Site and timing of synthesis of tubulin and other proteins during oogenesis in Drosophila melanogaster

DEVELOPMENTAL

BIOLOGY

86, 272-284 (1981)

Site and Timing of Synthesis of Tubulin and Other Proteins during Oogenesis in Drosophila melanogaster JOSEPH E. LOYD,’

C. RAFF, AND RUDOLF A. RAFF’

ELIZABETH

Program in Molecular, Cellular and Developmental Biology, Department of Biology, Indiana Received October 6, 1980; accepted in revised

form March

University,

Bloomington, Indiana 47405

26, 1981

Protein synthetic patterns during oogenesis in Drosophila melanogaster were examined; in particular the site, time, and rate of tubulin synthesis and accumulation during oogenesis were determined. Ovarian proteins were labeled with [35S]methionine in vivo or in organ culture in vitro, and the proteins synthesized in egg chambers of specific developmental stages displayed by two-dimensional gel electrophoresis. A dissection technique was devised to examine proteins synthesized in each of the three cell types present in stage 10B egg chambers. The majority of proteins which were resolved by two-dimensional gel electrophoresis, including tubulin and actin, were synthesized throughout oogenesis and, at least to some extent, in each of the stage 10B cell types. Protein synthesis specific to developmental stage and/or cell type was also observed; for example, two nonchorion proteins were synthesized only in follicle cells and primarily at stage 10. A sensitive and specific radioimmune assay was developed in order to quantitate tubulin accumulation. Synthesis of several a-tubulin subunits and one fl-tubulin subunit was observed. The tubulin content per egg chamber increased from 3 ng in stage 9 to 17 ng in stage 14, a period of about 13 hr. An accumulation rate of 1 ng/hr suggests that tubulin mRNA can account for about 4% of the total, nonmitochondrial, poly(A)+ RNA of the egg. Analysis of separated cell types at stage 10B revealed that both the follicle and nurse cells synthesize and accumulate appreciable amounts of tubulin. The stage 10B oocyte contains relatively little tubulin but actively synthesizes it. These two complementary analyses demonstrate that the tubulin present in the egg is synthesized within the oocyte-nurse cell syncytium, first in the nurse cells and later in the oocyte. INTRODUCTION

The basic unit of Drosophila oogenesis is the egg chamber, composed of the oocyte with its 15 syncytial nurse cells and an encapsulating monolayer of follicle cells. From the division of the stem cell, oogenesis requires about a week, during which time the size of the oocyte is increased lOO,OOO-fold. The site of much of the macromolecular synthetic activity within the egg chamber is not the oocyte itself, but its mitotic-sister nurse cells. The nurse cells synthesize nearly all of the RNA ultimately found in the egg and inject it, along with the entire cytoplasmic contents into the oocyte through specialized cytoplasmic interconnections called ring canals. The Drosophila oocyte nucleus itself remains condensed and synthetically inactive throughout most of oogenesis (Mahowald and Tiefert, 1970; Nokkula and Puro, 1976). At the time of laying, tubulin, the structural protein of microtubules, constitutes approximately 3% of the total Drosophila egg protein, or about 10% of the nonyolk proteins (Green et aZ.,1975,1979; Loyd, 1979). The oogenetic origin of the egg tubulin is of particular interest. Tubulin accumulation during oogenesis has been ’ Present address: Department of Zoology Research Building, 1117 W. Johnson St., University of Wisconsin, Madison, Wise. 53’706. * To whom reprint requests should be addressed. 272 0012-1606/81/120272-13$02.00/O Copyright All rights

0 1981 by Academic Press, Inc. of reproduction in any form reserved.

studied in Urechis caupo (Miller and Epel, 1973), Xenopus laevis (Pestell, 1975), and Ambystoma mexicanum (Raff, 1977). In all three cases, the total tubulin of the oocyte was found to increase in proportion to the total protein content of the oocyte. The pattern of oogenesis in these species is quite different from that in Drosophila; oogenesis is relatively protracted (up to months or years in the amphibians), and the oocyte nucleus is active in transcription of oogenetic mRNA. In Drosophila there are at least three possible sites for the synthesis of the tubulin that accumulates during oogenesis. Yolk, the chorion proteins, and acid phosphatase are representative of three different strategies which have been identified for accumulation of proteins of the egg or its coverings during oogenesis. Most of the yolk proteins are synthesized and secreted by the extraovarian fat body and taken into the oocyte by micropinocytosis during a 15-hr period (Kambysellis, 1977; Telfer, 1975; Giorgi and Jacob, 1977; Postlethwait and Handler, 1978; Warren and Mahowald, 1979). The chorion proteins are synthesized by the follicle cells and secreted onto the surface of the oocyte (Petri et al., 1976; Spradling and Mahowald, 1979; Waring and Mahowald, 1979). Acid phosphatase is synthesized within the oocyte-nurse cell syncytium (Postlethwait and Gray, 1975; Giorgi, 1974). In this study, the synthetic patterns of tubulin and

LLOYD, RAFF, AND RAFF

Oogenesis in Drosophila

other proteins during Drosophila oogenesis were determined. Two-dimensional gel electrophoresis was used to analyze newly synthesized proteins. A sensitive and specific radioimmune assay was developed in order to quantitate tubulin accumulation and a dissection technique was devised to analyze separately the three cell types of the egg chamber. MATERIALS

AND METHODS

Drosophila stocks. Eggs and adult flies of Drosophila melanogaster Oregon R, P-2 strain, came from population cages maintained by Dr. A. P. Mahowald. Labeling of ovary proteins. Ovary proteins were labeled in situ by injecting female flies with 0.1 ~1 of [35S]methionine (1000-1360 Ci/mmole, 5-8 mCi/ml), as described by Spradling and Mahowald (1979). After isotope incorporation, flies were anesthetized with CO2 and ovaries dissected in either PBS3 (150 mM NaCl, 3 mM NaN3, 50 mM sodium phosphate, pH 7.4) or in dissection medium (70 mM NaCl, 50 mM KCl, 20 mM MgSO,, 20 mM NaHzP04, 10 mM CaClz, 0.15% autoclaved Difco yeast extract, 0.2% glucose, 10 mg/ml phenol red, NazC03 to adjust to pH 6.6, sterilized by filtration through a 0.45~pm-pore Millipore filter) (Loyd, 1979). Alternatively, ovary proteins were labeled in organ culture by incubating isolated ovaries in the tissue culture medium “ZD” of Wyss and Bachmann (1976), made up without serum, cystine, or methionine. Fresh unlabeled cystine and [?S]methionine (to 5-10 mCi/ml) were added just before use. After either labeling protocol, ovaries were washed twice at 4’C and held at 4°C for dissection of egg chambers or further processing. Gel electrophoresis. Two-dimensional (2-D) polyacrylamide gel electrophoresis was carried out with non equilibrium pH gradients from pH 4.6 to 8.7 in the first dimension and SDS gels in the second dimension, by the procedure of O’Farrell et al. (1977) as modified by Waring et al. (1978). One-dimensional SDS-gel electrophoresis was performed by the method of Laemmli (1970). Fluorography was performed by the method of Bonner and Laskey (1974). Peptide mapping. The peptides obtained after specific limited proteolysis of tubulin and other proteins were analyzed by the method of Cleveland et al. (1977). Pur$cation and estimation of tub&in. Tubulin was purified from Drosophila eggs and embryos by self-as3 Abbreviations used: PBS, phosphate-buffered saline; 2-D, two-dimensional; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; BLTH buffer, 0.5% BSA, 0.05% Lubrol PX, 0.05% sodium heparin, 5 mA4 EDTA, 3 mAf NaNa, 150 mAf NaCl, 50 mM Tris-HCl, pH 7.4; BLT buffer, BLTH buffer minus heparin; LST buffer, BLT buffer minus BSA; RIA, radioimmunoassay.

melanogaster

273

sembly in vitro according to the method of Green et al. (1975,1979), slightly modified to allow for more efficient recovery of tubulin after multiple cycles of assemblydisassembly and for use in microscale preparations (Loyd, 1979). Further purification was achieved by phosphocellulose chromatography according to the procedure of Weingarten et al. (1975). Protein concentrations were estimated by the method of Lowry et al. (1951). Tubulin gave only 86% as intense a response in the Lowry assay as an equal weight of BSA; tubulin determinations were appropriately corrected. Colchicine binding assay. Tubulin solutions were adjusted to 100 mM sodium glutamate, 20 mMmagnesium acetate, and 10 mM sodium phosphate, pH 6.8, [3H]colchicine was added, and the capped assay tubes were incubated at 30°C for 120 min. Under these conditions the binding reaction reached equilibrium for solutions containing at least 0.4 mg tubulin/ml and at least 15 PM colchicine. Assay tubes were then chilled in an ice bath and aliquots were spotted onto moist DE81 filter disks, batch washed as described by Raff (1977), and counted in a scintillation counter. Before they were used in Scatchard (1949) plot analyses, raw colchicinebinding data were corrected for first-order decay of colchicine-binding activity prior to assay (measured for each tubulin preparation; the average half-life at 0°C was 35 hr [Loyd, 19791); 87% filter binding efficiency (determined by comparison with gel filtration on Sephadex G-150 [Loyd, 19791); and 30% counting efficiency of tritium. Antibody preparation. Young, female New Zealand White rabbits were injected subcutaneously or intradermally at approximately monthly intervals with 1 mg of highly purified Drosophila embryo tubulin emulsified in Freund’s adjuvant. Serum was made 15 mM in NaN3, filtered through a 0.45-pm-pore Millipore filter, and stored at 4°C. The antiserum used for all radioimmunoassays came from one rabbit and was found to specifically recognize only the a-tubulin complex and &tubulin by a procedure similar to that of Burridge (1978), employing a sandwich overlay of SDS-gels with ‘=I-labeled goat anti-rabbit IgG (Loyd, 1979). Protein iodination. Tubulin was iodinated by a modification of the methods of Greenwood et al. (1963) and Sonoda and Schlamowitz (1970). Thirty micrograms of highly purified tubulin was mixed with 100 ~1 of 0.5 M sodium phosphate buffer, pH 7.0, containing 1 mCi of Na’%I. The solution was stirred rapidly at 25°C as 100 ~1 of chloramine-T (5 mg/ml) and 100 ~1 of sodium metabisulfite (10 mg/ml) were added about 10 set apart. Separation of the unbound isotope from the iodinated protein was done by chromatography on Sephadex G25. The most active fractions of the iodinated tubulin peak were pooled, made to 10 mg BSA/ml, and stored

274

DEVELOPMENTALBIOLOGYVOLUME86,1981

radiogram corresponded with most of the proteins visualized on the gel by Coomassie blue staining (i.e., the synthesis of the major accumulated protein species was continuing during the labeling period); (2) the general correspondence between the autoradiographic spot intensities and the corresponding stained spot intensities (i.e., the relative rates of synthesis of major proteins were sustained during the labeling period); and (3) the absence of major autoradiographic spots that did not correspond to stained spots on the 2-D gel (i.e., major novel species were not induced by the experimental procedure). A large number of proteins are resolved. Most of the labeled proteins have relatively acidic isoelectric points and apparent molecular weights between 50 and 100 kdalton. Tubulins were initially identified by coelectrophoresis of labeled ovary proteins with tubulin purified from Drosophila eggs by three cycles of assembly in vitro. We could identify several tubulins. The a-tubulin subunits comprised a complex of at least two spots of somewhat variable resolution. The second class of tubulins, the /3-tubulins, are represented in ovaries by a single subunit, /?I (Kemphues et al., 1979). Tubulins were further characterized by peptide analysis, as shown in Fig. 2. Another protein, designated as X in Fig. 1, was further investigated because its electrophoretic mobility is similar to that of the &tubulin subunit specific to testis (Kemphues et al., 1979). This protein was not observed in any tissue other than ovary; it neither coassembled with authentic tubulins nor did it precipitate with vinblastine sulfate. The possibility that it might constitute an inactive form of /3-tubulin was eliminated by comparison of the peptide map of X with that of both /3r- and &tubulins from Drosophila (Fig. 2). Several other proteins in addition to the tubulins were identified as indicated in Fig. 1 and followed during this study. Actin (A) was identified by its comigration on 2-D gels with actin prepared from Drosophila embryos by the method of Clark and Merriam (1978). As previously observed by Storti et a2. (1978) and Fyrberg and Donady (1979), three forms of actin are synthesized. Yolk (Y), also composed of three major proteins (Warren and Mahowald, 1979), is a prominent synthetic component of labeling in situ. Two other proteins, whose identity is at present unknown, also served RESULTS as convenient reference markers. These are N97 and P, proteins of 97,000 and 87,000 daltons, respectively, Ovarian Proteins Synthesized in Viva prominent in both stained and labeled patterns. The Ovarian proteins could be readily labeled with latter is of particular interest since its labeling is per[35S]methionine in vivo and resolved on 2-D gels, as sistent; that is, it is both synthesized and accumulated shown in Fig. 1A. That the labeling thus achieved rep- at 25°C at all stages of oogenesis, and it is also one of resents an essentially normal pattern of synthesis is the principal proteins labeled in ovaries under condibased upon three criteria: (1) The spots on the auto- tions of heat shock. at -40°C in the absence of reducing agents. Although the tubulin was appreciably degraded by this protocol, it was still specifically recognized by the antitubulin antiserum and was effective in the radioimmunoassay. Radioimmunoassay for tub&n. Experimental tissue samples and dechorionated embryos which served as standards were sonically disrupted in BLTH buffer (0.5% BSA, 0.05% Lubrol PX, 0.05% sodium heparin, 5 mM EDTA, 3 rnM NaN3, 150 mM NaCl, 50 mM TrisHCl, pH 7.4) and centrifuged at 30009 for 3 min, and the resulting supernatants were used for the assay. The amount of tubulin in the experimental extract was determined by the relative competition with a known amount of iodinated tubulin compared to that in the extract made from a counted number of embryos. The radioimmunoassay was patterned after that of Van de Water and Olmsted (1978). Twenty-microliter aliquots of the standard and experimental extracts were mixed with 20 ~1 of serum at a dilution of l/1600 to l/3200 in BLT buffer (BLTH buffer without heparin). A l/1600 dilution of antitubulin serum precipitated about 45% of the immunoprecipitable counts. Sufficient extra normal rabbit IgG was added to the diluted serum to bind at equivalence with the staphylococcal immunoadsorbent reagent [a 10% suspension of Staphylococcus aureus, Cowan I cells, killed and fixed by the procedure of Kessler (1975)] to be added later. After 70 to 180 min at 24”C, 20 ~1 of ‘%I-tubulin containing 4 X lo5 cpm of immunoprecipitable material was added. After an additional 60 min at 24”C, the tubes were placed in an ice bath and 20 ~1 of staphylococcal reagent was added. After 10 min, 1.8 ml of LST buffer (BLT buffer without BSA) was added and the tubes were centrifuged for 10 min at 3OOOgat 4°C to pellet the immunoadsorbant. Supernatants were withdrawn and discarded without disturbing the pellets, which were then suspended in 1 ml of ice-cold LST buffer, collected on 0.45-pm-pore Millipore filter disks, and washed with eight l-ml aliquots of ice-cold LST buffer. The filters were then dried and ‘%I radioactivity was determined by scintillation counting. Each assay was performed in duplicate or triplicate and for each extract, the average activity retained by nonimmune serum was subtracted from that retained by the antitubulin serum.

LLOYD,RAFF,ANDRAFF

Oogenesis in Drosophila melanogoster

275

FIG. 1. Tubulin synthesis in vivo in ovaries and in vitro in staged chambers. Panel A: Autoradiographic patterns of the proteins from four ovaries which were labeled with [%]methionine for 1 hr in vivo and displayed on a 2-D gel. The first-dimension, nonequilibrium pH gradient electrophoretic migration was from left (acidic) to right (basic). Second (SDS)-dimension migration was from top to bottom. The positions and molecular weights, in kilodaltons of six standard proteins which were run in a flanking lane, are marked in the left margin. Protein designations: (Y,the cu-tubulins; 8, &-tubulin; X, a nontubulin protein of approximately 50 kdalton; Y, three yolk proteins; A, the actins; P and N97, unidentified proteins (see text). Panels B-F: Tubulin synthesis in staged chambers. Four ovaries were incubated in “ZD” medium with added [%]methionine for 1 hr at 25°C. The labeled egg chambers were dissected, sorted into groups by their oogenetic stage, and the proteins were displayed on 2-D gels from which the fluorograms shown were made: (B) egg chambers of stages 1-9, (C) stage 10; (D) stages 11 and 12; (E) stage 13; and (F) stage 14. All panels employ the axis conventions and protein designations of panel A.

Ovarian Proteins Sgnthesixed in Vitro: The Time of Tubulin and Other Protein Synthesis during Oogenesis The ability to obtain normal protein labeling patterns in isolated egg chambers is important in deciding whether the origin of any particular protein is auton-

omous to the egg chamber or, as is the case with yolk proteins, is synthesized in some other tissue and transported to the egg chamber. Large numbers of dissected ovaries could be labeled with [35S]methionine in organ culture without substantially altering the normal labeling pattern obtained in vivo. Egg chambers from ovaries labeled for 1 hr in organ

276

DEVELOPMENTAL BIOLOGY

A

B

C

D

E

VOLUME 86, 1981

moved during dissection. Little can be said about the site before stage 10 from this experiment, however, since the pooled stages l-9 (Fig. 1B) included many other ovarian cell types besides the egg chambers. The three actin forms were synthesized about equally (relative to each other) at all of the stages examined. The yolk peptides are weakly labeled in organ culture. This may be the result of secretion of yolk by ovaries in culture (Postlethwait et al., 1980). Developmentally Specific Protein Synthesis

FIG. 2. Peptides obtained after proteolysis by Staphylococcus aureus protease (1.25 fig/ml) by method of Cleveland et al. (197’7). (A) Coomassie blue stained pattern of peptides from p,-tub&n purified from Oregon R-strain P2 eggs and embryos by vinblastine sulfate precipitation followed by one-dimensional gel electrophoresis; (B) fluorogram of the gel lane in A, showing copurified [“Slmethionine-labeled Oregon R ovary fir-tubulin; (C) autoradiogram of [?3]methionine-labeled Oregon R testis-specific &-tubulin purified by coassembly in vitro with egg and embryo tubulin followed by one-dimensional gel electrophoresis; (D, E) fluorograms of [%]methionine-labeled ovary protein X from Oregon R and Oregon R-strain P2 females, respectively, purified by 2-D gel electrophoresis. Peptide patterns of a-tubulins (not shown) under the same conditions were of a characteristic and reproducible pattern, distinct from those of j3-tubulin (see Kemphues et al., 1979).

culture were manually dissected and sorted into groups of specific developmental stage according to the staging series of King (1970). Proteins from staged egg chambers were then displayed on 2-D gels and fluorograms were prepared, as shown in Figs. lB-F. The appearance of a novel protein (S) of 70 kdalton apparent molecular weight is of concern. Other investigators have reported that ovarian tissues labeled in culture synthesize heatshock RNAs (Petri et al., 1977; Spradling and Mahowald, 1979). Protein S coincides in electrophoretic mobility with one of the major heat-shock proteins, suggesting that labeling of ovaries in organ culture evoked a mild heat-shock response. However, no noticeable changes occurred in the synthesis of any of the major proteins normally synthesized in vivo. Thus, labeling in organ culture could be validly used to assess normal ovarian protein synthetic patterns. The a-tubulins, &-tubulin, actin, as well as proteins X and P were major products of normal protein synthesis in egg chambers during all stages examined. The synthesis detected in this experiment during stages lo14 certainly occurred within the egg chambers, since the ovariolar epithelial sheaths, which are the tissues most likely to contaminate the egg chambers, were re-

The experiments shown in Fig. 1 revealed some changes in protein synthetic patterns related to developmental stage. Two spots, designated FllO and F80, that appear in stages l-9 (Fig. lB), become major spots in stage 10 (Fig. 1C) and disappear by stages 11-12 (Fig. 1D). Another such protein is N97, which appeared in early egg chamber stages (Fig. 1B) but at no other stage. Other, more subtle changes are also present. The development-related changes in chorion-protein synthesis are not seen in this experiment because the major chorion proteins are methionine poor (Petri, et al., 1976; Waring and Mahowald, 1979). Sites of Synthesis of Tub&n and Other Proteins in Drosophila Egg Chambers Egg chambers contain three discrete cell types, the oocyte, the nurse cells, and the follicle cells. We devised the dissection technique shown in Fig. 3 to separate and recover the three cell types with little cross-contamination. Stage 10B was chosen for dissection because at this stage the nurse cells reach their maximum size (just before their contents are injected into the oocyte), and the majority of follicle cells form a compact cap around the oocyte. Stage 10 egg chambers from flies labeled in vivo with [%]methionine were dissected from ovaries at 0°C and placed in a 5-mm-deep, ZO-mm-diameter well of a cold glass slide in about 20 ~1 of PBS. Chambers were cut with a pair of tungsten needles crossed at the level of the interface between the nurse chamber and the oocyte; when the needles were drawn past one another with a quick, flicking motion, the chamber was sheared and the two halves were propelled to the opposite sides of the dissection well (Figs. 3A, B). Nurse chambers were removed in small groups before they leaked much of their contents into the medium; in cold dissection solutions, the cytoplasm was very viscous and leakage was minimal. The ooplasm leaked from the oocytes as the medium warmed or could be extruded by gentle prodding (Figs. 3B, C), leaving the follicle cell caps intact (Fig. 3D). After the follicle cell caps were removed

LLOYD, RAFF, AND RAFF

FIG. oocyte at the follicle

Oogenesis in Drosophila mdanogaater

3. Dissection of stage 10B egg chambers into their component cell types: Panel A shows (0), thick cap of clear follicle cells (F), and cluster of nurse cells (N) with their giant, clear level marked by the arrow and “CUT,” giving the two halves seen in panel B. Panel C is cell half with its ooplasm leaking out. Panel D shows the shiny follicle cell cap after the

from the well, the residual fluid constituted the ooplasm fraction. Both the nurse chamber and the follicle cell cap were recovered essentially intact. The ooplasm, however, was diluted into the dissection medium and was in contact with the glass bottom of the dissection well; the efficiency of its recovery was thus less certain. Experiments designed to gauge the recovery of proteins from the dissection well by addition of known amounts of radioactively labeled proteins to the wells revealed that

277

a stage 1OB egg chamber with its opaque nuclei. Egg chambers were sheared in half a higher-magnification view of an oocyteooplasm had leaked out.

both ooplasmic proteins and tubulin were recovered from the dissection well in 70-95’S yield (Loyd, 1979). Proteins synthesized in the three cell types were displayed on 2-D gels, as shown in Fig. 4. Reproducible differences were observed in the patterns of proteins in the different cell types. The yolk protein spots are virtually absent from the nurse cell proteins (Fig. 4A) both on the stained gel and on its fluorogram. Conversely, both the ooplasm (Fig. 4B) and the follicle cell (Fig. 4C) gels yielded prominent yolk bands on both

278

DEVELOPMENTAL BIOLOGY

VOLUME 86, 1981

FIG. 4. Protein synthesis in specific cell types: fluorographic patterns of (A) nurse cells, (B) oocytes, and (C) follicle cells, from ovaries labeled for 3 hr in vivo with [%]methionine. The axis conventions and protein spot designations are the same as those in previous figures; N69, an unidentified protein used as a reference spot.

their stained gels and on their fluorograms. All three forms of actin were heavily labeled in short-term incubations in all cell types. Tubulin accumulated label in both nurse cells and oocytes, but the level of tubulin labeling in follicle cells was low. In addition, the follicle cell pattern differs strikingly in the relative prominence of spots FllO and F80 in comparison with the nurse cell

and ooplasm patterns. The FllO and F80 spots were previously noted as proteins synthesized primarily during stage 10. The experiment shown in Fig. 4 confirms their synthesis in stage 10 and further suggests that they are synthesized mainly, if not exclusively, in the follicle cells. The peptides produced by heat-shocked stage 10B follicle cells were clearly distinguishable from

LLOYD, RAFF, AND RAFF

Oogenesis in Bosophila

melanogaster

279

FIG. I.-Continued

both FllO and F80 (data not shown). Proteins F80 and FllO are probably not chorion proteins, since they do not appear in preparations of isolated chorions. Their migration over a range of isoelectric points suggests that F80 and FllO may represent families of proteins rather than single species. Some cross-contamination of the three cell types does occur. In order to evaluate both cross-contamination and time-dependent developmental changes in the patterns, selected spots on the fluorograms were scanned on a densitometric gel scanner. Integrated peak areas from each fluorogram were normalized to the area of spot N69, a non-heat-shock protein which appeared to be representative of the synthesis of many of the major spots in all three cell types and which was identifiable and easily scanned on all of the fluorograms. Normalization compensated for differences in total label content, in exposure times to the X-ray film, and in the distribution of label in different cell types. These experiments indicate that the degree of cross-contamination between cell types is too low in most dissections to account for the amount of label present in the major proteins observed in the isolated cell types. The levels of yolk, FllO, and F80 in nurse cells suggest that yolk protein does not enter the oocyte from the nurse cells and that neither ooplasm nor follicle cell material contaminates the nurse cell fraction by more than 2%. Based upon the amount of F80 in oocyte patterns, follicle cell proteins constitute less than 15% of

the material on the oocyte gels. Conversely, if the yolk present in the follicle cell patterns derives exclusively from ooplasmic contamination, then ooplasmic contamination is generally less than 10% in the follicle cell patterns. The level of yolk labeling by follicle cells (Fig. 4C) is consistent with the observation of Brennan et al. (1981) that follicle cells are a major site of vitellogenin synthesis. The normalized data from fluorograms of nine gels (three of which are shown in Fig. 4) indicate that /3tubulin, actin, and N-69 are synthesized in all cell types, whereas yolk accumulates only in oocytes, and FllO and F80 are the products of the follicle cells. Accumulation

of Tub&in during Oogenesis

Because of the small amount of material available from hand-dissected ovaries, a sensitive radioimmune assay (RIA) was used to quantitate tubulin. Purified egg tubulins and tubulins present in supernatant fractions did not exhibit equivalent antigenic behaviors in the RIA (Loyd, 1979). Since we wished to measure tubulin concentrations in supernatants from ovary homogenates, we used embryo supernatants, which did have the same antigenic behavior as the experimental samples, as the source of standard amounts of competitor in the assay. The tubulin content in such supernatants, quantitated by Scatchard (1949) plot analysis of colchine binding, was found to be 29 ng tubulin

280

DEVELOPMENTALBIOLOGY

per average embryo. Extracts of Drosophila eggs, embryos, and egg chambers, particularly at stage lOB, were found to cause high levels of nonspecific binding of tracer to the adsorbent in nonimmune serum control reactions. Since egg chambers are rich in basic ribosomal and chromosomal proteins and have a high DNA content, agents known to bind to basic proteins or to nucleic acids were added to homogenates of vitellogenic egg chambers. The addition of the polyanionic polysaccharide heparin eliminated the nonspecific binding (Loyd, 1979). In the presence of heparin at an optimal concentration of 200 pg/ml, the RIA could be used with great sensitivity to quantitate tubulin content of small samples at concentrations between 2 and 500 ng per assay. A typical RIA standard curve is shown in Fig. 5. The RIA assay was used to determine the amount of tubulin in egg chambers of specific development stages; Fig. 6 displays the data pooled from seven experiments. The means that differ from one another at the 95% confidence level are those for stages 9 or 10A; 10B; 14; and embryos. The data in Fig. 6 indicate that egg chambers accumulate more than 80% (14 of 17 ng) of their total tubulin during the last 10 hr of oogenesis and that an apparent burst of tubulin synthesis during stage 10 may account for more than half of that accumulation. If none of the accumulated tubulin is degraded during oogenesis, the minimum average rate of tubulin synthesis

E

’ . 0.6

21.. 3

5

.

.

10

20

. . . 50

.

100

.*.. 300

l0.J

1000

COMPETITOR TUBULIN (ng) FIG. 5. A radioimmunoassay standard curve for Drosophila tubulin. Dilutions of supernatants from counted embryos sonically disrupted in measured volumes of BLTH buffer (competitor tubulin) were assayed in the presence of 200 ng heparin/ml. A tubulin content of 29 ng per embryo was determined from colchicine-binding data. The specifically bound cpm equal the difference in cpm of ‘%I-labeled tubulin bound by antitubulin serum and that bound by preimmune serum in the presence of each concentration of competitor tubulin. The B/B,, ratio is the quotient of the activity bound at a particular competitor tubulin level by the activity bound in the absence of competitor. A serum dilution of 112133 was used in this particular RIA.

VOLUME 86,1981

r

l-

TIME

(hr)

FIG. 6. Tubulin content of egg chambers and embryos. The abscissa is graduated in hours after the entry of the stage 1 egg chamber into the vitellarium, according to the data of David and Merle (1968). The time scale is telescoped to accommodate embryogenesis, which takes about 22 hr at 25°C (Bownes, 1975). The arrow marks the time of ovulation, fertilization, and laying. The width of each bar represents the duration of the stage designated within it. The developmental stage for each experimental group is given on each bar. No data are reported for stage 11, which occupies the short gap between stages 10B and 12. Stage 10 is arbitrarily divided in half because the durations of suhstages 10A and 10B are not known. The top of each bar represents the mean tubulin content per egg chamber or embryo determined for that stage; error bars represent one standard deviation of the mean. All values were determined by the RIA, except for the value for embryos, which was determined by colchicine binding.

between stages 9 and 14 is 1 ng/hr, but the rate may exceed 2 ng/hr during stage 10. Since tubulin continues to accumulate between stages 10B and 14, its synthesis is almost certainly occurring in the oocyte on mRNA molecules that were transcribed in the nurse cells and injected into the oocyte during stage 11. The additional increase of 12 ng of tubulin between stage 14 and the middle of embryogenesis is probably an underestimate, since any follicle cell tubulin would be included in the value determined for the stage 14 egg chambers but not in the value determined for dechorionated embryos. The tubulin contents of the three compartments of stage 10B egg chambers, dissected as described for Fig. 3, were also determined by RIA. The means of four determinations for each cell type from two separate experiments as well as the data from the experiments shown in Fig. 6, are tabulated in Table 1. Stage 10B nurse cells contain about half of the total tubulin in the chamber, but the follicle cells account for most of the rest, leaving the oocyte with only about a twentieth of the total tubulin. The mean value for the sum of the parts from each dissection is lower than the mean value

LLOYD, RAFF, AND RAFF

281

Oogenesis in Drosophila melonogoster TABLE 1

TUBULIN CONTENT OF EGG CHAMBERS, DECHORIONATED EMBRYOS, AND STAGE 10B CELL TYPES

Stage or cell type

Mean tubulin content“ (rig/chamber or embryo)

95% Confidence limits*

9 10A 10B 12 13 14 Embryos, chorions removed

2.87 5.12 12.4’ 13.2 14.3 17.7 29.0

* 0.83 * 2.11 k 3.85 + 12.4 + 17.6 + 2.5 f 3.3

6 7 15 4 3 8 5

0.59 3.32 4.61

k 0.95 + 2.69 f 1.62

4 4 4

9.4d 13.9”

f 3.6 * 5.7

10

10B oocytes 10B follicle cells 10B nurse cells 10B (sum of cell types) 10B (intact egg chambers)

Number of determinations

5

a All values were determined by radioimmunoassay except that for embryos, which was determined by Scatchard analysis. * Confidence limits were determined by Student’s t test using n-l & ‘This mean was determined from values for the sum of the dissected cell types as well as the values for intact egg chambers. See footnotes d and e. d Values on this line derive from the sums of the values for the cell types from five dissections. One of the five was visibly cross-contaminated, so, data from it were not used in calculation of the means and confidence limits for the separated cell types, but the sum of the values was still considered to be a valid determination of the total tubulin in the stage 10B egg chamber. eValues on this line derive from determinations of total tubulin from unfractionated stage 10B egg chambers.

for intact stage 10B egg chambers, but a t test for 2 means revealed that the totals were not statistically different (0.4 > P > 0.2). Experimental results described previously demonstrated that recovery of tubulin from the dissection well was between 70 and 95%) and crosscontamination of the cell types was generally less than 15%. We consistently recovered comparable amounts of [35S]methionine-labeled proteins in the nurse cell and oocyte fractions (Fig. 4) and found strong labeling of the tubulin spots on fluorograms from 2-D gels of ooplasm. Thus, the difference between the mean values for whole stage 10B egg chambers and for the sum of the values for the cell types probably did not arise from preferential loss from any single compartment. DISCUSSION

The data reported here show that during oogenesis in Drosophila, the majority of proteins resolvable on 2-D gels are synthesized throughout oogenesis but modulations in relative synthesis, as well as developmentally specific synthesis also occur. Our results are in agreement with those in a recent study of Drosophila oogenesis and early embryogenesis by Gutzeit and Gehring (1979), who also observed some protein synthesis specific to a particular time or region of the egg chamber or embryo. We examined proteins synthesized both in vivo and

in organ culture of ovaries in vitro. Both methods gave essentially identical labeling patterns, except that ovaries labeled in vitro also synthesized S, a major novel component which appears to be a heat-shock protein, hsp70, shown to be induced even at 26°C (Ashburner and Bonner, 1979). However, we did not find that labeling in vitro induced severe heat shock, as has been reported (Petri et al., 1977; Spradling and Mahowald, 1979). This may be due to differences in culturing conditions or to the fact that the observations by Petri et al., (1977) were made on separated egg chambers, which we have found to be more susceptible to heat shock than are intact ovaries. A second protein, P, which is synthesized in ovaries labeled either in viva or in vitro, also appears to be a heat-shock protein, hsp82. However, this protein is prominent in the stained pattern of proteins from unlabeled ovaries, and appears in labeled protein patterns obtained by other investigators in unshocked ovaries (Lewis et aZ., 1975; Kuo and Garen, 1978; Waring and Mahowald, 1979), tissue culture cells (Mirault et al., 1978), and testes (K. J. Kemphues, personal communication). Ashburner and Bonner (1979) showed that hsp82, like hsp70, has a low temperature threshold for induction. Furthermore, in an analysis of heat-shock RNA synthesis, Spradling et a2. (1977) could not rule out the possibility that one or both of the two largest RNA species were synthesized in unshocked cells. The proteins encoded by those two RNAs were later found

282

DEVELOPMENTAL BIOLOGY

to be hsp82 and hsp70. All of these observations indicate that hsp82 is a normal, major product of unshocked ovaries. The dissection of stage 10B egg chambers exploited the natural segregation of the cell types at this stage. Nurse cells and oocytes actively synthesized tubulin and actin, while follicle cells synthesized relatively less tubulin and other proteins than actin and a few follicle cell-specific proteins. This observation is consistent with the specialization of follicle cells to the synthesis of large amounts of vitelline membrane components during stages 9 and 10 (King, 1970; Mahowald and Kambysellis, 1980). Follicle cells in fact showed the most striking example of developmentally specific protein synthesis, in the FllO and F80 proteins, synthesized largely, if not exclusively, in the follicle cells of stage 10. Another large protein, designed N97, is synthesized mainly in the oocyte-nurse cell syncytium, and only in egg chambers of very early stages, prior to stage 10. The actin family of peptides actively incorporated [35S]methionine in all three cell types of stage 10B and in egg chambers from stages 9 to 14. Since actin and tubulin contain similar percentages of methionine residues (Stephens and Linck, 1969) and since actin is invariably more heavily labeled than tubulin, its synthesis rate must exceed the tubulin synthesis rate in all stages. Tubulin accumulates roughly in parallel with the volume increase of the egg chamber, and it is probably synthesized from mRNA molecules that were transcribed in the highly polyploid nuclei of the nurse cells. A similar origin is indicated for the actin present in the egg. Approximately 15 ng of tubulin accumulates per egg chamber during the last 13 hr of oogenesis, for an average rate of 1 ng/hr. At stage lOB, a large fraction of the total tubulin of the egg chamber was present in the follicle cells, but it probably never reaches the oocyte due to the impermeability of the vitelline membrane. Most of the tubulin present in the oocyte-nurse cell syncytium at this stage was localized in the nurse cells. This result seemed paradoxical because 2-D gel analysis revealed comparably active tubulin synthesis in both the oocyte and the nurse chamber. The lesser accumulation of tubulin in oocytes than in nurse cells suggests that sufficient mRNA and ribosomes have been injected to allow a high rate of tubulin synthesis, but that little tubulin has yet accumulated. This explanation is consistent with the current knowledge of the dynamics of the injection process: Injection is continuous at a low rate until stage 11, when the remaining nurse cell cytoplasm is injected rapidly (Mahowald and Kambysellis, 1980).

VOLUME 86.1981

The parameters described for tubulins and their mRNAs from Drosophila and other systems allows the calculation, by methods described by Palmiter (19’75), of the amount of tubulin mRNA necessary for tubulin synthesis in late oogenesis. Our assumptions are that the mRNAs for D. melanogaster tubulin are poly(A)+, as in rat and chick brain (Saborio et aZ.,1978; Cleveland et aZ., 1978); that they contain about 2000 nucleotides, as in chick and rat brain (Cleveland et al., 1978; Saborio et al., 1978; Bryan et al., 1978); and that they have an average ribosome packing number of 15 ribosomes per message (as estimated from the data of Saborio et al., 1978; Lovett and Goldstein, 1977; Anderson and Lengyel, 1979; Lamb and Laird, 1976). Using a peptide elongation rate of 100 residues per minute at 25°C (Conconi et al., 1966) combined with the value of 430 amino acid residues per tubulin subunit, the method of Palmiter (1975) gives an estimate of 0.086 fmole, or 0.055 ng of tubulin mRNA per egg chamber. If 1% of the 0.15 g of total RNA of an egg is nonmitochondrial, poly(A)+ RNA (Anderson and Lengyel, 1979; Limbourg and Zalokar, et al., 1974), then tubulin mRNA 1973; Fausto-Sterling accounts for approximately 4% of the total nonmitochondrial, cytoplasmic poly(A)+ RNA of the egg, or about 2% of total poly(A)+ RNA. This estimate agrees with the 1.6% of the total ooplasmic protein that tubulin constitutes at stage 14 and seems to be reasonable in light of the intensity of labeling of the tubulin spots on fluorograms of 2-D gels. We wish to express our gratitude to L. Van de Water for help with the radioimmunoassay and to A. P. Mahowald, A. C. Spradling, L. H. Green, and K. J. Kemphues for valuable discussions of the work. This study was supported by NSF Grant PCM 78-10218 to E.C.R. During the study J.E.L. was a predoctoral trainee in biochemistry supported by NIH Training Grant 5TOl-GM1046 and R.A.R. was a recipient of USPHS Career Development Award KDO4-HD47. REFERENCES ANDERSON, K. V., and LENGYEL, J. A. (1979). Rates of synthesis of major classes of RNA in Drosophila embryos. Develop. Biol. 70, 217-231. ASHBURNER, M., and BONNER, J. J. (1979). The induction of gene activity in Drosophila by heat shock. Cell 17,241-254. BONNER, W. M., and LASKEY, R. A. (1974). A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 83-88. BRENNAN, M. D., WEINER, A., GORALSKI, T., and MAHOWALD, A. P. (1981). Follicle cells are a major site of vitellogenin synthesis in Drosophila melanogaster. In preparation. BRYAN, R. A., CU?TER, G. A., and HAYOSHI, M. (1978). Separate mRNAs code for tubulin subunits. Nature (London) 272,81-83. BURRIDGE, K. (1978). Direct identification of specific glycoproteins and antigens in sodium dodecylsulfate gels. In “Methods in Enzymology” (V. Ginsburg, ed.), Vol. 50, pp. 54-64. Academic Press, New York.

LLOYD, RAFF, AND RAFF

Oogenesis in Drosophila melanogaster

CLARK, T. G., and MERRIAM, R. W. (1978). Actin in Xenopus oocytes. I. Polymerization and gelation in vitro. J. Cell Biol. 77, 427-438. CLEVELAND,D. W., FISCHER,S. G., KIRSCHNER,M. W., and LAEMMLI, U. K. (1977). Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Chem. 252, 1102-1106. CLEVELAND,D. W., KIRSCHNER,M. W., and COWAN,N. J. (1978). Isolation of separate mRNAs for a- and @-tubulin and characterization of the corresponding in vitro translation products. Cell 15, 10211031. CONCONI,F. M., BANKS, A., and MARKS, P. A. (1966). Polyribosomes and control of protein synthesis: Effects of sodium fluoride and temperature on reticulocytes. J. Mol. Biol. 19,525-540. FAUSTO-STERLING,A., ZHEUTLIN,L. M., and BROWN,P. R. (1974). Rate of RNA synthesis during early embryogenesis in Drosophila melanogaster. Develop. Biol. 40, 78-83. FYRBERG,E. A., and DONADY, J. J. (1979). Actin heterogeneity in primary embryonic culture cells from Drosophila melanogaster. Develop. Biol. 68, 487-502. GIORGI, F. (1974). Acid phosphatase localization in ovarian cells of Drosophila melanogaster. Role of the Golgi apparatus. Histochem. J. 6, 71. GIORGI,F., and JACOB,J. (1977). Recent findings on oogenesis in Drosophila melanogaster. I. Ultrastructural observations on the developing ooplasm. J. Embryol. Exp. Morphol. 138, 125-138. GREEN,L. H., BRANDIS,J. W., TURNER,F. R., and RAFF, R. A. (1975). Cytoplasmic microtubule proteins of the embryo of Drosophila melanogaster. Biochemistry 14,4487-4491. GREEN, L. H., RAFF, E. C., and RAFF, R. A. (1979). Tubulin from a developing insect embryo undergoing rapid mitosis: factors regulating in vivo assembly of tubulin from Drosophila melanogaster. Insect Biochem. 9, 489-495. GREENWOOD,F. C., HUNTER, W. M., and GLOVER,J. S. (1963). The preparation of 1311-labeledhuman growth hormone of high specific radioactivity. Biochem. J. 89, 114-123. GUTZEIT, H. O., and GEHRING, W. J. (1979). Localized synthesis of specific proteins during oogenesis and early embryogenesis in L?rosophila melanogaster. Wilhelm Roux Arch. 187. 151-165. KAMBYSELLIS,M. P. (1977). Genetic and hormonal regulation of vitellogenesis in Drosophila Amer. 2001. 17, 535-549. KEMPHUES, K. J., RAFF, R. A., KAUFMANN, T. C., and RAFF, E. C. (1979). Mutation in a structural gene for a &tubulin specific to testis in Drosophila melanogaster. Proc. Nat. Acad. Sci. USA 76, 3991-3995. KESSLER,S. W. (1975). Rapid isolation of antigens from cells with a Staph&coccal protein A-antibody absorbent: Parameters of the interaction of antibody-antigen complexes with protein A. J. Zmmunol. 115,1617-1624. KING, R. C. (1970). “Ovarian Development in Drosophila melanogaster.” Academic Press, New York. KUO, C. H. and GAREN, A. (1978). Analysis of the coding activity and stability of messenger RNA in Drosophila oocytes. Develop. Biol. 667,237-242. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. LAMB, M. M., and LAIRD, C. D. (1976). The size of poly(A) containing RNA’s in Drosophila melanogaster embryos. Biochem. Genet. 14, 357-371. LEWIS, M., HELMSING, P. J., and ASHBURNER,M. (1975). Parallel changes in puffing activity and patterns of protein synthesis in salivary glands of Drosophila. Proc. Nat. Acad. Sci USA 72.36043608.

283

LIMBOURG,B., and ZALOKAR,M. (1973). Permeabilization of Drosophila eggs. Develop. Biol. 35, 382-387. LOVETT, J. A., and GOLDSTEIN,E. S. (1977). The cytoplasmic distribution and characterization of poly(A)+ RNA in oocytes and embryos of Drosophila. Develop. Biol. 61, 70-78. LOWRY,0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193,265-275. LOUD,J. E. (1979). “Tubulin Synthesis and Accumulation during Oogenesis in Drosophila melanogaster.” Ph.D. dissertation, Indiana University. MAHOWALD,A. P., and KAMBYSELLIS,M. P. (1980). Oogenesis. In “The Genetics and Biology of Drosophila” (M. Ashburner and T. R. F. Wright, eds.), Vol. 2D, pp. 141-224. Academic Press, New York. MAHOWALD, A. P., and TIEFERT, M. (1970). Fine structural changes in the Drosophila oocyte nucleus during a short period of RNA synthesis. Wilhelm Roux Arch. 165, 8-25. MILLER, J. H., and EPEL, D. (1973). Studies of oogenesis in Urechis caupo. II. Accumulation during oogenesis of carbohydrate, RNA, microtubule protein and soluble, mitochondrial and lysosomal enzymes. Develop. Biol. 32. 331-334. MIRAULT, M. E., GOLDSCHMIDT-CLERMONT, M., MORAN, L., ARRIGO, A. P., and TISSIERES,A. (1978). The effect of heat shock on gene expression in Drosophila melanogaster. Cold Spring Harbor Sump. Qua&. Biol. 42,819-827. NOKKULA, S., and PuRO, J. (1976). Cytological evidence for a chromocenter in Drosophila melanogaster oocytes. Hereditas 83, 265268. O’FARRELL,P. Z., GOODMAN,H. M., and O’FARRELL,P. H. (1977). High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12. 1133-1142. PALMITER, R. A. (1975). Quantitation of parameters that determine the rate of ovalbumin synthesis. Cell 4,189-197. PESTELL, R. Q. W. (1975). Microtubule protein synthesis during oogenesis and early embryogenesis in Xenopus lawis. B&hem. J. 145, 527-534. PETRI, W. H., WYMAN, A. R., and HENIKOFF, S. (1977). Synthesis of heat shock mRNA by Drosophila melanogaster follicle cells under standard organ culture conditions. Drosophila Z&rm. Serv. 52,80. PETRI, W. H., WYMAN, A. R., and KAFATOS,F. C. (1976). Specific protein synthesis in cellular differentiation. III. The egg shell proteins of Drosophila melanogaster and their program of synthesis. Develop. Biol. 49, 185-199. POSTLETHWAIT,J. H., and GRAY, P. (1975). Regulation of acid phosphatase activity in the ovary of Drosophila melanogaster. Develop. Biol. 47. 196-205. POSTLETHWAIT,J. H., and HANDLER, A. M. (1978). Nonvitellogenic female sterile mutants and the regulation of vitellogenesis in Drosophila melanogaster. Develop. Biol. 67, 202-213. POSTLETHWAIT,J. H., BOWNES,M., and JOWE?T,T. (1980). Sexual phenotype and vitellogenin synthesis in Drosophila melanogaster. Develop. Biol. 79. 379-387. RAFF, E. C. (1977). Microtubule proteins in axolotl eggs and developing embryos. Develop. Biol. 58, 56-75. SABORIO,J. L., PALMER, E., and MEZA, I. (1978). In vivo and in vitro synthesis of rat brain cr- and j3-tubulins. Exp. Cell Res. 114, 365373. SCATCHARD,G. (1949). The attractions of proteins for small molecules and ions. Ann. N. Y. Acad. Sci. 51, 660-672. SONODA,S., and SCHLAMOWITZ,M. (1970). Studies of ‘%I trace labeling of immunoglobulin G by chloramine-T. Immunochemistry 7. 885898.

284

DEVELOPMENTALBIOLOGY

SPRADLING,A. C., and MAHOWALD, A. P. (1979). Identification and genetic localization of mRNA’s from ovarian follicle cells of Drosqphila melanogoster. Cell 16.589-598. SPRADLING,A. C.. PARDUE, M. L., and PENMAN, S. (1977). Messenger RNA in heat-shocked Drosophila cells. J. Mol. Biol. 109,559-587. STEPHENS,R. E., and LINCK, R. W. (1969). A comparison of muscle actin and ciliary microtubule protein in the mollusk, Aequipecten irradians. J. Mol. Biol. 40.497501. STORTI,R. V., HOROVITCH,S. J., SCOTP,M. P., RICH, A., and PARDUE, M. L. (1978). Myogenesis in primary cell cultures from Drosophila melanogastw Protein synthesis and actin heterogeneity during development. CeU 13.589-598. TELFER, W. H. (1975). Development and physiology of the oocytenurse cell syncytium. Advan. Insect. Physiol. 11, 223-319. VAN DE WATER, L., III, and OLMSTED,J. B. (1978). Quantitation and

VOLUME 86,198l

characterization of antibody binding to tubulin. J. Biol. Chum. 253, 5980-5984. WARING, G. L., ALLIS, C. D., and MAHOWALD,A. P. (1978). Isolation of polar granules and the identification of polar granule-specific protein. Develop. Biol. 66,197-206. WARING, G. L., and MAHOWALD,A. P. (1979). Identification and time of synthesis of chorion proteins in Drosophila melanogaster. Cell 16.599-607. WARREN, T. G., and MAHOWALD,A. P. (1979). Isolation and partial characterization of three major yolk polypeptides from Drosophila melanogaster. Develop. Biol. 68. 130-139. WEINGARTEN,M. D., LOCKWOOD,A. H., Hwo, S.-Y., and KIRSCHNER, M. W. (1975). A protein factor essential for microtubule assembly. Proc. Nat. Acad. Sci. USA 72.1858-1862. WYSS,C., and BACHMANN,G. (1976). Influence of amino acids, mammalian serum and osmotic pressure on the proliferation of Drosophila ceil lines. J. Insect. Physiol. 22, 1581-1586.