microtubule protein pools in early development

MICROTUBULE PROTEIN POOLS IN EARLY DEVELOPMENT *

Rudolf A. Raff, John W. Brandis, Lorrence H. Green, John F. Kaumeyer, and Elizabeth C. Raff

Department of Zoology Indiana University

Bloornington, Indiana 4740 I

The process of early embryonic development is an ordered sequence of exquisitely timed events. In many cases the preparation for the sequential steps have taken place much earlier, either during oogenesis or at a preceding stage in development itself. In the sea urchin, the existence of maternal messenger RNA, which is transcribed during oogenesis and stored for utilization after fertilization, is well documented.', * Microtubule protein is one product of such oogenetically transcribed mRNA.:j-" Proteins themselves are also stored in the egg until needed in embryogenesis; there exists a soluble pool of microtubule protein subunits in the sea urchin egg, from which the mitotic apparatus s. and later the cilia are formed.U It is not known how the maternal message is unmasked by the developing embryo after the events of fertilization, nor what triggers or controls the assembly of microtubules into the spindle fibers and associated structures. In the latter case it might be the synthesis of some essen- tial component other than tubulin or the formation of an organizing center. In many cases the centriole, itself a microtubule-containing organelle, appears to be the focal point for the organization of spindle fibers and asters, but the tubules of these are not continuous with the centrioles as are the ciliary or flagellar tubules with the tubules of the basal body from which they arise. The basal body appears to be the same as a centriole, with the addition of complex transitional structures at its junction with the ciliary shaft.IO In addition, mitotic apparatuses without centrioles or asters are well known (for example, all those of higher plants). The rate of cell division early in embryogenesis is very rapid, and the mitotic apparatus is thus the most important microtubule-containing organelle in early development. The cell cycle in early Drosophila embryos is only 10 min long, a doubling time similar to that in prokaryotes.Il And yet with each cycle, the mitotic apparatus is dismantled and reassembled in the daughter cells.

In the sea urchin embryo, as development proceeds microtubules play several roles in addition to their part in the mitotic apparatus. At the blastula stage, basal bodies develop and cilia grow from them; the cilia are of two types: the normal motile cilia responsible for the swimming of the embryo, and the nonmotile, longer cilia of a sensory organ, the apical or animal tuft, which appears at the animal pole of the embryo.'? The cells derived from the micro- meres of the 16cell-stage embryo do not become ciliated at all. Gibbins et al.13 suggested, on the basis of electron micrographs of sectioned embryos, that they

* This work was supported by United States Public Health Service Grant HD 6902 and National Science Foundation Grant GB 41633. This is contribution number 954 from the Department of Zoology.

304

Raff et al.: Protein Pools in Early Development 305

grow and then resorb cilia, but observations of intact embryos, both living and by light and scanning electron microscopy, have shown that they never grow cilia, and they constitute a bare patch at the vegetal pole of the These cells, however, become the primary mesenchyme cells, which eventually migrate into the blastocoel and lay down the embryonic skeleton. Cytoplasmic microtubules in these cells produce the differentiation in cell shape that causes the orientation necessary for skeletal formation.lR, IG* li Likewise, during the development of Drosophilu, arrays of cytoplasmic microtubules play a signifi- cant role in the determination of embryonic organization.lx

Thus, in the events of early development, a single protein or group of very closely related proteins participates in the formation of several structures of differing orders of complexity. Two orders of assembly are involved: first, the assembly of the protein subunits into microtubules, and second, the assembly of the microtubules into organelles. These organelles in turn are of different types: labile structures (such as the mitotic apparatus or cytoplasmic microtubule arrays) or stable structures (such as cilia, flagella, basal bodies, and centrioles). As expected, the accessory structures for the stable microtubule- containing organelles are greater both in number and degree of complexity than those for the labile organelles. This set of relationships in turn raises many questions. Do all of these microtubule-containing organelles share a common pool of subunit proteins, so that their differing nature is accounted for by different accessory proteins and differing cellular environments? If so, then how do the subunit proteins interact with other proteins to achieve the differ- ential states, and how are the interactions regulated? What keeps the centriole from growing a cilium? Or are the subunit pools for the various microtubule- containing organelles compartmentalized, and if so, how is the compartmentali- zation achieved? There has been some evidence for the presence of pools of insoluble forms of microtubule protein, which may perhaps be i n v o l ~ e d . ~ ~ Finally, there is the problem of the regulation of the timing of the formation of these organelles, and of how the organizing events for them differ. While assembly of microtubules in vitro has shown that the first level of assembly is by self-assembly or p ~ l y r n e r i z a t i o n , ~ ~ - ~ ~ there is evidence that even in vitro nucleation centers are 23. 2’ No evidence is as yet available on the nature of the second level of assembly, into structured organelles.

By studying the chemical nature of microtubule proteins, the nature and behavior of the pools of these proteins, and their synthesis in early development in various organisms, we hope to begin to answer some of these questions.

MATERIALS A N D METHODS

Culture of Embryos

Sea urchin embryos were cultured as described 2 5 Zlyunussa obsoleta were obtained from the Supply Department of the Marine Biological Laboratory, Woods Hole, Massachusetts, and the snails were maintained in seawater aquaria with Instant Ocean salts. Egg capsules were collected and handled according to the method of Costello et a1.,28 and embryos were cultured in Millipore@-filtered artificial seawater that contained penicillin and strepto- mycin (0.1 g/liter each). Drosophilu rnelunoguster were raised in population cages. Agar and molasses trays covered with yeast were used to feed the flies

3 06

and to collect eggs. Eggs from the trays were washed through a United States Standard Sieve series (W. S. Tyler Co.) of 841 p, 420 p, and 125 p. Eggs were collected with a 0.01% Triton@ X-100 solution from the 125 p sieve and de- chorionated by gentle stirring in 2.75% sodium hypochlorite for 5 min. The eggs were then allowed to settle through cold 0.01 % Triton several times, until contaminating feces and debris were removed.?'

Annals New York Academy of Sciences

Preparation of Microtubule Proteins

Vinblastine precipitation, in vivo labeling of microtubule proteins, and poly- acrylamide gel electrophoresis (SDS/ urea system) methods have been detailed elsewhe~e.~. 2 5 , 28 Microtubule proteins were purified from Drosophila embryos by homogenization at 4 ° C in an equal volume of the Weisenberg buffer,2n which consists of 0.1 M 2-(N-morpholino)ethane sulfonic acid (MES), pH 6.4, 1 mM ethylenediaminetetraacetic acid (EDTA) or bis( aminoethyl) glycol- ether-N,N,N',N'-tetraacetic acid (EGTA) , 1 mM guanosine triphosphate (GTP), and 0.5 mM MgCI,; and they were centrifuged at 150,000 X g for 90 min. The supernatant was diluted with an equal volume of buffer that contained 8 M glycerol,23 and incubated for 30 min at 25" C. After incubation, samples were centrifuged at 100,000 X g for 60 min at 25" C. Pellets from this first assembly step were resuspended in one-half the original volume of homogenization buffer at 4°C and incubated for 30 min, then centrifuged at 150,000 x g for 90 min at 4" C. Further purification could be obtained by repetition of the assembly procedure.

Assay Methods

Colchicine binding to protein fractions was done according to the DEAE- impregnated filter paper method of Weisenberg et a1.29 For binding studies, embryos were homogenized at 0" C in PMg buffer (0.01 M sodium phosphate, pH 7.0, that contained 0.02 M magnesium acetate), and centrifuged at 150,000 X g for 60 min. The supernatant fraction was used for assays. Protein concentrations were determined by the method of Lowry et al.30

RESULTS AND DISCUSSION

Isolation of Microtubule Proteins f r o m Embryos

Small amounts of microtubule protein were routinely obtained from sea urchin eggs and embryos by precipitation from high-speed supernatants with vinblastine s ~ l f a t e . ~ In combination with electrophoretic techniques, this has provided us with a rapid and convenient method for studying the synthesis and other characteristics of the soluble microtubule proteins of sea urchin embryos. The data discussed below in the section on synthesis of microtubule proteins were obtained by this procedure. More recently, we have prepared soluble microtubule proteins from sea urchin embryos by precipitation with antibodies prepared against sperm tail tubules.31

We can precipitate microtubule proteins from high-speed supernatants pre-


pared from Drosophila embryos with vinblastine. We have found, however, that we can prepare relatively pure microtubule proteins in high yield by use of the procedures described by Weisenberg?O and by Shelanski et al.23 for in vitro self-assembly of brain microtubules. This provides us with a very good method for preparing large amounts of microtubule proteins from Drosophila embryos. The microtubules obtained from the first assembly step are shown in

FIGURE 1. Microtubules from the first assembly step in vitro from a 150,000 x g (90 min) supernatant of Drosopliila embryos. The microtubule pellet was fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate (pH 7.2), followed by 1% OsO, in 0.1 M sodium cacodylate (pH 7.2), and stained with uranyl acetate and lead citrate. ( x 133,000. Taken by F. R. Turner.)

FIGURE 1. These are complete tubules with closed, circular cross-sections. The tubules produced by the second assembly step contain a large proportion of abnormal products, with “(2”- and “S”-shaped cross-sections. An electrophoretic gel of Drosophila microtubule proteins purified by assembly is presented in FIGURE 2. The amount of contaminating protein in the preparation is low. A plot of the migration of molecular weight standards on a duplicate gel is drawn in the upper panel of the figure. We have found the behavior of the Drosophila

308 Annals New York Academy of Sciences

microtubule proteins in the alkaline SDS/urea gel system to be like that of the sea urchin proteins; two proteins are resolved with apparent molecular weights of 53,000 and 56,000. We have also found that purified Drosophila proteins bind [3H]colchicine. Our amino acid composition data from the purified proteins are similar to those for other microtubule proteins.:’?

s E 0 o . 1 5 0 ~

7 - 6 - 5 -

4 -

3 -

2 -

I -

0 0”5D 1 2 3 4 5 8 7 8

FIGURE 2. SDS/urea acrylamide gel electrophoresis of Drosophila microtubule proteins purified by in vitro assembly. Molecular weight as a function of migration is plotted for standards (bovine serum albumin, ovalbumin, carbonic anhydrase, and myoglobin) .

Embryonic Microtubule Protein Pools

The absolute sizes of the embryonic microtubule pools have been measured for several organisms. The results are summarized in TABLE 1. The organisms studied represent diverse phyla: Arbacia is an enchinoderm, Drosophila an insect, Spisula a clam, and Urechis an echiuroid worm. The sizes of the microtubule pools in the eggs and embryos of these organisms comprise about 0.5 to 3% of the total protein; this may be contrasted with chick brain, which contains a tenfold larger pool.

Raff et al.: Protein Pools in Early Development

160

140

120

loo

80

60-

40-

309

- - - - -

TABLE 1 SOLUBLE MICROTUBULE PROTEIN POOLS IN EGGS AND EMBRYOS

-

Size of Pool Amount in Percentage of

Organism Embryo Total Protein Reference

Pg Arbacia prtrictrrlala 1 . 2 ~ lo-' 0.4 Raff and Kau-

meyer ?' Drosoplt ila rrielarrogaster l o x 1c3 2 0.4 this paper Spisrila solidissirria 2.2 x 10-4 3.3 Burnside et a].= Urechis carcpo 4.3 x lo-' 2 0.8 Miller and Epel 5(

Chick brain (9-17 days) - 24 Bamburg et a].=

The methods used to determine these pool sizes in eggs and embryos differ from one another as greatly as the organisms used. The Arbacia pool was determined by estimating the amount of microtubule protein by vinblastine precipitation. This was done by electrophoresis on gels, followed by staining, which was quantitated with known amounts of sperm tail microtubule proteins.25 The value for Drosophila embryos represents the recovery of microtubule protein f rom our mass scale preparations, for which we employed the in vitro assembly technique. The pool of Spisula embryos was determined by a n isotope dilution procedure in which chemically labeled microtubule protein was added to homogenates and copurified with the endogenous The Urechis pool was estimated by measurement of [3H]colchicine binding.34 The chick brain pool was determined by quantitative disc gel electrophoresis of total soluble pr0tein.3~

We have monitored the behavior of the soluble pool during early development of both the sea urchin and Drosophila embryos by determining the [3H]colchicine-binding activity of high-speed supernatants f rom various stages. The sea urchin results are presented in FIGURE 3. Three stages of development

FIGURE 3. Binding of [3Hl- colchicine to Arbacia egg and embryo proteins. Embryos were homogenized at the indicated stages in PMg buffer, and a 150,000 x g (60 min) supernatant was prepared. Three separate cultures of normal embryos (a) and two cultures of embryos cultured in the presence of 20 pg/ml actinomycin D ( A ) were used. The error bars indicate the range. The developmental stages diagrammed are unfertilized egg, 16 cell, and ciliated blastula. The cultures developed synchronously.

0

i t

P P H n

P P

2 2 0 1 , , , , , , , , , , , U F 2 u l 4 8 I632 MORULA BIAS'MA

STAGE


are diagrammed above the data: the unfertilized egg, which is essentially quiescent; the 16-cell stage, which is in the midst of the active cleavage period, with associated rapid mitosis; and the blastula stage, in which mitosis has slowed but ciliary assembly is occurring. The soluble microtubule pool is constant in size during this span of development (about 10 hours in Arbacia), both in normal embryos and in those in which transcription is abolished by actinomycin D. This constant level is probably maintained by a steady-state balance of synthesis and removal of protein from the pool, since synthesis is well doc~mented ,~ . 2 5 and there is evidence both for turnover and for the entry 0 *'#

4 P n A

0 4

0

4

I 2 3 4 5

TIME OF DEVELOPMENT f h d

FIGURE 4. Binding of [sH]colchicine to Drosophila embryo proteins. Embryos at the indicated times were homogenized in PMg, and a 150,000 x g (90 min) supernatant was prepared. The symbols represent four separate sets of determinations. The relative binding values are plotted because of the differences between specific activities in the various determinations. The developmental stages diagrammed are newly fertilized egg, early cleavage, blastema stage, blastoderm stage, and gastrulation. Drosophila development is described in detail by Sonnenblick." In one experiment ( A ) , there was a 30-min spread in the age of embryos. In the other experiments the spread of developmental ages was +60 min.

of newly synthesized microtubule proteins into cilia (which are long-lived structures) .3* 3G

Drusophilu development is extremely rapid (FIGURE 4). In the first 2 hours at 25" C the embryo completes 9 nuclear division cycles, to become a plas- modium that comprises about 500 nuclei. Most of the nuclei migrate to the edge of the embryo, and complete 3 more nuclear divisions; this is followed by formation of the blastoderm, and immediately after by gastrulation and organo- genesis. There is no investment of microtubule protein into cilia. During blastoderm formation, however, cytoplasmic microtubules are involved in the determination of the shape and orientation of the elongating nucleus.18 As in


the case of the sea urchin embryo, we found that the microtubule pool remains constant during this early period of development, as shown in FIGURE 4.

A similar constancy has been observed by Burnside et al.33 for Spisula stages from egg to gastrula (about 5 hours of development).

The adequacy of the measured pools for such functions has been nicely illustrated with sea urchin embryos. Sea urchin zygotes treated with 45% D,O have been observed to show a twofold increase in spindle birefr ingen~e.~ ' Similar effects have been noted with embryos treated with glycols,38 and with increases in temperature.:'!' These data indicate that an available microtubule protein pool exists in excess of that assembled into structures, under normal conditions. By counting microtubules in sections of the first mitotic apparatus of Arbncin, Cohen and Rebhun ' O calculated that the spindle microtubules constitute 0.067% of total egg protein (0.1% if astral rays are included). Our estimate that microtubule protein constitutes about 0.4% of Arbacia egg protein suggests that there is a four- to fivefold excess of subunits in the pool. Stephens:'t; estimated that the cilia grown at blastula contain a n amount of microtubule protein equal to about 0.1% of the total embryo proteins. His labeling data from cilia regeneration experiments are consistent with the presence of a pool of subunits three to four times greater than is needed for one round of ciliogenesis.

Synthesis of Microtiibirle Proteins in Early Developrnent

Microtubule protein synthesis is readily detectable in all stages of early embryonic development in sea urchins.3* ' 9 2 f i , l 1 FIGURE 5 shows the labeling of microtubule proteins in unfertilized eggs, newly fertilized eggs, and early cleavage stage embryos of Arbncia when incubated with [:iH]leucine. Micro- tubule proteins were isolated by vinblastine precipitation and analyzed by gel electrophoresis. In this and similar experiments, low but detectable levels of microtubule protein synthesis were found in eggs. Quantitation of microtubule protein synthesis in unfertilized eggs is difficult, because of high background incorporation. Incorporation of labeled amino acids into microtubule proteins rises rapidly after fertilization, however, and by midcleavage the microtubule protein peaks stand well above the background incorporation level. Micro- tubule proteins are thus synthesized at a low level in eggs and at an increasing level in embryos. The absolute rate of microtubule protein synthesis increases concomitantly with the increase in overall protein synthesis observed in the period after fertilization and prior to first cleavage.-l. As is shown in TABLE 2, however, labeling studies conducted with later embryonic stages cultured both in the presence and in the absence of actinomycin, indicate that the rate of microtubule protein synthesis in fact increases relative to the rate of overall protein synthesis during cleavage.5. x

A probable role for microtubule proteins synthesized early in development is in the mitotic apparatus. It was noted by Gross and Cousineau,.12 Stafford and Iverson,':' and Mangan et a1.I' in autoradiographic studies of sea urchin embryos exposed to labeled amino acids as zygotes, and fixed at first metaphase, that there is a concentration of silver grains over the mitotic apparatus. Auto- radiography of isolated mitotic apparatuses from such embryos also showed silver grains. Gross If, has analyzed the distribution of silver grains in electron microscopic autoradiograms of the labeled mitotic apparatus. His analysis


indicated that grains were located over microtubules more frequently than would be allowed by a random distribution. These results certainly suggest that newly labeled microtubule proteins are incorporated in the mitotic apparatus. This conclusion has weaknesses, however. As was pointed out by Gross,15 the radioactive species located on the spindle fibers could be a catalytic or accessory protein necessary for the polymerization of preexisting microtubule protein


TABLE 2

PERCENTAGE OF TOTAL INCORPORATED BY Arbacia EMBRYOS INCORPORATION OF [RHILEUCINE INTO MICROTUBULE PROTEINS:

Experiment Number <‘

Stage at Labeling

Percentage of Total Incorpo-

rated by Microtubule

Protein i

1 unfertilized egg 0.18 1 fertilized egg 0.39 1 4-8 cell 0.73 2 $ 8 cell 0.18 2.4 16 cell 0.3 1 3 16 cell-morula 0.27 3 early gastrula 0.90

Each experiment number represents one batch of cggs or embryos divided into several cultures for labeling. Labeling was done with from 0.5 to 3.0 pCi [“H]leucine/ ml, for periods of 30 min to 2 hours.

’i These figures are corrected for 50% recovery by vinblastine precipitation. $ Embryos were cultured in 20 pg actinomycin D/ml.

FIGURE 5. Synthesis of microtubule proteins by unfertilized eggs and early cleavage stages of the sea urchin embryo, Arbacia punctulata: (A) in unfertlized eggs, (B) in newly fertilized eggs, and (C) at the 4-8 cell stage. The cross-hatching indicates the protein labeling that corresponds to the microtubule protein peaks. (-) =Optical density; (0-0) = 3I-cpm per slice.


subunits. It is unlikely, however, that microtubule protein synthesis in the sea urchin zygote is necessary for formation of the first mitotic apparatus. The microtubule protein pool of eggs is more than adequate to assemble the first mitotic apparatus.

Bibring and Baxandall 18 very convincingly demonstrated that the microtubule proteins of the first mitotic apparatus could be extracted from isolated apparatuses with the organic mercurial, meralluride, and displayed by poly- acrylamide gel electrophoresis. This approach could profitably be employed to determine unequivocally whether newly synthesized microtubule protein enters the mitotic apparatus.

While the source of microtubules in the mitotic apparatus is still uncertain, a role for newly synthesized microtubule protein molecules in ciliogenesis has been clearly demonstrated. Cilia can be readily removed from blastulae and gastrulae, without damage to the embryos." The cilia are easily recovered for gel electrophoresis. Hatching blastulae that are in the process of ciliation, and gastrulae that are either augmenting or turning over cilia that are already

TABLE 3 EVIDENCE THAT MICROTUBULE PROTEIN SYNTHESIS

IN SEA URCHIN EMBRYOS UTILIZES OOGENETIC mRNA

1. Labeling is abolished by emetine. 2. Synthesis occurs in unfertilized eggs. 3. Synthesis occurs in embryos cultured in actinomycin D. 4. Synthesis occurs in activated enucleate half eggs. 5. Synthesis occurs in embryos cultured in actinomycin plus ethidium bromide.

present, were shown to incorporate labeled amino acids into ciliary micro- t ~ b u l e s . ~

Stephens 31i directly tested whether microtubule protein molecules labeled early in development are utilized for the eventual assembly of cilia by blastulae of the sea urchin, Strongylocentrotus droebachiensis. Labeling pulses were per- formed at 3-hour intervals, starting immediately after fertilization, until 1 1 batches of embryos had been so labeled. At 30 hours, cilia were removed from all batches and analyzed by gel electrophoresis. Stephens' data demonstrated that microtubule proteins (as well as several other ciliary proteins) are synthesized during cleavage, several hours prior to ciliogenesis, persist in the embryo, and are utilized in the assembly of cilia.

Oogenetic tnRNA for Microtubule Proteins

In sea urchins and other organisms, the general increase in protein synthesis at fertilization is dependent upon the mobilization of stored oogenetic mRNA.l* Several experiments indicate that microtubule protein mRNAs are of oogenetic The relevant data are summarized in TABLE 3. The


abolition of the labeling of microtubule proteins in embryos by emetine indicates that de novo protein synthesis occurs, not merely the enzymic modification of pool proteins. The synthesis of microtubule proteins in unfertilized eggs indicates not only the presence of preformed mRNA in eggs that are inactive in transcription, but also the availability of limited amounts of this mRNA for translation. Eggs preincubated for 1-2 hours in 20 pg actinomycin/ml (a dose sufficient to abolish transcription in sea urchin embryos 4i) and then cultured for several hours in the presence of the same dose of actinomycin show nearly normal incorporation of [3H]leucine into microtubule proteins. This alone, however, is not sufficient to prove that oogenetic mRNAs are involved, since a small amount of high-molecular-weight RNA is labeled in the presence of actinomycin. Microtubule protein mRNA could quite conceivably represent a special region of the DNA, whose transcription is unaffected by actinomycin. This possibility was tested with nucleate and enucleate sea urchin egg halves prepared by centrifugal cleavage:IN~ .19 Artificially activated halves (both nucleate and enucleate) incorporated labeled amino acids into microtubule proteins. Thus both chemical and physical enucleation indicate that synthesis of microtubule protein mRNA in the nucleus is not required after fertilization. Further, the lack of inhibition of microtubule protein synthesis by ethidium bromide clearly indicated that microtubule protein mRNA was not a product of mitochondria1 transcription in early development. The sum of the evidence indicates that microtubule protein mRNAs, which are certainly present in the unfertilized egg, are most probably synthesized during oogenesis and stored in the egg cytoplasm.

At this time, unfortunately, very little information is available on microtubule protein synthesis in embryos other than that of the sea urchin. We are investigating one spiralian embryo (that of the snail, Ilyanmsa), and have found that this embryo does synthesize microtubule proteins (FIGURE 6). As in sea urchin embryos, this synthesis is insensitive to actinomycin D, and during development it increases in relation to the total protein synthesis.5o These results suggest that microtubule protein synthesis is probably subject to similar regulation mechanisms in the early development of both protostomes and deutero- stomes.

SUMMARY

Microtubule protein pools have been demonstrated to exist in unfertilized eggs and the early embryonic stages of several organisms. The microtubule pool of the sea urchin embryo is constant in size (about 0.4% of the total embryo protein) throughout early development. Protein withdrawn from this pool for organelle assembly is replaced by new synthesis. Eggs and embryos of Drosophila similarly contain a pool of microtubule proteins (20.4% of the total embryo protein, ~ 3 % of the soluble protein), which is constant in size throughout early development. The Drosophila egg microtubule proteins are easily purified by self-assembly in vitro of microtubules, and are similar to microtubule proteins from other organisms in molecular weight and other properties. Synthesis of microtubule proteins in sea urchin embryos is supported by oogenetic mRNA. This appears also to be the case in molluscan (Ilyanussa) embryos. It is not known whether Drosophilu embryos synthesize microtubule proteins during the early stages of development.


I600

9 1400 g 2.0 - u,

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200

I I

I

/ ! j . I

I I

I I

, 1 1 1 1 1

10 20 30 40 SO 60 70 80 90 100 IK) 120 130 140 SLICE NUMBER

FIGURE 6. Synthesis of microtubule proteins by 2-day-old embryos of Ilyanassa. A thousand embryos were labeled for 14 hours with 40 pCi [8H]leucine/ml, washed 3 times with seawater and once with buffer that contained 0.01 M Tris (pH 7.0), 0.02 M magnesium acetate, and 0.25 M sucrose, and homogenized with 0.3 ml packed sea urchin eggs to provide carrier microtubule proteins, Purification was by vinblastine precipitation. (-) =Optical density; (- 0 -) = [3H]leucine incorporation.

ACKNOWLEDGMENTS

We thank Dr. A. P. Mahowald for generously allowing the use of his Drosophilu culture facility, and Dr. F. R. Turner for taking electron micrographs. We also thank Mrs. Carolyn Huffman for her expert technical assist- ance.

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