colchicine (2 to 40 x 10e6 M), and 1 x 10s3 M unlabeled colchicine were incubated in the same manner. Bound colchicine was determined by a modification (supra vide) of the method of Sherline et al. (1974). Associa- tion constants were determined by means of Scatchard plots (Scatchard, 1949).

Inhibition of colchicine binding by podophyllotoxin. Aliquots of 50,OOOg A. suum embryo supernatants (280 pg soluble embryo protein/100 ~1 assay volume) were incubated with various concentrations (2 to 10 X 10F6 M) of [3H]colchicine in the absence and presence of 2 x lo-” M podophyllotoxin for 6 hr at 37°C. Podophyllo- toxin (1 X lo-” M) was dissolved in ethanol and the final concentration of ethanol in the reaction mixture was 0.02%. Bound colchicine was determined by a modification (supra vide) of the method of Sherline et al. (1974). The inhibition constant was determined from a double reciprocal plot (Se- gal, 1975).

Chemicals. Colchicine (ring C-methoxyl- 3H) with specific activities of 2.4 or 4 Ci/ mmole was purchased from Amersham. Po- dophyllotoxin was purchased from Aldrich Chemical Company, Inc. Unlabeled colchi-

Ascaris suum embryonic tubulin 49

tine, GTP, and vinblastine sulfate were purchased from Sigma. All other chemicals were reagent grade.

RESULTS

Embryogenesis and synchrony. Eggs of A. suum are inseminated as they pass from the oviduct into the uterus, and remain in the uncleaved state while in the uterus. A chitinous shell and an inner lipid layer are produced by the egg as it moves down the uterus. The lipid layer is responsible for the high degree of impermeability found in inseminated eggs (Fairbairn, 1957). The male and female pronuclei remain separated at 12- to 24-l-u postinsemination. Further development is arrested until eggs are ovipos- ited into favorable environmental conditions.

Embryogenesis was prevented in the laboratory by storage of eggs at 4°C. Further development was initiated by incubation of uncleaved eggs (defined as 0 days of incubation) at 30°C. Male and female pronuclei approach after 10 hr and fuse at 38 hr (Fairbairn, 1957).

The first cleavage occurred at 48 hr (Fig. 1C) and the schedule of subsequent developmental stages was as indicated in Figs. 1D to I. The percentage of synchronously dividing embryos ranged from 73 to 94% as shown in Table 1.

Tub&in levels during embryogenesis. A. suum eggs were incubated at 30°C and har- vested at specific times during the course of development from separate cultures. Rel- ative tubulin levels, expressed as dpm per microgram of soluble embryo protein, were determined by incubating cytosolic fractions from various stages with 5 x lop6 M [3H]colchicine at 37°C for 90 min (Fig. 2). During early development, one- to eight- cell stage, apparent tubulin levels were low and relatively constant averaging 7.3 dpm per microgram of soluble embryo protein. At blastula, tubulin levels increased approximately twofold above early embryo levels. Tubulin levels were maximal during late development, gastrula to first larval


FIG. 1. Developmental sequence of Ascaris suum at 30°C. (A) Uncleaved, 0 days of incubation; (B) initiation of the first cleavage, 1 day of incubation; (C) 2-cell stage, 2 days of incubation; (D) 4-cell stage, 3 days of incubation; (E) 8-cell stage, 4 days of incubation; (F) blastula formation, 5 days of incubation; (G) gastrula formation, 8 days of incubation; (H) formation of the fast larval stage, 9 days of incubation; (I) motile first stage larvae, 10 days of incubation. X 480.

stage, and averaged 22 dpm per microgram volved in the regulation of tubulin levels of soluble embryo protein. This increase in during A. suum embryogenesis, we have tubulin represented an apparent threefold characterized tubulin from early and late increase in tubulin levels during develop- embryonic stages. We have found that the ment. To determine the mechanism in- apparent increases in tubulin levels during

FRIEDMAN, PLATZER, AND CARROLL Ascaris suum embryonic tubulin 51

embryogenesis were a result of changes in affinity of tubulin for colchicine.

Tubulin characterization in early A. suum embryos. Tubulin in cytosolic extracts of early stage embryos (Fig. 1E) was described on the basis of the known properties of the tubulin-colchicine interaction as outlined by Wilson and Bryan (1974). Formation of a tubulin-colchicine complex is characterized by time dependence and lack of binding at O”C, first order decay and stabilization by GTP and vinblastine sulfate, the binding constant for colchicine, and competitive inhibition by podophyllotoxin.

TABLE 1

EMBRYOGENESIS AND SYNCHRONY OF Ascaris suum AT 30°C

Days of incu- Developmental Percent- Percent- bation stage age via- age syn-

bility” chronyb

0 l-Cell - - 1 l-Cell - -

2 2-Cell 66 85 3 4-Cell 77 86 4 8-Cell 80 73

The formation of the tub&n-colchicine complex at 37°C was slow and reached pseudosaturation at 6 hr (data not shown). The K, of early embryo tubulin for colchi- tine was 4.35 X lo4 M-’ (Table 2, Fig. 3). At extrapolated infinite colchicine concentration 17.8 x lo-l5 moles of colchicine were bound per microgram of soluble embryo protein (Table 2). A more complete characterization of early embryo tubulin was prevented by the extremely low association constant.

30

1

1 I 0 2 4 6 6 IO 12 14

5 Blastula 83 83 8 Gastrula 82 82

DAYS AT 3O’C

10 L’ 86 76 FIG. 2. Relative tubulin levels, expressed as dpm/

13 LI’ 86 94 microgram of soluble embryo protein, during Ascaris suum embryogenesis at 3O’C. Samples of embryo cy-

’ Percentage of cleaving embryos. tosolic fractions were incubated with 5 x lo-’ M b Percentage of synchronously dividing embryos. [3H]colchicine (specific activity 4 Ci/mmole) for 90 ’ Fit larval stage present. min at 37°C. Error bars indicate f 2 standard errors.

TABLE 2

ASSOCIATION CONSTANTS AND SPECIFIC ACTIVITIES OF EARLY AND LATE Ascaris suum EMBRYO TUBULINS

Embryo stage Ratio”

8-Cell Gastrula

Kc, Of-‘) Experiment 1 5.54 x ld(5)b 1.48 x 105(5)

Experiment 2 3.15 x 104(3) 2.23 x 105(5)

Average Specific activity’

4.35 x lo4 1.86 x lo5 4.3

Moles colchicine bound/pg soluble embryo protein at in&&e colchicine concentration

Experiment 1 20.1 x 10-15 Experiment 2 15.5 x lo-l5

Average 17.8 x lo-l5

D Ratio of values of 8-day embryos:l-day embryos. * Number of data points fitted by regression analysis for Scatchard plots. ’ Specific activity of embryo cytosolic fractions.

13.1 x lo-l5 14.5 x WL5

13.8 x lo-l5 0.78

DEVELOPMENTAL BIOLOGY VOLUME 76,198O 52

5

4 \ K,= 3 15xlO’I,lcrr/mole

\, 0 4 8 12 16

6) I IO”

FIG. 3. Scatchard plot: binding of colchicine to early Ascaris suum embryo tubulin at 37°C. The final concentrations of colchicine were 1-4 x 10e5 M (specific activity 4 Ci/mmole). The K, was calculated from the slope of the line. The specific activity of embryo cytosolic fractions, determined from the intercept of the abscissa, provided an estimate of the tubulin concentration. v, moles of colchicine bound per microgram of soluble embryo protein.

Tubulin characterization in late A. suum embryos. Tubulin in cytosolic extracts of gastrula stage embryos (Fig. 1G) was also described on the basis of the known properties as outlined by Wilson and Bryan (1974).

Embryo cytosolic fractions maintained at 37°C exhibited significant colchicine binding reaching pseudosaturation at 6 hr (Fig. 4). No binding was detected in samples incubated for 8 hr at 0°C (data not shown).

Colchicine binding activity of embryo cytosolic fractions in the absence of GTP or vinblastine sulfate decayed with first-order kinetics with a half life (t& of 377 min and a decay constant (iz) of 1.84 x lop3 min-’ (Fig. 5). In the presence of 1 X 10e3 M GTP or 5 x 10d4 M vinblastine sulfate, the tu-

0 2 4 6 6

TIME (HOURS)

FIG. 4. Binding kinetics of 1 x 10m6 M [3H]colchi- tine (specific activity 4 Ci/mmole) to late Ascaris suum embryo cytosolic fractions at 37°C.

I

0 2 4 6

PREINCUBATION TIME (HOURS)

FIG. 5. First-order decay of colchicine binding activity in late Ascaris suum embryo cytosolic fractions upon aging at 37°C in the absence (0) and presence of 1 x 10e3 M GTP (U) or 5 x 1O-4 M vinblastine sulfate (A). Loss of colchicine binding activity was determined by the time-decay assay described under Materials and Methods. The final concentration of colchicine was 4 x 10m6 M (specific activity 4 Ci/mmole).

bulin-colchicine interaction was stabilized (Table 3). Extrapolation of first order decay lines to 90 min from the zero time, indicated the initial binding capacity of the cytosolic fractions when incubated with 4 x 1O-6 A4 [3H]colchicine. In the absence and presence

FRIEDMAN, PLATZER, AND CARROLL Ascaris suum embryonic tub&in 53

TABLE 3

STABILIZATION OF COLCHICINE-BINDING ACTIVITY OF LATE Ascaris suum EMBRYO TUBULIN BY GTP

AND VINBLASTINE SULFATE

Addition t1/2 kx min 1om3

min-’

None 377 1.84 1.0 mM GTP 563 1.23 0.5 mkf vinblastine sulfate 877 0.79

of GTP or vinblastine sulfate, the initial binding capacities were nearly identical. These findings indicated that GTP or vinblastine sulfate affected the decay rate of the tubulin-colchicine interaction and not the affinity of tubulin for colchicine.

The K, of soluble embryo tubulin for colchicine was 1.86 x lo5 M-’ (Table 2, Fig. 6). At extrapolated infinite colchicine concentration 13.8 x lo-l5 moles of colchicine were bound per microgram of soluble embryo protein (Table 2). These data indicated that the affinity of early embryo tubulin for colchicine was approximately

3 h

Kp= 2 23~10~ Ilters/mole

I I \ , 0 4 8 I2 16

(9 x d5

FIG. 6. Scatchard plot: binding of colchicine to late Ascaris suum embryo tubulin at 37°C. The final concentrations of colchicine were 2-10 x lo-” M (specific activity 4 Ci/mmole). The K, was calculated from the slope of the line. The specific activity of embryo cytosolic fractions, determined from the intercept of the abscissa, provided an estimate of the tubulin concentration. v, moles of colchicine bound per microgram of soluble embryo protein.

fourfold less than the affinity of late embryo tubulin for colchicine. However, the specific activities, (moles colchicine bound per microgram of soluble embryo protein extrapolated to infinite colchicine concentration) of early and late embryo cytosolic fractions were equal (Table 2). At extrapolated infinite colchicine concentration, the tubulin pool was estimated to be 0.3% of the soluble embryo protein. This value was calculated by correction for decay of the tubulin colchicine interaction (t1,2 = 377 min) and on the assumption of one colchicine binding site per tubulin dimer with a molecular weight of 110,000. Colchicine binding was competitively inhibited by podophyllotoxin with a Ki of 1.1 x 10e6 M (Fig. 7).

Mechanism of tub&in affinity changes. A series of mixing and dilution experiments were used to determine if the observed developmental tubulin-colchicine affinity changes were due to the presence of two different tubulins at early and late stages. Cytosolic fractions of early and late embryos were mixed in different proportions based on protein concentration (100% = 280 pg soluble embryo protein/100 ~1 assay volume) and incubated with 5 x 10e6 M [3H]colchicine (specific activity 2.4 Ci/ mmole) at 37°C for 90 min (Table 4). In addition, early and late embryo cytosolic

FIG. 7. Competitive inhibition of colchicine binding to late Ascaris suun embryo tubuiin by podophyllotoxin. Samples were incubated at 37°C in the absence (0) and presence (0) of 2 x lo-” M podophyllotoxin as described under Materials and Meth- ods. The final concentrations of colchicine were 2-10 x IO-” M (specific activity 4 Ci/mmole). V, moles of colchicine bound per microgram of soluble embryo protein.

54 DEVELOPMENTAL BIOLOGY VOLUME 76.1980

fractions were prepared at 33 and 66% of maximum protein concentration, diluted to volume with PMgG buffer, and incubated under similar conditions (Table 5). If the early and late embryo tubulins are differ-

TABLE 4

THE EFFECTS OF MIXING EARLY AND LATE Ascaris suum EMBRYO CYTOSOLIC FRACTIONS ON

COLCHICINE-BINDING ACTIVITIES

Direct mix- Independ- ing” (dpm) ent dilution

and summation*

(dpm)

33% E/66% L Experiment 1 Experiment 2

1240 947 1307 733

Average 66% E/33% Ld

Experiment 1 Experiment 2

1274 840

955 393 938 453

Average 947 424

a Cytosolic fractions of early and late embryos were mixed in different proportions based on protein concentration, 100% = 280 pg protein/100 ~1 assay volume.

b Early and late cytosolic fractions were prepared at 33 and 66% of maximum protein concentration and diluted to volume with PMgG. Values are the sum of independent dilutions: data from Table 5.

’ 33% of the protein from early embryos; 66% of the protein from late embryos.

d 66% of the protein from early embryos; 33% of the protein from late embryos.

ent, we would predict that the colchicine binding activities of mixed cytosolic fractions and the summation of activities of independently diluted cytosolic fractions would be equal. However, mixed cytosolic fractions had greater activity than independent dilutions (Table 4). A mixture containing 33% early embryo protein and 66% late embryo protein had a greater activity than fractions independently diluted and summed (1274 vs 840 dpm). A mixture containing 66% early embryo protein and 33% late embryo protein had a greater activity than fractions independently diluted and summed (947 vs 424 dpm). These results suggested an alternate hypothesis: one tubulin is present during development and that its affinity for colchicine is modified by an inhibitor(s) in early embryos or by an activator(s) in late embryos. Results of the dilution experiments have enabled us to distinguish between these hypotheses. Early embryo cytosolic fractions showed a constant specific activity upon dilution with PMgG buffer and averaged 1.8 dpm per microgram of soluble embryo protein (Ta- ble 5). In contrast, the specific activity of undiluted late embryo cytosolic fractions was 5.6 dpm per microgram of soluble embryo protein (Table 5). The specific activity of late embryo cytosolic fractions decreased

TABLE 5

THE EFFECTS OF DILUTION OF EARLY AND LATE Ascaris suum EMBRYO CYTOSOLIC FRACTIONS ON COLCHICINE-BINDING ACTIVITY

100%” 66% 33%

dpm S.A.h dpm S.A. dpm S.A.

Early embryos Experiment 1 535 1.9 313 1.7 171 1.9 Experiment 2 529 1.9 244 1.3 173 1.9

Average 532 1.9 279 1.5 172 1.9 Late embryos

Experiment 1 1400 5.0 776 4.2 80 0.9 Experiment 2 1718 6.1 560 3.0 209 2.3

Average 1559 5.6 668 3.6 145 1.6

a 100% = 280 pg soluble embryo protein/100 ~1 assay volume; 66% = 187 pg soluble embryo protein/NO /.d assay volume; 33% = 93 pg soluble embryo protein/100 ~1 assay volume. Samples were adjusted to volume with PMgG.

b Specific activity, dpm/pg protein.

FRIEDMAN, PLATZER, AND CARROLL Ascaris suum embryonic tub&in 55

linearly with dilution. At 33% dilution the specific activity of late embryo cytosolic fractions was nearly equal to the specific activity of early embryo cytosolic fractions. These results suggest the presence of an activator(s) of tubulin-colchicine affinity in late embryo cytosolic fractions. The positive effects of the activator(s) on tubulin affinity can be eliminated through dilution, since dilution would favor dissociation of a tubulin-activator complex.

DISCUSSION

Many studies on early development uti- lizing cytosolic fractions have shown that a pool of soluble tubulin exists in the unfertilized egg and remains fairly constant during embryogenesis. The importance of the tubulin pool during embryogenesis has been reviewed (Raff, 1975; Raff et al., 1975; Raff and Raff, 1978). Relatively constant tubulin pools have been demonstrated in embryos of Drosophila (Green et al., 1975), sea urchin (Raff et al., 1971; Raff and Kau- meyer, 1973; Raff, 1975), Spisula (Burnside et al., 1973), and axolotl (Raff, 1977). In contrast, the relative size of the tubulin pool in A. suum embryos appeared to increase during the course of development. Tubulin levels during embryogenesis of A. suum were determined at subsaturating concentrations of colchicine. This apparent increase in tubulin levels may have resulted from increased tubulin synthesis or from a change in the association constant of tubulin for colchicine. To determine the mechanisms involved in the regulation of tubulin levels during A. suum embryogenesis, we have characterized tubulin from early and late embryonic stages.

We have found that the soluble colchi- tine binding activities in cytosolic fractions of early and late embryos of A. suum were similar to those reported for eggs and embryos of other animal species. Comparisons of tubulins were based on the thermody- namic properties of the tubulin-colchicine interaction, stability of tubulin, and the affinities of tubulin for podophyllotoxin and

colchicine (Wilson and Bryan, 1974). Formation of a tubulin-colchicine com-

plex at 37°C in early and late A. suum embryo cytosolic fractions was slow and did not form at 0°C. Similar results have been reported for tubulin from cytosolic fractions of eggs of axolotl (Raff, 1977) and the Echiuroid worm Urechis caupo (Miller, 1973) and for tubulin in cytosolic fractions of embryos of sea urchin (Raff and Kau- meyer, 1973; Pfeffer et al., 1976a) and Dro- sophila (Green et al., 1975). The first order decay rate of colchicine binding activity of late A. suum embryo cytosolic fractions was comparable to decay rates of other orga- nisms. Unstabilized cytosolic fractions incubated at 37°C decayed with tljz of 6.8 hr for Urechis eggs (Miller, 1973), 6.6 hr for Drosophila embryos (Green et al., 1975), 4.2 hr for sea urchin eggs (Pfeffer et al., 1976a), and 6-7 hr for axolotl eggs (Raff, 1977). Podophyllotoxin competitively inhibited colchicine binding to late A. suum embryo tubulin. Competitive inhibition of colchicine binding by podophyllotoxin has also been demonstrated with sea urchin egg tubulin with a Ki of 6.6 x low7 M (Pfeffer et al., 1976a). The affinity of early A. suum embryo tubulin for colchicine was fourfold less than the affinity of late A. suum embryo tubulin for colchicine. The association constant of late A. suum embryo tubulin for colchicine was in the range of association constants determined for tubulin in cytosolic fractions of axolotl eggs, K, = l-4 X lo5 M-’ (Raff, 1977) and sea urchin eggs, K, = 2.9 X lo5 M-’ (Pfeffer et al., 1976b). The specific activities, extrapolated to infinite colchicine concentrations, of early and late A. suum embryo cytosolic fractions were equal (Table 2). From these results we predicted that the size of the tubulin pool remained relatively constant during A. suum embryogenesis. We have estimated the size of the tubulin pool to be 0.3% of the soluble embryo protein. In other species the percentage of total protein existing as a soluble tubulin pool was 0.4% for Dro- sophila (Green et al., 1975), 0.4-1.7% for


sea urchin (Raff and Kaumeyer, 1973; Raff et al., 1975; Pfeffer et al., 1976a), 3.3% for Spisula (Burnside et al., 1973), 0.8% for Urechis (Miller, 1973), and 0.27% for the axolotl (Raff, 1977).

We have demonstrated that there is a preexisting tubulin pool in A. suum which remained constant during embryogenesis. However, tubulin from early A. suum embryos had a lower affinity for colchicine than late A. suum embryo tubulin. As a result, only a portion of the tubulin pool was detected by experiments which utilized subsaturating concentrations of colchicine.

Differences in colchicine binding affinities between sea urchin egg tubulin and tubulin from sea urchin sperm tail outer doublet has been reported by Pfeffer et al. (1976b). However, we are the first to report on different colchicine binding affinities of tubulin prepared from embryonic stages of the same organism. Our findings suggest that the affinity of A. suum embryonic tubulin for colchicine is developmentally reg- ulated. The schedule of tubulin affinity regulation coincides with the completion of the blastula stage and regulation continued through gastrulation and formation of the first larval stage. Although cell division be- gins to slow down at this time, tubulin is important in cell differentiation, cell migra- tion, and organogenesis (Tilney, 1968). Dur- ing these developmental events, tubulin is incorporated into labile structures, e.g., cytoplasmic microtubules and stable structures, e.g., neuronal microtubules. A high colchicine affinity tubulin may be important in the regulation of microtubule po- lymerization. The interaction of the colchi- tine binding site and tubulin-tubulin binding sites has been described (Wilson and Meza, 1972; Margolis and Wilson, 1977).

In this study we provided evidence for the presence of an activator(s) in late embryo cytosolic fractions which enhances the affinity of late embryo tubulin for colchi- tine. The positive effects of the activator(s) can be eliminated through dilution since dilution would favor dissociation of a tu-

bulin-activator complex. Lockwood (1979) and Sherline et al. (1979) have recently demonstrated the presence of endogenous cytoplasmic factors that interact with the colchicine site on mammalian tubulin and have suggested their importance in the regulation of microtubule assembly. We antic- ipate that our findings may facilitate further studies on the importance of tubulin and the regulation of tubulin function during embryogenesis.

The authors thank Dr. R. Olsen for providing tech- nical advise. We also thank Farmer John Clougherty Packing Company of Los Angeles for providing us with Ascaris suum.

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