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The cytoskeletal framework of sea urchin eggs and embryos: Developmental changes in the association of messenger RNA
DEVELOPMENTAL
BIOLOGY
95,
44’7-458
(1983)
The Cytoskeletal Framework of Sea Urchin Eggs and Embryos: Developmental Changes in the Association of Messenger RNA RANDALL ‘Marine Biological Laboratory, Washington 98195; $Department Zo&gy, Ohio State University,
T. MooN,‘~*~~ ROBERTO F. NICOSIA,*$ CHERIE OLSEN,*~ MERRILL B. HILLE,~ AND WILLIAM R. JEFFERY*~#
Woods Hole, Massachusetts of Pathology, The Medical Columbus, Ohio &9210; and Received
June
025qS; TDepartment of Zoology NJ-G, University of Washington, Seattle, College of Pennsylvania, Philadelphia, Pennsylvania 191U; 5Department of #Department of Zoology, University of Texas at Austin, Austin, Texas 78712
25, 1982; accepted
in revised
form September
17, 1982
Extraction of sea urchin eggs and embryos with Triton X-100 generated a cytoskeletal framework (CSK) composed of a cortical filamentous network and an internal system of filaments associated with ribosomes. The CSK contained only lo-20% of the cellular protein, RNA, and lipid. A specific subset of proteins was enriched in the CSK. Several lines of evidence suggest that mRNA is a component of the CSK of both eggs and embryos. First, the CSK contained poly(A) sequences ,which hybridized with [3H]poly(U). Second, the CSK contained polyribosomes. Finally, RNA extracted from the CSK showed translational activity in an in vitro system. The nonhistone messages present in the CSK were qualitatively similar to those solubilized by detergent, as determined by separation on polyacrylamide gels of the products of in vitro translation. In the unfertilized egg, most mRNA was present as nonpolyribosomal messenger ribonucleoprotein complexes which, along with monoribosomes, were efficiently extracted by Tritop X-100. The converse was found in blastulae, as most of the mRNA was present as polyribosomes associated with the CSK, although monoribosomes were still efficiently extracted by detergent. These results indicate a correlation between the activation of protein synthesis in eggs and the association of polyribosomes with the CSK.
INTRODUCTION
Our perception of the intracellular conditions under which RNA is synthesized, transported, and translated has recently undergone marked changes. The discovery that mRNA was associated with proteins to form messenger ribonucleoprotein (mRNP) complexes led to studies showing that mRNA is present as mRNP from the time of its synthesis until its ultimate degradation (Spirin, 1978). A current revolution in our understanding of RNA metabolism centers around the issue of whether nuclear ribonucleoprotein complexes and cytoplasmic polyribosomes are associated with cellular filaments constituting, respectively, the nuclear skeleton (Long et al., 1979; van Eekelen and van Venrooij, 1981) and the cytoskeleton (Lenk et al., 1977; Lenk and Penman, 1979; Fulton et al., 1980; Cervera et al., 1981). That polyribosomes could be associated with a cellular matrix raises the fundamental question of whether their interaction can se:rve as a mechanism of translational control. Experiments to answer this question are feasible, since cytoskeleton preparations can readily be prepared by extraction of cells with nonionic detergents such as Triton X-3.00. The detergent quickly solubilizes most of the lipi’d, tRNA, and monoribosomes, ’ Present address: Division of Biology, California Institute nology, Pasadena, Calif. 91125. To whom correspondence addressed.
of Techshould be
leaving a detergent-insoluble cytoskeletal framework (CSK) containing microfilaments, microtubules, and other filaments (e.g., Brown et al., 1976; Heuser and Kirschner, 1980; Goldman et al., 1981), up to 200 different proteins (Schliwa et al., 1981) including an mRNA 5’ cap-binding protein (Zumb6 et al., 1982), and most of the polyribosomes (Cervera et al., 1981). A possible role for the cytoskeleton in regulating translation has been suggested by studies demonstrating that HeLa cell CSKs selectively bind translationally active viral mRNA and exclude host mRNA which is not being translated (Lenk and Penman, 1979). Embryonic systems may be useful for investigating the role of the cytoskeleton in translation, since developing sea urchins (Wells et al., 1981) and surf clams (Rosenthal et al., 1980) exhibit translational control of protein synthesis. In sea urchin eggs, fertilization elicits a rapid increase in the rate of protein synthesis (reviewed by Raff, 1980). The templates for this synthesis are maternal messages synthesized during oogenesis and stored in the egg as nonpolyribosomal mRNPs. Although the rate of polypeptide chain elongation is slower in the unfertilized egg than in the developing embryo, the primary block to translation in the unfertilized egg appears to be some step in the binding of 40 S ribosomal subunits to mRNPs. This block in the recruitment of stored mRNPs into polyribosomes may be due to the associ-
447
0012-1606/83/02044'7-12$03.00/O Copyright All rights
0 1983 by Academic Press. Inc. of reproduction in any form reserved.
448
DEVELOPMENTAL
BIOLOGY
ation with the mRNA of a phenol-soluble inhibitor of translation (Jenkins et al., 1978; Ban and Ilan, 1978), though recent data on the translational efficiencies of egg mRNPs do not support this mechanism of translational control (Moon et al., 1982). The studies on the selective binding of viral mRNA to the host cytoskeleton, mentioned above, however, raise the possibility that the low level of protein synthesis in unfertilized sea urchin eggs may be due to a lesion in the interaction of the stored mRNPs with the egg CSK. The postfertilization polymerization of actin or other cytoskeletal filaments (reviewed by Vacquier, 1981) may be indicative of a restructuring of the egg CSK which promotes its interaction with stored mRNPs and increases the rate of translation. To test this possibility we have isolated the CSK from sea urchin eggs and embryos by detergent extraction and examined its composition, particularly with respect to the distribution of nonhistone mRNAs and polyribosomes. MATERIALS
AND
METHODS
Arbacia punctulata were collected at Woods Hole, Massachusetts; Lqtechinus pi&us were obtained from Pacific Biomarine Laboratories, Venice, California; and Str~~~lo~ntr~tus borate were obtained both from Pacific Biomarine and by collection from the Strait of Juan de Fuca in Washington. Ripe adults were spawned by intracoelomic injection of 0.55 M KCl. L. pictus eggs were dejellied by physical agitation, while A. punctulata and S. purpuratus eggs were dejellied by treatment with pH 5 seawater for 2 min, at which time 1 M Tris-HCl (pH 8.0) was added to return the pH of the seawater to 8.0. The dejellied eggs were fertilized in seawater which had been filtered through nitrocellulose filters with a pore size of 0.45 ym (FSW) and which contained 1 n&f 3-amino-1,2,&triazole to prevent hardening of the fertilization membrane (Showman and Foerder, 1979). Embryos were cultured with gentle agitation at a density of 1% {v/v) in FSW containing 100 mg/liter penicillin and 100 mg/liter streptomycin. All embryo cultures displayed at least 95% elevation of the fertilization membrane and normal cleavages. Preparation
of the CSK for Electron
VOLUME
95, 1983
procedure, the vitelline layer was removed from some dejellied eggs by treatment with 5 mM dithiothreitol in pH 9.0 FSW for 5 min (Epel et al., 1970). Eggs were then fertilized to show the absence of the fertilization membrane. In order to remove the unhardened fertilization membrane from zygotes which had been fertilized in the presence of 3-amino-1,2,4-triazole they were poured through nitex mesh (55 pm pore size for A. ~WZtulata and 63 pm pore size for L. pi&us and S. purpuratus).
We extracted eggs and zygotes lacking extracellular coats with a number of extraction buffers in order to obtain the best-preserved CSKs for electron microscopy. The extraction buffer for SEM (SEMEB) was similar to a buffer used by Cervera et al. (1981) to prepare HeLa cell CSKs and contained 100 m&f KCl, 5 m2M Mg (OAc), 300 mM sucrose, 5 mM EDTA (or EGTA where noted), 20 mM 3-(iV-morpholino)propanesulfonic acid (MOPS), 0.5% (w/v) (Triton X-100, and 5 m&f phenylmethylsulfonyl fluoride (pH 6.7 for unfertilized eggs and pH 7.3 for zygotes). After removal of the extracellular coats, cells were washed by settling through FSW, then through Ca’+-free seawater (Marine Biological Laboratory formula), and finally through two changes of SEMEB lacking Triton X-100. The washed cells were then extracted with approximately 20 vol of SEMEB for 1 hr at 20°C in Syracuse watch glasses (5.5 mm diameter). The cells were intermittently mixed by gentle agitation of the dishes and at the end of the extraction period the soluble material was removed with a Pasteur pipet. The CSKs were then washed by the addition to the dishes of SEMEB without Triton X-100. Washed CSKs were fixed in 2% gluteraldehyde in 0.1 M phosphate buffer (pH 7.2) for 30 min at 2O”C, washed in three changes of 0.1 ~phosphate buffer at 4”C, and postfixed with 1% 0~0, for 5 min at 20°C. The CSKs were dehydrated, critical point dried, sputter coated, and examined by SEM. Eggs and zygotes to be viewed by TEM were handled as described above but the CSKs were extracted in the same buffer used for biochemical analyses (see below). The cells were fixed with 2% gluteraldehyde in 0.1 M phosphate buffer (pH 7.2), washed with three changes of 0.1 M phosphate buffer, postfixed with 1% 0~04 for 1 hr at 20°C, washed with phosphate buffer, dehydrated, and embedded in Epon.
Microscopy
It was necessary to remove the extracellular coats from the eggs and embryos for examination by scanning electron microscopy (SEM). The vitelline layer of dejellied eggs was removed by a brief treatment with 25 @g/ml protease after which the eggs were extensively washed with FSW (Epel, 1970). As a control for this
Preparatirm of SOL and CSK for B~~hern~e~l Analyses
Whereas the methods described above were designed to maximize the retention of cellular structure, the following procedures were adopted to arrest ribosomes on polyribosomes and to maintain ionic conditions which
MOON
ET AL.
C#x&eletal
yield intact mRNPs (Moon et a!., 1980). Unhatched embryos cultured in 1 mM 3-amino-1,2,4-triazole were concentrated to a density of 10% (v/v) by low-speed centrifugation then poured through nitex mesh to remove the fertilization membrane, as described above. These embryos were then cultured for 20 min to allow the reformation of polyribosomes, which tend to decrease in number and size during ~entrifu~ation of the embryos. Dejellied eggs were not treated with protease or dithiothreitol, since control experiments demonstrated that the presence of the vitelline layer did not affect the proportion of poly(A) released during the extraction and since these methods might increase ribonuclease activity. Protein synthesis was arrested by plunging the suspensions of eggs and ‘embryos into 15 vol of cold FSW (-4”C), The eggs and embryos were prepared for extraction by pelleting at 500~ for 30 set (0°C) then pelleting through 100 vol of Caz+-free seawater (O*C). The extraction buffer used for biochemical analyses (BEB) consisted of 220 mM K+, 75 mM, Cl-, 145 mM OAc-, 300 mMsucrose, 5 mMMgzi, 20 mMMOPS, 0.5% Triton X100, and 05 mg/ml soybean trypsin inhibitor (pH 6.9 for both eggs and embryos). After transfer to sterile 15-ml polycarbonate centrifuge tubes, the cells were washed twice with 15 vol of BEB lacking Triton X-100 (0°C) by centrifugation at 200~ for 30 sec. The pellets were gently resuspended in 5-10 vol of BEB by inversion of the tube and extracted on ice fur 20 min, with mixing accomplished by inversion of the tube every 5 min. The CSK was pelleted by centrifugation at 200~ fur 45 set and the su~rnatant was removed. The CSK pellets were again washed by gentle resuspension in 2-4 vol of BEB for 5 min (O’C) followed by recentrifugation and removal of the supernatant. The combined supernatants were designated the SOL. Potyribosome
mRNA
in Sea Urchins
449
hollowing centrifugation the gradients were either pumped through an Isco Model UA-5 absorbance monitor or fractions of 1.5 ml were collected into 3 ml of ice-cold ethanol in sterile polypropylene test tubes (12 X 75 mm, obtained through BioRad, Richmond, Calif.) and precipitated at -80°C.
The ethanol precipitates of the sucrose gradient fractions were collected by centrifugation of the polypropylene test tubes in a Sorvall HS4 rotor at 5000~ for 45 min at -10°C. The drained pellets were dissolved in 0.5 ml of 0.11M Tris-HCl (pH 9.0), 0.5% SDS. An equal volume of buffer-saturated redistilled phenol was added and the samples were mixed twice by vortex at 1-min intervals. After the addition of 0.5 ml of CHC& each tube was mixed by vortex and centrifuged for 15 min at 5000~ in the HS4 rotor (15°C). The organic phase was removed with a pipet and discarded; the aqueous phase was reextracted with 0.5 ml of CHCb, and the samples were recentrifuged. The aqueous phases were transferred to siliconized glass test tubes, brought to 0,l 1M NaCl, and precipitated with 2 vu1 of ethanol (-SO°C). The ethanol precipitates were collected as described above, dissolved in 0.5 ml of 0.24 A4 NH*OAc, and reprecipitated with 2 vol of ethanol at -80°C. The precipitate was collected by centrifugation and dissolved in 20-50 ~1 of sterile water. RNA was extracted quantitatively from the CSK and SOL by the CsCl centrifugatiun method of Glisin et al. (f914), as modified by Speith and Whiteley (1981). Although DNA does not pellet during this procedure, the resulting RNA samples were precipitated from 2n/rlrLiCl (24 hr at O’C) to ensure DNA removal. The percentage of RNA in the SOL and CSK was determined by absorbance at 260 nm.
Anal&s
The CSK was resuspended in 100 rnM NaCl, If) mM piperazine-N,N’-bis(2-ethanesulfonic acid) (Pipes, pH 7.0), 1% Tween 40, 0.5% deoxycholate (Penman, 1966) to the same volume as the total SOL. The CSK was then disrupted by three passages through a 22-gauge needle. The disrupted CSK and the SOL were then centrifuged at 20,000~ for 6 min at 0°C in a Sorvall SS34 rotor to remove a small amount of particulate material and nuclei. Half of each sample was brought to 30 mM EDTA with a 0.5 M stock solution Samples of 0.4 ml were then layered over 11-ml, lo-40% (w/v) linear sucrose gradients which contained a l-ml cushion of 60% sucrose and final salt concentrations of 220 mM K’, 145 rnM OAc-, 5 mM Mg+‘, 20 mM MOPS (pH 6.9), and 1 mM dithiothreitol. The gradients were centrifuged in a Beckman SW41 rotor at ~90~000~~~~ for 70 min at 1°C.
Aliquots of purified RNA were hybridized to t3H]poly(U) as previously described (Moon et al., 1930) to quantitate the amount of poly(A) in the SOL and CSK. In Vitro Translation Total RNA from the SOL and CSK, as well as from sucrose gradient fractions, was translated in a nuclease-treated message-dependent rabbit reticulocyte lysate supplemented with ~~5S]methionine (Rosenthal & GIN., 1980$,After incubation for 60 min at 30°C the reaction was stopped by the addition of 5 vol of SDSpolyaerylamide gel electrophoresis sample buffer (Laemmli, 1970) to each tube.
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DEVELOPMENTAL BIOLOGY
VOLUME 95, 1983
FIG. I,. Dark-field light micrographs of norm al and Triton X-100 extracted S: purpz w&useggs. (A) Untreated egg, 100X. (B) Extracted em3 with fat :us on islets of pigment granules spars6$1~distributed at the surface of the CS#K, 100~. (C) Extracted egg focused to show the corl:ical rim, 175X.
Eggs were fractionated into SOL and CSK after treatment with Triton X-100 in BEB as described above. The SOL and CSK were extracted by shaking for 5 min in petroleum ether and the extracts were dried and analyzed for lipid content by the method of ZSllner and Kirsch (1962). Protein Anal@
tion. Since the detergent-extracted CSK of sea urchin eggs had not been previously described in detail, as an initial step in this research we analyzed the morphology of the CSK using a combination of light microscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM).
and Gel Electrophwesis
The protein content of the SOL and CSK was determined according to Bradford (1976) using a commercially available assay (Bio-Rad, Richmond, Calif.). Proteins were separated in 10% polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS), then stained with Coomassie brilliant blue (Moon et al., 1980). Up to 25 ~1 of rabbit reticulocyte lysate in sample buffer was subjected to electrophoresis without prior heating of the sample. The gel was then processed for fluorography using EnHance (New England Nuclear, Boston, Mass.) and exposed to Kodak XAR-5 X-ray film at -80°C for 2-4 days, then for 20-40 days to ensure the validity of the interpretations. RESULTS
AND DISCUSSION
The purpose of this study was to examine the possible association of mRNA with the eytoskeleton of sea urchin eggs and embryos and to assess whether any changes in this association occurred during development. Our approach was to extract eggs and embryos with Triton X-100 and to separate the soluble material from the detergent-insoluble residue, which we define as the cytoskeleton. We then measured the distribution of translatable nonhistone mRNA and polyribosomes between the cytoskeletal fraction and the soluble frac-
Dark-field micrographs of normal eggs and eggs extracted with Triton X-100 are shown in Fig. 1. The eggs became transparent during the extraction, leaving a CSK which consisted of a cortical rim and opaque internal islets containing pigment granules. TEM showed that the CSK of eggs was less complex than that of one-cell zygotes or blastulae (Fig. 2). The extracted eggs were largely depleted of cell structures except for a plasma membrane lamina (Ben Ze’ev et al., 19’79), components which may be the remnants of cortical granules, pigment granules, and scattered internal patches of filaments associated with ribosomes (Figs. 2A and C). After fertilization a dense filamentous network appeared in the cortex and the internal portions of the CSK became richer in filaments associated with ribosomes (Fig. 2B). As shown in Fig. 2D the internal filaments with associated ribosomes were more abundant in the CSK of blastula cells than in the CSK of eggs. This suggests that an increase in the interaction of ribosomes with the CSK occurs during early development. The nature of the surface of the CSK was further studied by SEM. Figures 3 and 4 show the cortical filamentous network of A. pun&data and L. pictus eggs. The surface of the unextracted egg was covered with short microvilli (Fig. 3A), whereas the unextracted zygote was covered with long microvitli as previously re-
FIG. 2. TEM of Triton X-100 extracted eggs and embryos of A. p~nct~lata. (A) The cortical region of an egg CSK showing the plasma membrane lamina (downward facing arrow), possible remnants of cortical granules (upward facing arrows), and pigment granules, 7100X. (B) The cortical region of a fertilized egg CSK showing a dense cortical filamentous network [arrow) and increased compfexity in the internat regions, 1100x. (Cf Internal region of an unfertilized egg CSK showing the sparse distribution of filaments with associated ribosomes, 12,500X. (D) Internal region of the CSK of a hatched hlastula cell showing abundance of filaments associated with ribosomes, 12,500X. The horizontal bars represent 1 pm.
ported by Schroeder (1979) (Fig. 3B). In contrast, the surface of the CSK of unfertilized eggs was characterized by spherical structures averaging about 0.8-1.0 pm in diameter (Fig. 3C). The spherical structures appeared to be positioned in rowa one layer thick and were eonnetted to an underlying network of filaments. The num-
ber of these structures exposed on the surface of the cytoskeleton of L p&us eggs was calculated to be approximately 24,000. The spherical structures are similar in size and number to cortical granules (Schroeder, 1979). As expected of putative cortical granules, these structures were not observed in the CSK of fertilized eggs
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DEVELOPMENTAL BIOLOGY
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FIG. 3. SEM of the surface of normal and Triton X-100 extracted eggs and one-cell zygotes of L. pi&us. (A) An untreated egg with short microvilli. (B) An untreated zygote with long mierovilli. (C) An egg CSK with exposed cortical granules. (D) A zygote CSK with exposed cortical filamentous network. (E) An egg CSK treated with Cazf to rupture cortical granules and expose the underlying filamentous network. All bars represent 2 hm.
(Fig. 3D, Fig. 4). The putative cortical granules were also not observed after treatment of the CSK with 1 m&l Ca’* (Vacquier, 1975), which revealed an underlying network of filaments (Fig. 3E). SEM analysis revealed a filamentous cortical network in the CSK of fertilized eggs (Fig. 3D, Fig. 4). This network probably includes the F-a&in CSK previously observed in the cortex of fertilized sea urchin eggs by electron microscopy (Vacquier, 1981). The filamentous network cannot consist entirely of actin filaments, however, because the network also exists in the CSK of unfertilized eggs in which little F-actin is known to be present (Vacquier, 1981). The other filamentous elements of the cortical rim of the CSK may be identical to the heterogeneous network of filaments recently described by Kidd and Mazia (1980). BiodaernicaZ Composition of the CSK
The proportion of total protein lipid, RNA, and poly(A) remaining in the CSK after eggs and developing embryos were extracted with Triton X-100 is shown in
Table 1. It can be seen that only 16% of the total lipid, lo-20% of the total protein, and 9-25% of the total RNA remained in the CSK. The percentage of total RNA in the CSK appeared to increase slightly after fertilization, from 9% in the unfertilized egg to 17% in the fourcell embryo, and 25% in hatched blastulae. The amount of total RNA in the CSK of the developing embryo may be an underestimate of actual values, since any “runoff” of ribosomes from polyribosomes would contribute to the RNA mass in the SOL because free monoribosomes are not retained in the CSK (described below). A large portion of poly(A)-containing RNA remained in the CSK of eggs and embryos, and this amount gradually increased during early embryogenesis (Table 1). RNAs from Since some prevalent poly(A)-containing sea urchin eggs cannot be translated in vitro and represent mitochondrial RNA (Wells et al., 1982), the percentage of poly(A) retained in the CSK cannot be directly correlated with the amount of translationally active cytoplasmic mRNA in this fraction. In order to determine whether the proteins retained in the CSK represent a subset of the total proteins of
MOON
FIG. 4. SEM of a Triton is 4 pm. (B) The horizontal
X-1013 extracted bar is 1 pm.
ET AL.
A. pm&data
Cytoskeletal
zygote
showing
intact eggs and embryos, the CSK and SOL were subjected to SDS-polyacrylamide gel electrophoresis. The gels showed that distinct sets of major proteins were present in the CSK and SOL (Fig. 5). The CSK of unfertilized eggs, four-cell embryos, hatched blastulae, and prism-stage embryos were enriched relative to the SOL in major proteins of 44, 46, 49, 53, 72, and 94 X lo3 molecular weight (M,) at a.11stages. The protein composition of the CSK changed during early embryogenesis. Proteins of 23,24,65, and 180 X 103, major components of the CSK from unfertilized eggs and four-cell embryos, were absent or present in considerably reduced levels in the CSK of hatched blastula- and prism-stage embryos. Moreover, 15-20 proteins of 20-41 X 10” M, increased in the CSK during embryogenesis. These proteins probably represent ribosomal proteins, a possibility which is consistent with the visuaiization of ribosomes in the CSK by TEM (Figs. ZC, D) and the identification of polyribosomes in the CSK by the biochemical studies discussed below. and CSK Messenger
RNAs
The presence of poly(A) in the CSK at all developmental stages suggests that it contains mRNA. Therefore, we tested whether SOL and CSK RNAs were translatable in a message-dependent rabbit reticulocyte lysate. We also determined whether SOL and CSK RNAs coded for qualitatively different polypeptides by separating their translation products on polyacrylamide gels, followed by fluorography. RNAs extracted from the SOL and CSK of unfertilized eggs, four-cell embryos, and hatched blastulae all contained translatable mRNAs,
mRNA
in
Sea
the filamentous
Urchins
network
of the cortical
rim.
(A)
The horizontal
bar
as evidenced by the in vitro synthesis of [3”S]methioninelabeled products (Fig. 6). Comparing the products synthesized by SOL and CSK mRNAs at any one developmental stage revealed no qualitative differences which could be resolved by gel electrophoresis. In this regard our results support those of van Venrooij et aE. (1981), who have made similar observations for detergent-extracted human KB cells. Cervera et al. (1981), however, have shown that the CSK of mammalian cells contains a distinct subset of prevalent mRNAs. Since our gels did not resolve histones, it is not known whether histone
TABLE 1 BIOCHEMICAL COMPOSITION OF THE CYTOSKELETAL FRAMEWORK SEA URCHIN EGGS AND DEVELOPING EMBRYOS Percentage Developmental stage’ Unfertilized
egg
Zygote
OF
in eytoskeleton
Lipid
Protein
RNA
16
13
9
42 (1)” 44 (2)
-
-
15
43 (1)
Poly(A)
Four-cell
embryo
-
10
17
53 (1) 39 (21
Hatched
biastula
-
12
25
69 (1)
60 (2) Prism
-
20
-
87 (2)
aS. purpuratus eggs were employed for lipid analysis. All other analyses were done with A. punctulata~. In experiment (1) the poly(A) content of RNA was determined after purification of the RNA by CsCl centrifugation. In experiment (2) the purified RNA was precipitated with 2 df LiCl before hybridization.
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95, 1983
in that it contained only 15-30% of the free monoribosomes (Fig. ?A and Table 2).
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Increased Associatim
scscscsc EGG
HBI
PRiSM
CL
FIG. 5. Total proteins of the SOL and CSK of A. ~n~~~o~. In lanes 2-9 S designates soluble proteins and C designates CSK proteins. Proteins of the egg (lanes 2,3), four-cell-stage (lanes 4,5) hatched hlastula (lanes 6, 7). and prism-stage (lanes 8, 9) were separated on a 10% polyacrylamide gel and stained with Coomassie blue. Lane 1 contains molecular weight standards (muscle myosin, 225,000 M,; actinin, 105,000 M,; phosphorylase B, 94,000 M,; transferrin, 78,000 M,; bovine serum albumin, 67,000 M;; actin, 42,000 M,; coneanavalin A, 2’7,000 nCr,). The 22,700 &fr protein in lanes 4, 6, and 8 is soybean trypsin inhibitor, added to the extraction buffer. Proteins discussed in the text are indicated by a dot and their calculated molecular weights are given X10e3.
of Messages with
The presence of mRNA (Fig. 6) and polyribosomes (Fig. 7) in the embryonic CSK and the apparent increase in poly(A)-containing RNA in the CSK after fertilization (Table 1) suggest that the postfertilization recruitment of stored mRNPs into polyribosomes may be correlated with increased interaction of the mRNPs with the CSK. If this phenomenon exists in vz’vo, we would expect the stored nonpolyribosomal mRNPs of unfertilized eggs to be located predominantly in the SOL. Since nonpolyribosomal mRNPs are not detectable in absorbance tracings such as those in Fig. 7, we used an alternative technique to detect the mRNPs. Aliquots of the SOL and CSK of unfertilized eggs and early blastulae (6 hr, 2O*C) of A. ~~et~la~ were centrifuged through sucrose gradients similar to those in Fig. 7. We then fractionated the gradients, extracted the RNA from each fraction, translated the RNA in the message-dependent rabbit reticulocyte lysate, and visualized the products of translation after separation of the proteins on SDS-polyacrylamide gels (Fig. 8 and Fig. 9).
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messages display a preferential the SOL or the CSK.
localization
in either
Recent studies with cultured mammalian cells indicate that all polyribosomes, but not monoribosomes, are associated with the CSK (Lenk et al., 1977; Cervera et al., 1981). These studies would predict, therefore, that some or most of the mRNA detected in the CSK of sea urchin embryos would be present as polyribosomes. This possibility was investigated by sucrose gradient analysis of aliquots of SOL and disrupted CSK prepared from unfertilized A. punctulata eggs and hatched blastulae (9 hr, 2O’C). Tracings of the absorbance of these gradients at 260 nm are presented in Fig. 7, and quantitated in Table 2. As predicted, approximately 90% of the EDTA-sensitive polyribosomes, but only 20% of the monoribosomes, were associated with the CSK of hatched blastulae (Fig. 7B and Table 2). Although the low levels of polyribosomes present in unfertilized eggs (Humphreys, 1971) could not be quantitated by this method, the egg CSK was similar to the hatched blastula CSK
ESCSCSC EGG
HBI CL
FIG. 6. Qualitative comparison of mRNAs in the SOL and CSK. RNA extracted from the SOL (S, lanes 2,4, and 6) and CSK (C, lanes 3, 5, and 7) of eggs (lanes 2, 3), four-cell embryos (lanes 4, 5), and hatched blastulae (lanes 6, 7) were translated at saturating concentrations of message in the rabbit reticulocyte lysate and the products separated on a 10% polyacrylamide gel followed by fluorography. Endogenous (E) products are shown in lane 1.
MOON ET AL.
Cytoskeletal mRNA in Sea Urchins
We translated sucrose gradient fractions of blastula SOL and CSK to confirm that embryonic polyribosomes were present in the CSK. The peak of translational activity in blastulae was, as expected, in the polyribosome region of the CSK sucrose gradient (Fig. 8A, fractions 1 and 2). The polyribosomal nature of the RNA in fractions 1 and 2 of Fig. 8A was suggested by the displacement with EDTA of the polyribosomal mRNA to the region of the gradient known to contain free mRNPs (fractions 5-7 of Fig. SC)1(Jenkins et al., 1978; Moon et al., 1980). Little translational activity was detected in any of the fractions of the blastula SOL gradient (Fig. 8B), although after treat.ment of the SOL with EDTA translationally active mRNA was detected in fractions 5-7 (Fig. 8D). This result. suggests that small amounts of translationa~ly competent SOL polyribosomal mRNA were distributed between many gradient fractions, and the diluted mRNA was only detectable after treatment with EDTA shifted the mRNA to primarily one fraction (Fig. 8D). The detection of mRNA in the SOL gradient does not, however, definitively establish that these messageswere present in polyribosomes, or that these messageswere free in the cytoplasm. These results support the analyses based upon the absorbance of the gradients at 260 nm and, taken together, indicate that more polyribosomes were present in the CSK than in the SOL of the blastula. TranslationaIly active mRNA was, however, present in both fractions. In contrast to the results obtained with blastulae, most of the translational activity of eggs (Fig. 9) was present in the SOL gradient and divided between fraction 1 (the polyribosome region) and fractions 4-7 (the small polyribosome and free mRNP region) (Fig. 9B). Fraction 6 probably contains the highest concentration of the nonpolyribosomal mRNP complexes of eggs. These mRNPs sediment with a broad distribution averaging about 60 S (Kaumeyer et al., 1978; Moon et al., 1980). TABLE 2 MONORIBOSOME AND POLYRIBOSOME DISTRIBUTION IN THE CYTOSKELETAL FRAMEWORK OF A. punctulata EGGS AND HATCHED BLASTULAE Percentage in cytoskeleton” Developmental Unfertilized
stage
eggs
Hatched blastulae
Monoribosomes 30 (1) 16 (2) 19
Polyribosomes undetectable 90
a The percentage of monoribosomes and polyribosomes in the CSK was calculated from the area of the peaks shown in Fig. ‘7.The baseline was established by comparison of the absorbance tracing of EDTAtreated samples and blank gradients.
3. Blaslula
d A
2
“1,
4
6
8 Fraction
,
2
,
,
4
)
,
6
,
8
Number
FIG. 7. Segregation of monoribosomes and polyribosomes between the SOL and the CSK of (A) eggs and (B) hatched blastula embryos. The absorbance at 254 nm is shown for SOL and CSK centrifuged on lo-40% sucrose gradients and pumped through an absorbance monitor with the top of the gradient (right) as the leading edge f-). Identical samples were pretreated with 30 mM EDTA before centrifugation (- - -). In panel A the dashed line merges with the solid line. For clarity, the dashed line in panel B is shown in the postmonoribosome region only. The discontinuity in the absorbance in panel B is due to a twofold change of scale. Fractions similar to those used for RNA purification and in vitro translation are indicated.
After treatment with EDTA, all of the translatable mRNA of the egg SOL was detected in fractions 5-7 of the gradient (Fig. 9C). The sensitivity to EDTA of the mRNA present in gradient fractions 1 and 4 of Fig. 9A implies that the mRNA in these fractions was associated with detergent-extracted polyribosomes which were not part of the CSK. The small amount of translational activity present in the egg CSK was located in fraction 6, suggesting that it represented nonpolyribosomal mRNP (Fig. 9A). Our results show that most of the mRNA in eggs sediments in sucrose gradients as free mRNP and is present in the SOL whereas most of the mRNA in blastulae occurs as polyribosomes in viva and is present in the CSK. The retention of polyribosomes in the CSK of embryos was not species specific or dependent upon the use of the BEB extraction buffer. When A. punctulatahatched blastulae were extracted with SEMEB (without EGTA or EDTA), most polyribosomes were retained in the CSK. The SOL and CSK of S. purpurutus eggs and hatched blastulae were also examined for translationally active mRNA across sucrose gradients (data not shown). Consistent with the data presented here for A. punctuhta, most of the translatable mRNA in the eggs of 5’. purpwatus was present in the nonpolyribosomal mRNP region of the SOL, and most of the translatable
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not allow precise quantitation, a rough estimate is that very little translatable mRNA is retained in the CSK of the unfertilized egg, and that approximately 50% or more of the translatable mRNA is present in the CSK of blastulae. The association of egg mRNA with the CSK could occur shortly after fertilization at the same time as the recruitment of stored messages into polyribosomes, though this awaits further analysis. Our finding that mRNA-~ytoskeletal interactions change during development is consistent with the observation that viral polyribosomes, but not host messages, are associated with the CSK in infected HeLa cells (Lenk and Penman, 1979). The correlation noted here between the low level of
FIG. 8. Translation across sucrose gradients of blastula CSK and SOL. A. ~~ct~~~~ blastulae (6 hr, 2O“C after fertilization) CSK (panels A and C) and SOL (panels B and D) were centrifuged on lo-40% sucrose gradients similar to those in Fig. 7 with (panels C and D) or without (panels A and B) pretreatment with 30 mM EDTA. Gradient fractions corresponding approximately with those in Fig. 7 (top of the gradients on the right), and pelleted material (lane B), were extracted and the RNA was translated in the rabbit reticulocyte lysate. The fluorograph of the 10% ~olyacrylam~de gel localized sea urchin messages in the sucrose gradients.
mRNA in hatched blastulae was present in the polyribosome region of the CSK after extraction of the cells with S~MEB containing 5 mfl/l EGTA. Since the retention of polyribosomes in the CSK was not species specific, the next step is to determine whether or not the apparent association of polyribosomes with the CSK involves specific sites on both the polyribosomes and the CSK. In summary, the results of the present investigation show that a marked increase occurs in the percentage of translatable mRNA which is retained in the CSK during development. Although the in vitro translation method used to detect CSK-associated messages does
FIG. 9. Translation across sucrose gradients of egg CSK and SOL. A&a& pun&da CSK (panels A and C) and SOL (panels B and D) were centrifuged on lo-40% sucrose gradients similar to those in Fig. 7 with (panels C and D) or without (panels A and B) pretreatment with 30 mM EDTA. The direction of centrifugation was from right to left. Gradient fractions correspond approximately with those in Fig. 7, and fraction B contained pelleted material. Fraction 8 of panel C was lost. RNA extracted from the sucrose gradient fractions was translated in the rabbit reticulocyte lysate and the products directed by sea urchin or endogenous (E) messages were separated on a 10% polyacrylamide gel followed by fluorography.
MOON
ET AL.
Cytoskeletal
mRNA-CYK interactions and the relative translational inactivity of the egg raiseis the possibility that the postfertilization recruitment of mRNAs for translation may be dependent upon association of the message with cytoskeletal elements. In eggs and blastulae, however, a small yet detectable amount of translatable mRNA, some of which may have been polyribosomal, was present in the SOL. Thus, at present it must be concluded that although there appears to be a correlation between activation of protein synthesis and the retention of mRNAs in the CSK, this apparent association may not be absolutely required for translation. Alternatively, it is possible that the association of mRNAs with the CSK enhances the efficiency of translation after fertilization, or that this association is a mechanism which promotes a nonrandom spatial distribution of maternal mRNA during early development.
mRNA
in
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revealed in platinum replicas of freeze-dried cytoskeletons. J. Cell Biol. 86.212-234. HUMPHREY& T. (1371). Measurements of messenger RNA entering polyrihosomes upon fertilization of sea urchin eggs. Ltev Biol. 26, 201-208. ILAN, J., and ILAN, J. (1978). Translation of maternal messenger ribonucleoprotein particles from sea urchin in a cell-free system from unfertilized eggs and product analysis. Dev. Biol. 66, 375-385. JENKINS, N. A., KAUMEYER, J. F., YOUNG, E. M., and RAFF, R. A. (1378). A test for masked message: The template activity of messenger ribonucleoprotein particles isolated from sea urchin eggs. Dev. Bill 63,273-238.
KAUMEYER, J. F., JENKINS, N. A., and RAFF, R. A. (1378). Messenger ribonucleoprotein particles in unfertilized sea urchin eggs. Deu. Biol 63,266-278.
KIDD, P., and MAZIA, D. isolated from fertilized J. struts. Res. 70, LAEMMLI, U. K. (1370). assembly of the head
(1380). The ultrastructure of surface layers and chemically stimulated sea urchin eggs. 58-63. Cleavage of structural proteins during the of bacteriophage T4. Nature (Lmdm) 227,
680-685.
We thank E. Rosenthal for provision of rabbit reticulocyte lysate and hefpful suggestions; L. Hyman, Rr. 3. Ruderman, and Dr. T. Hunt, for supplies and equipment; and Dr. M. A. Rankin, for performing the lipid analysis. S. Houghton provided technical assistance in eleetron microscopy. This work was supported by NIH Grants HD-13970 (W.R.J.), HD-11070 (M.B.H.), NRSA HD-07183 (R.T.M.), and 323HD07098 (Embryology course, Marine Biological Laboratory, Woods Hole, Mass.) and American Cancer Society Institutional Grant IN 26U (R.T.M.). REFERENCES BEN ZE’EV, A., DUERR, A., SOLOMON, F., and PENMAN, S. (1979). The outer boundary of the cytoskeleton: A lamina derived from plasma membrane proteins. C& 17,853-865. BRADFORD, M. (1376). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Bz’mhem. 72.248-254. BROWN, S., LEVINSON, W., and SPUDICH, J. A. (1976). Cytoskeletal elements of duck embryo fibroblasts revealed by detergent extraction. J. supramd .%%ct. 5,113-130. CERVERA, M., DREYFUSS, G., and PENMAN, S. (1981). Messenger RNA is translated when associated with the cytoskeletal framework in normal and VSV-infected HeLa cells. CeEE 23, 113-120. EPEL, D. (1970). Methods for removal of the vitelline layer of sea urchin eggs. II. Controlled exposure to trypsin Lo eliminate postfertilization clumping of embryos. Exp. Cell Res. 61, 69-70. EPEL, D., WEAVER, A. M., and MAZIA, D. (1970). Methods for removal of the vitelline layer of sea urchin eggs. I. Use of dithiothreitol (Cleland reagent). Exp. CeZl Res. 61, 64-68. FULTON, A. B., WAN, K. M., and PENMAN, S. (1980). The spatial distribution of polyribosomes in 3T3 cells and the associated assembly of proteins into the skeletal framework. Cell 20, 849-857. GLISIN, V., CRKVENJAKOV, R., and BYUS, C. (1974). Ribonueleic acid isolation by cesium chloride centrifugation. Biochemistry 13,26332637.
GOLDMAN, R. D., CHOJNACKI, B., GOLDMAN, A. E., STARGER, J., STEINERT, P., TALIAN, J., WHITMAN:. M., and ZACKROFF, R. (1381). Aspects of the cytoskeleton and cytomusculature of nonmuscle cells. Neurosci Res. F’rog. Bull. 19, 59-82. HEUSER, J. E., and KIRSCHNER, M. W. (1380). Filament. organization
LENK, R., RANSOM, L., KAUFMANN, Y., and PENMAN, S. (1977). A cytoskeletal structure with associated poiyribosomes obtained from HeLa cells. CeU 10, 6’7-78. LENK, R., and PENMAN, S. (1979). The eytoskeletal framework and poliovirus metabolism. Cell 16, 289-301. LONG, B. H., HUANG, C. Y., and POGO, A. 0. (1979). Isolation and characterization of the nuclear matrix in Friend erythroleukemia cells: Chromatin and hnRNA interactions with the nuclear matrix. CeEl 18.1079-1090. MOON, R. T., MOE, K. D., and HILLE, M. B. (1980). Polypeptides of nonpolyribosomal messenger ribonucleoprotein complexes of sea urchin eggs. Biochemistry 19,2723-2730. MOON, R. T., DANILCHIK, M. V., and HUE, M. B. (1382). An assessment of the masked message hypothesis: Sea urchin egg messenger ribonucleoprotein complexes are efficient templates for in vitro protein synthesis. Dev. BioL 93.389-403. PENMAN, S. (1966). RNA metabolism in the HeLa cell nucleus. 2 Mot Bid 17,117-130. RAFF, R. A. (1980). Masked messenger RNA and the regulation of protein synthesis in eggs and embryos. In “Cell Biology” (M. R. Prescott, ed.,) Vol. 4, pp. 107-136. Academic Press, New York. ROSENTHAL, E. T., HUNT, T., and RUDERMAN, J. V. (1980). Selective translation of mRNA controls the pattern of protein synthesis during early development of the surf clam Spisuta solidissime CeU 20, 487-434.
SCHLIWA, M., VAN BLERKOM, J., and PORTER, K. R. (1981). Stabilization of the cytoplasmic ground substance in detergent-opened cells and a structural and biochemical analysis of its composition. Proc. Nat. Acad. Sci. USA 78,4323-4333. SCHROEDER, T. E. (1379). Surface area change in fertilization: Resorption of the mosaic membrane. Deu. Biol 70, 306-326. SHOWMAN, R. M., and FOERDER, C. A. (1379). Removal of the fertilization membrane of sea urchin embryos employing aminotriazole. Exp. Cell Res. 120, 253-255. SPEITH, J., and WHITELEY, A. H. (1981). Polyribosome formation and poly(A)-containing RNA in embryos of the sand dollar, Dendraster exce~~~c~ Wilhelm Row’s Arch. 190, 111-117. SPIRIN, A. S. (1978). Eukaryotic messenger RNA and informosomes. FEBS I&t. 88, 15-17. VACQUIER, V. D. (1975). The isolation of intact cortical granules from sea urchin eggs: Calcium ions trigger granule discharge. Dev. BioL 43,62-74.
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VACQUIER, V. D. (1981). Dynamic changes of the egg cortex. Dev. Biol 84. l-26. VAN EEKELEN, C. A. G., and VAN VENROOIJ, W. J. (1981). hnRNA and its attachment to a nuclear protein matrix. J. CeU BioL 88,554-563. VAN VENROOIJ, W. J., SILLEKENS, P. T. G., VAN EEKELEN, C. A. G., and REINDERS, R. J. (1981). On the association of mRNA with the cytoskeleton in uninfected and adenovirus-infected human KB cells. Exn.
Cell Res. 135.79-91.
WELLS, D. E., SHOWMAN, R. M., KLEIN, W. H., and RAFF, R. A. (1981). Delayed recruitment of maternal histone H3 mRNA in sea urchin embryos. Nature (Lack 292,477-478.
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WELLS, D. E., BRUSKIN, A. M., O’BROCHTA, D. A., and RAFF, R. A. (1982). Prevalent RNA sequences of mitochondrial origin in sea urchin embryos. Dev. Biol. 92, 557-562. ZOLLNER, N., and KIRSCN, K. (1962). Uber die quantitative Bestimmung von Lipoiden (Mikromethode) mitteis der vielen naturlichen Lipoiden (allen bekantiten Plasmalipoiden) gemeinsamen Sulphophosphovanillen-reaction. 2. Ges. Exp. Med. 135, 545-561. ZUMB$, A., STAHLI, C., and TRACHSEL, H. (1982). Association of a M, 50,966 cap-binding protein with the cytoskeleton in baby hamster kidney cells. Proc. Nut. Amd. Sci USA 79, 2927-2931.
BIOLOGY
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(1983)
The Cytoskeletal Framework of Sea Urchin Eggs and Embryos: Developmental Changes in the Association of Messenger RNA RANDALL ‘Marine Biological Laboratory, Washington 98195; $Department Zo&gy, Ohio State University,
T. MooN,‘~*~~ ROBERTO F. NICOSIA,*$ CHERIE OLSEN,*~ MERRILL B. HILLE,~ AND WILLIAM R. JEFFERY*~#
Woods Hole, Massachusetts of Pathology, The Medical Columbus, Ohio &9210; and Received
June
025qS; TDepartment of Zoology NJ-G, University of Washington, Seattle, College of Pennsylvania, Philadelphia, Pennsylvania 191U; 5Department of #Department of Zoology, University of Texas at Austin, Austin, Texas 78712
25, 1982; accepted
in revised
form September
17, 1982
Extraction of sea urchin eggs and embryos with Triton X-100 generated a cytoskeletal framework (CSK) composed of a cortical filamentous network and an internal system of filaments associated with ribosomes. The CSK contained only lo-20% of the cellular protein, RNA, and lipid. A specific subset of proteins was enriched in the CSK. Several lines of evidence suggest that mRNA is a component of the CSK of both eggs and embryos. First, the CSK contained poly(A) sequences ,which hybridized with [3H]poly(U). Second, the CSK contained polyribosomes. Finally, RNA extracted from the CSK showed translational activity in an in vitro system. The nonhistone messages present in the CSK were qualitatively similar to those solubilized by detergent, as determined by separation on polyacrylamide gels of the products of in vitro translation. In the unfertilized egg, most mRNA was present as nonpolyribosomal messenger ribonucleoprotein complexes which, along with monoribosomes, were efficiently extracted by Tritop X-100. The converse was found in blastulae, as most of the mRNA was present as polyribosomes associated with the CSK, although monoribosomes were still efficiently extracted by detergent. These results indicate a correlation between the activation of protein synthesis in eggs and the association of polyribosomes with the CSK.
INTRODUCTION
Our perception of the intracellular conditions under which RNA is synthesized, transported, and translated has recently undergone marked changes. The discovery that mRNA was associated with proteins to form messenger ribonucleoprotein (mRNP) complexes led to studies showing that mRNA is present as mRNP from the time of its synthesis until its ultimate degradation (Spirin, 1978). A current revolution in our understanding of RNA metabolism centers around the issue of whether nuclear ribonucleoprotein complexes and cytoplasmic polyribosomes are associated with cellular filaments constituting, respectively, the nuclear skeleton (Long et al., 1979; van Eekelen and van Venrooij, 1981) and the cytoskeleton (Lenk et al., 1977; Lenk and Penman, 1979; Fulton et al., 1980; Cervera et al., 1981). That polyribosomes could be associated with a cellular matrix raises the fundamental question of whether their interaction can se:rve as a mechanism of translational control. Experiments to answer this question are feasible, since cytoskeleton preparations can readily be prepared by extraction of cells with nonionic detergents such as Triton X-3.00. The detergent quickly solubilizes most of the lipi’d, tRNA, and monoribosomes, ’ Present address: Division of Biology, California Institute nology, Pasadena, Calif. 91125. To whom correspondence addressed.
of Techshould be
leaving a detergent-insoluble cytoskeletal framework (CSK) containing microfilaments, microtubules, and other filaments (e.g., Brown et al., 1976; Heuser and Kirschner, 1980; Goldman et al., 1981), up to 200 different proteins (Schliwa et al., 1981) including an mRNA 5’ cap-binding protein (Zumb6 et al., 1982), and most of the polyribosomes (Cervera et al., 1981). A possible role for the cytoskeleton in regulating translation has been suggested by studies demonstrating that HeLa cell CSKs selectively bind translationally active viral mRNA and exclude host mRNA which is not being translated (Lenk and Penman, 1979). Embryonic systems may be useful for investigating the role of the cytoskeleton in translation, since developing sea urchins (Wells et al., 1981) and surf clams (Rosenthal et al., 1980) exhibit translational control of protein synthesis. In sea urchin eggs, fertilization elicits a rapid increase in the rate of protein synthesis (reviewed by Raff, 1980). The templates for this synthesis are maternal messages synthesized during oogenesis and stored in the egg as nonpolyribosomal mRNPs. Although the rate of polypeptide chain elongation is slower in the unfertilized egg than in the developing embryo, the primary block to translation in the unfertilized egg appears to be some step in the binding of 40 S ribosomal subunits to mRNPs. This block in the recruitment of stored mRNPs into polyribosomes may be due to the associ-
447
0012-1606/83/02044'7-12$03.00/O Copyright All rights
0 1983 by Academic Press. Inc. of reproduction in any form reserved.
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ation with the mRNA of a phenol-soluble inhibitor of translation (Jenkins et al., 1978; Ban and Ilan, 1978), though recent data on the translational efficiencies of egg mRNPs do not support this mechanism of translational control (Moon et al., 1982). The studies on the selective binding of viral mRNA to the host cytoskeleton, mentioned above, however, raise the possibility that the low level of protein synthesis in unfertilized sea urchin eggs may be due to a lesion in the interaction of the stored mRNPs with the egg CSK. The postfertilization polymerization of actin or other cytoskeletal filaments (reviewed by Vacquier, 1981) may be indicative of a restructuring of the egg CSK which promotes its interaction with stored mRNPs and increases the rate of translation. To test this possibility we have isolated the CSK from sea urchin eggs and embryos by detergent extraction and examined its composition, particularly with respect to the distribution of nonhistone mRNAs and polyribosomes. MATERIALS
AND
METHODS
Arbacia punctulata were collected at Woods Hole, Massachusetts; Lqtechinus pi&us were obtained from Pacific Biomarine Laboratories, Venice, California; and Str~~~lo~ntr~tus borate were obtained both from Pacific Biomarine and by collection from the Strait of Juan de Fuca in Washington. Ripe adults were spawned by intracoelomic injection of 0.55 M KCl. L. pictus eggs were dejellied by physical agitation, while A. punctulata and S. purpuratus eggs were dejellied by treatment with pH 5 seawater for 2 min, at which time 1 M Tris-HCl (pH 8.0) was added to return the pH of the seawater to 8.0. The dejellied eggs were fertilized in seawater which had been filtered through nitrocellulose filters with a pore size of 0.45 ym (FSW) and which contained 1 n&f 3-amino-1,2,&triazole to prevent hardening of the fertilization membrane (Showman and Foerder, 1979). Embryos were cultured with gentle agitation at a density of 1% {v/v) in FSW containing 100 mg/liter penicillin and 100 mg/liter streptomycin. All embryo cultures displayed at least 95% elevation of the fertilization membrane and normal cleavages. Preparation
of the CSK for Electron
VOLUME
95, 1983
procedure, the vitelline layer was removed from some dejellied eggs by treatment with 5 mM dithiothreitol in pH 9.0 FSW for 5 min (Epel et al., 1970). Eggs were then fertilized to show the absence of the fertilization membrane. In order to remove the unhardened fertilization membrane from zygotes which had been fertilized in the presence of 3-amino-1,2,4-triazole they were poured through nitex mesh (55 pm pore size for A. ~WZtulata and 63 pm pore size for L. pi&us and S. purpuratus).
We extracted eggs and zygotes lacking extracellular coats with a number of extraction buffers in order to obtain the best-preserved CSKs for electron microscopy. The extraction buffer for SEM (SEMEB) was similar to a buffer used by Cervera et al. (1981) to prepare HeLa cell CSKs and contained 100 m&f KCl, 5 m2M Mg (OAc), 300 mM sucrose, 5 mM EDTA (or EGTA where noted), 20 mM 3-(iV-morpholino)propanesulfonic acid (MOPS), 0.5% (w/v) (Triton X-100, and 5 m&f phenylmethylsulfonyl fluoride (pH 6.7 for unfertilized eggs and pH 7.3 for zygotes). After removal of the extracellular coats, cells were washed by settling through FSW, then through Ca’+-free seawater (Marine Biological Laboratory formula), and finally through two changes of SEMEB lacking Triton X-100. The washed cells were then extracted with approximately 20 vol of SEMEB for 1 hr at 20°C in Syracuse watch glasses (5.5 mm diameter). The cells were intermittently mixed by gentle agitation of the dishes and at the end of the extraction period the soluble material was removed with a Pasteur pipet. The CSKs were then washed by the addition to the dishes of SEMEB without Triton X-100. Washed CSKs were fixed in 2% gluteraldehyde in 0.1 M phosphate buffer (pH 7.2) for 30 min at 2O”C, washed in three changes of 0.1 ~phosphate buffer at 4”C, and postfixed with 1% 0~0, for 5 min at 20°C. The CSKs were dehydrated, critical point dried, sputter coated, and examined by SEM. Eggs and zygotes to be viewed by TEM were handled as described above but the CSKs were extracted in the same buffer used for biochemical analyses (see below). The cells were fixed with 2% gluteraldehyde in 0.1 M phosphate buffer (pH 7.2), washed with three changes of 0.1 M phosphate buffer, postfixed with 1% 0~04 for 1 hr at 20°C, washed with phosphate buffer, dehydrated, and embedded in Epon.
Microscopy
It was necessary to remove the extracellular coats from the eggs and embryos for examination by scanning electron microscopy (SEM). The vitelline layer of dejellied eggs was removed by a brief treatment with 25 @g/ml protease after which the eggs were extensively washed with FSW (Epel, 1970). As a control for this
Preparatirm of SOL and CSK for B~~hern~e~l Analyses
Whereas the methods described above were designed to maximize the retention of cellular structure, the following procedures were adopted to arrest ribosomes on polyribosomes and to maintain ionic conditions which
MOON
ET AL.
C#x&eletal
yield intact mRNPs (Moon et a!., 1980). Unhatched embryos cultured in 1 mM 3-amino-1,2,4-triazole were concentrated to a density of 10% (v/v) by low-speed centrifugation then poured through nitex mesh to remove the fertilization membrane, as described above. These embryos were then cultured for 20 min to allow the reformation of polyribosomes, which tend to decrease in number and size during ~entrifu~ation of the embryos. Dejellied eggs were not treated with protease or dithiothreitol, since control experiments demonstrated that the presence of the vitelline layer did not affect the proportion of poly(A) released during the extraction and since these methods might increase ribonuclease activity. Protein synthesis was arrested by plunging the suspensions of eggs and ‘embryos into 15 vol of cold FSW (-4”C), The eggs and embryos were prepared for extraction by pelleting at 500~ for 30 set (0°C) then pelleting through 100 vol of Caz+-free seawater (O*C). The extraction buffer used for biochemical analyses (BEB) consisted of 220 mM K+, 75 mM, Cl-, 145 mM OAc-, 300 mMsucrose, 5 mMMgzi, 20 mMMOPS, 0.5% Triton X100, and 05 mg/ml soybean trypsin inhibitor (pH 6.9 for both eggs and embryos). After transfer to sterile 15-ml polycarbonate centrifuge tubes, the cells were washed twice with 15 vol of BEB lacking Triton X-100 (0°C) by centrifugation at 200~ for 30 sec. The pellets were gently resuspended in 5-10 vol of BEB by inversion of the tube and extracted on ice fur 20 min, with mixing accomplished by inversion of the tube every 5 min. The CSK was pelleted by centrifugation at 200~ fur 45 set and the su~rnatant was removed. The CSK pellets were again washed by gentle resuspension in 2-4 vol of BEB for 5 min (O’C) followed by recentrifugation and removal of the supernatant. The combined supernatants were designated the SOL. Potyribosome
mRNA
in Sea Urchins
449
hollowing centrifugation the gradients were either pumped through an Isco Model UA-5 absorbance monitor or fractions of 1.5 ml were collected into 3 ml of ice-cold ethanol in sterile polypropylene test tubes (12 X 75 mm, obtained through BioRad, Richmond, Calif.) and precipitated at -80°C.
The ethanol precipitates of the sucrose gradient fractions were collected by centrifugation of the polypropylene test tubes in a Sorvall HS4 rotor at 5000~ for 45 min at -10°C. The drained pellets were dissolved in 0.5 ml of 0.11M Tris-HCl (pH 9.0), 0.5% SDS. An equal volume of buffer-saturated redistilled phenol was added and the samples were mixed twice by vortex at 1-min intervals. After the addition of 0.5 ml of CHC& each tube was mixed by vortex and centrifuged for 15 min at 5000~ in the HS4 rotor (15°C). The organic phase was removed with a pipet and discarded; the aqueous phase was reextracted with 0.5 ml of CHCb, and the samples were recentrifuged. The aqueous phases were transferred to siliconized glass test tubes, brought to 0,l 1M NaCl, and precipitated with 2 vu1 of ethanol (-SO°C). The ethanol precipitates were collected as described above, dissolved in 0.5 ml of 0.24 A4 NH*OAc, and reprecipitated with 2 vol of ethanol at -80°C. The precipitate was collected by centrifugation and dissolved in 20-50 ~1 of sterile water. RNA was extracted quantitatively from the CSK and SOL by the CsCl centrifugatiun method of Glisin et al. (f914), as modified by Speith and Whiteley (1981). Although DNA does not pellet during this procedure, the resulting RNA samples were precipitated from 2n/rlrLiCl (24 hr at O’C) to ensure DNA removal. The percentage of RNA in the SOL and CSK was determined by absorbance at 260 nm.
Anal&s
The CSK was resuspended in 100 rnM NaCl, If) mM piperazine-N,N’-bis(2-ethanesulfonic acid) (Pipes, pH 7.0), 1% Tween 40, 0.5% deoxycholate (Penman, 1966) to the same volume as the total SOL. The CSK was then disrupted by three passages through a 22-gauge needle. The disrupted CSK and the SOL were then centrifuged at 20,000~ for 6 min at 0°C in a Sorvall SS34 rotor to remove a small amount of particulate material and nuclei. Half of each sample was brought to 30 mM EDTA with a 0.5 M stock solution Samples of 0.4 ml were then layered over 11-ml, lo-40% (w/v) linear sucrose gradients which contained a l-ml cushion of 60% sucrose and final salt concentrations of 220 mM K’, 145 rnM OAc-, 5 mM Mg+‘, 20 mM MOPS (pH 6.9), and 1 mM dithiothreitol. The gradients were centrifuged in a Beckman SW41 rotor at ~90~000~~~~ for 70 min at 1°C.
Aliquots of purified RNA were hybridized to t3H]poly(U) as previously described (Moon et al., 1930) to quantitate the amount of poly(A) in the SOL and CSK. In Vitro Translation Total RNA from the SOL and CSK, as well as from sucrose gradient fractions, was translated in a nuclease-treated message-dependent rabbit reticulocyte lysate supplemented with ~~5S]methionine (Rosenthal & GIN., 1980$,After incubation for 60 min at 30°C the reaction was stopped by the addition of 5 vol of SDSpolyaerylamide gel electrophoresis sample buffer (Laemmli, 1970) to each tube.
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FIG. I,. Dark-field light micrographs of norm al and Triton X-100 extracted S: purpz w&useggs. (A) Untreated egg, 100X. (B) Extracted em3 with fat :us on islets of pigment granules spars6$1~distributed at the surface of the CS#K, 100~. (C) Extracted egg focused to show the corl:ical rim, 175X.
Eggs were fractionated into SOL and CSK after treatment with Triton X-100 in BEB as described above. The SOL and CSK were extracted by shaking for 5 min in petroleum ether and the extracts were dried and analyzed for lipid content by the method of ZSllner and Kirsch (1962). Protein Anal@
tion. Since the detergent-extracted CSK of sea urchin eggs had not been previously described in detail, as an initial step in this research we analyzed the morphology of the CSK using a combination of light microscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM).
and Gel Electrophwesis
The protein content of the SOL and CSK was determined according to Bradford (1976) using a commercially available assay (Bio-Rad, Richmond, Calif.). Proteins were separated in 10% polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS), then stained with Coomassie brilliant blue (Moon et al., 1980). Up to 25 ~1 of rabbit reticulocyte lysate in sample buffer was subjected to electrophoresis without prior heating of the sample. The gel was then processed for fluorography using EnHance (New England Nuclear, Boston, Mass.) and exposed to Kodak XAR-5 X-ray film at -80°C for 2-4 days, then for 20-40 days to ensure the validity of the interpretations. RESULTS
AND DISCUSSION
The purpose of this study was to examine the possible association of mRNA with the eytoskeleton of sea urchin eggs and embryos and to assess whether any changes in this association occurred during development. Our approach was to extract eggs and embryos with Triton X-100 and to separate the soluble material from the detergent-insoluble residue, which we define as the cytoskeleton. We then measured the distribution of translatable nonhistone mRNA and polyribosomes between the cytoskeletal fraction and the soluble frac-
Dark-field micrographs of normal eggs and eggs extracted with Triton X-100 are shown in Fig. 1. The eggs became transparent during the extraction, leaving a CSK which consisted of a cortical rim and opaque internal islets containing pigment granules. TEM showed that the CSK of eggs was less complex than that of one-cell zygotes or blastulae (Fig. 2). The extracted eggs were largely depleted of cell structures except for a plasma membrane lamina (Ben Ze’ev et al., 19’79), components which may be the remnants of cortical granules, pigment granules, and scattered internal patches of filaments associated with ribosomes (Figs. 2A and C). After fertilization a dense filamentous network appeared in the cortex and the internal portions of the CSK became richer in filaments associated with ribosomes (Fig. 2B). As shown in Fig. 2D the internal filaments with associated ribosomes were more abundant in the CSK of blastula cells than in the CSK of eggs. This suggests that an increase in the interaction of ribosomes with the CSK occurs during early development. The nature of the surface of the CSK was further studied by SEM. Figures 3 and 4 show the cortical filamentous network of A. pun&data and L. pictus eggs. The surface of the unextracted egg was covered with short microvilli (Fig. 3A), whereas the unextracted zygote was covered with long microvitli as previously re-
FIG. 2. TEM of Triton X-100 extracted eggs and embryos of A. p~nct~lata. (A) The cortical region of an egg CSK showing the plasma membrane lamina (downward facing arrow), possible remnants of cortical granules (upward facing arrows), and pigment granules, 7100X. (B) The cortical region of a fertilized egg CSK showing a dense cortical filamentous network [arrow) and increased compfexity in the internat regions, 1100x. (Cf Internal region of an unfertilized egg CSK showing the sparse distribution of filaments with associated ribosomes, 12,500X. (D) Internal region of the CSK of a hatched hlastula cell showing abundance of filaments associated with ribosomes, 12,500X. The horizontal bars represent 1 pm.
ported by Schroeder (1979) (Fig. 3B). In contrast, the surface of the CSK of unfertilized eggs was characterized by spherical structures averaging about 0.8-1.0 pm in diameter (Fig. 3C). The spherical structures appeared to be positioned in rowa one layer thick and were eonnetted to an underlying network of filaments. The num-
ber of these structures exposed on the surface of the cytoskeleton of L p&us eggs was calculated to be approximately 24,000. The spherical structures are similar in size and number to cortical granules (Schroeder, 1979). As expected of putative cortical granules, these structures were not observed in the CSK of fertilized eggs
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FIG. 3. SEM of the surface of normal and Triton X-100 extracted eggs and one-cell zygotes of L. pi&us. (A) An untreated egg with short microvilli. (B) An untreated zygote with long mierovilli. (C) An egg CSK with exposed cortical granules. (D) A zygote CSK with exposed cortical filamentous network. (E) An egg CSK treated with Cazf to rupture cortical granules and expose the underlying filamentous network. All bars represent 2 hm.
(Fig. 3D, Fig. 4). The putative cortical granules were also not observed after treatment of the CSK with 1 m&l Ca’* (Vacquier, 1975), which revealed an underlying network of filaments (Fig. 3E). SEM analysis revealed a filamentous cortical network in the CSK of fertilized eggs (Fig. 3D, Fig. 4). This network probably includes the F-a&in CSK previously observed in the cortex of fertilized sea urchin eggs by electron microscopy (Vacquier, 1981). The filamentous network cannot consist entirely of actin filaments, however, because the network also exists in the CSK of unfertilized eggs in which little F-actin is known to be present (Vacquier, 1981). The other filamentous elements of the cortical rim of the CSK may be identical to the heterogeneous network of filaments recently described by Kidd and Mazia (1980). BiodaernicaZ Composition of the CSK
The proportion of total protein lipid, RNA, and poly(A) remaining in the CSK after eggs and developing embryos were extracted with Triton X-100 is shown in
Table 1. It can be seen that only 16% of the total lipid, lo-20% of the total protein, and 9-25% of the total RNA remained in the CSK. The percentage of total RNA in the CSK appeared to increase slightly after fertilization, from 9% in the unfertilized egg to 17% in the fourcell embryo, and 25% in hatched blastulae. The amount of total RNA in the CSK of the developing embryo may be an underestimate of actual values, since any “runoff” of ribosomes from polyribosomes would contribute to the RNA mass in the SOL because free monoribosomes are not retained in the CSK (described below). A large portion of poly(A)-containing RNA remained in the CSK of eggs and embryos, and this amount gradually increased during early embryogenesis (Table 1). RNAs from Since some prevalent poly(A)-containing sea urchin eggs cannot be translated in vitro and represent mitochondrial RNA (Wells et al., 1982), the percentage of poly(A) retained in the CSK cannot be directly correlated with the amount of translationally active cytoplasmic mRNA in this fraction. In order to determine whether the proteins retained in the CSK represent a subset of the total proteins of
MOON
FIG. 4. SEM of a Triton is 4 pm. (B) The horizontal
X-1013 extracted bar is 1 pm.
ET AL.
A. pm&data
Cytoskeletal
zygote
showing
intact eggs and embryos, the CSK and SOL were subjected to SDS-polyacrylamide gel electrophoresis. The gels showed that distinct sets of major proteins were present in the CSK and SOL (Fig. 5). The CSK of unfertilized eggs, four-cell embryos, hatched blastulae, and prism-stage embryos were enriched relative to the SOL in major proteins of 44, 46, 49, 53, 72, and 94 X lo3 molecular weight (M,) at a.11stages. The protein composition of the CSK changed during early embryogenesis. Proteins of 23,24,65, and 180 X 103, major components of the CSK from unfertilized eggs and four-cell embryos, were absent or present in considerably reduced levels in the CSK of hatched blastula- and prism-stage embryos. Moreover, 15-20 proteins of 20-41 X 10” M, increased in the CSK during embryogenesis. These proteins probably represent ribosomal proteins, a possibility which is consistent with the visuaiization of ribosomes in the CSK by TEM (Figs. ZC, D) and the identification of polyribosomes in the CSK by the biochemical studies discussed below. and CSK Messenger
RNAs
The presence of poly(A) in the CSK at all developmental stages suggests that it contains mRNA. Therefore, we tested whether SOL and CSK RNAs were translatable in a message-dependent rabbit reticulocyte lysate. We also determined whether SOL and CSK RNAs coded for qualitatively different polypeptides by separating their translation products on polyacrylamide gels, followed by fluorography. RNAs extracted from the SOL and CSK of unfertilized eggs, four-cell embryos, and hatched blastulae all contained translatable mRNAs,
mRNA
in
Sea
the filamentous
Urchins
network
of the cortical
rim.
(A)
The horizontal
bar
as evidenced by the in vitro synthesis of [3”S]methioninelabeled products (Fig. 6). Comparing the products synthesized by SOL and CSK mRNAs at any one developmental stage revealed no qualitative differences which could be resolved by gel electrophoresis. In this regard our results support those of van Venrooij et aE. (1981), who have made similar observations for detergent-extracted human KB cells. Cervera et al. (1981), however, have shown that the CSK of mammalian cells contains a distinct subset of prevalent mRNAs. Since our gels did not resolve histones, it is not known whether histone
TABLE 1 BIOCHEMICAL COMPOSITION OF THE CYTOSKELETAL FRAMEWORK SEA URCHIN EGGS AND DEVELOPING EMBRYOS Percentage Developmental stage’ Unfertilized
egg
Zygote
OF
in eytoskeleton
Lipid
Protein
RNA
16
13
9
42 (1)” 44 (2)
-
-
15
43 (1)
Poly(A)
Four-cell
embryo
-
10
17
53 (1) 39 (21
Hatched
biastula
-
12
25
69 (1)
60 (2) Prism
-
20
-
87 (2)
aS. purpuratus eggs were employed for lipid analysis. All other analyses were done with A. punctulata~. In experiment (1) the poly(A) content of RNA was determined after purification of the RNA by CsCl centrifugation. In experiment (2) the purified RNA was precipitated with 2 df LiCl before hybridization.
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in that it contained only 15-30% of the free monoribosomes (Fig. ?A and Table 2).
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Increased Associatim
scscscsc EGG
HBI
PRiSM
CL
FIG. 5. Total proteins of the SOL and CSK of A. ~n~~~o~. In lanes 2-9 S designates soluble proteins and C designates CSK proteins. Proteins of the egg (lanes 2,3), four-cell-stage (lanes 4,5) hatched hlastula (lanes 6, 7). and prism-stage (lanes 8, 9) were separated on a 10% polyacrylamide gel and stained with Coomassie blue. Lane 1 contains molecular weight standards (muscle myosin, 225,000 M,; actinin, 105,000 M,; phosphorylase B, 94,000 M,; transferrin, 78,000 M,; bovine serum albumin, 67,000 M;; actin, 42,000 M,; coneanavalin A, 2’7,000 nCr,). The 22,700 &fr protein in lanes 4, 6, and 8 is soybean trypsin inhibitor, added to the extraction buffer. Proteins discussed in the text are indicated by a dot and their calculated molecular weights are given X10e3.
of Messages with
The presence of mRNA (Fig. 6) and polyribosomes (Fig. 7) in the embryonic CSK and the apparent increase in poly(A)-containing RNA in the CSK after fertilization (Table 1) suggest that the postfertilization recruitment of stored mRNPs into polyribosomes may be correlated with increased interaction of the mRNPs with the CSK. If this phenomenon exists in vz’vo, we would expect the stored nonpolyribosomal mRNPs of unfertilized eggs to be located predominantly in the SOL. Since nonpolyribosomal mRNPs are not detectable in absorbance tracings such as those in Fig. 7, we used an alternative technique to detect the mRNPs. Aliquots of the SOL and CSK of unfertilized eggs and early blastulae (6 hr, 2O*C) of A. ~~et~la~ were centrifuged through sucrose gradients similar to those in Fig. 7. We then fractionated the gradients, extracted the RNA from each fraction, translated the RNA in the message-dependent rabbit reticulocyte lysate, and visualized the products of translation after separation of the proteins on SDS-polyacrylamide gels (Fig. 8 and Fig. 9).
1234567
messages display a preferential the SOL or the CSK.
localization
in either
Recent studies with cultured mammalian cells indicate that all polyribosomes, but not monoribosomes, are associated with the CSK (Lenk et al., 1977; Cervera et al., 1981). These studies would predict, therefore, that some or most of the mRNA detected in the CSK of sea urchin embryos would be present as polyribosomes. This possibility was investigated by sucrose gradient analysis of aliquots of SOL and disrupted CSK prepared from unfertilized A. punctulata eggs and hatched blastulae (9 hr, 2O’C). Tracings of the absorbance of these gradients at 260 nm are presented in Fig. 7, and quantitated in Table 2. As predicted, approximately 90% of the EDTA-sensitive polyribosomes, but only 20% of the monoribosomes, were associated with the CSK of hatched blastulae (Fig. 7B and Table 2). Although the low levels of polyribosomes present in unfertilized eggs (Humphreys, 1971) could not be quantitated by this method, the egg CSK was similar to the hatched blastula CSK
ESCSCSC EGG
HBI CL
FIG. 6. Qualitative comparison of mRNAs in the SOL and CSK. RNA extracted from the SOL (S, lanes 2,4, and 6) and CSK (C, lanes 3, 5, and 7) of eggs (lanes 2, 3), four-cell embryos (lanes 4, 5), and hatched blastulae (lanes 6, 7) were translated at saturating concentrations of message in the rabbit reticulocyte lysate and the products separated on a 10% polyacrylamide gel followed by fluorography. Endogenous (E) products are shown in lane 1.
MOON ET AL.
Cytoskeletal mRNA in Sea Urchins
We translated sucrose gradient fractions of blastula SOL and CSK to confirm that embryonic polyribosomes were present in the CSK. The peak of translational activity in blastulae was, as expected, in the polyribosome region of the CSK sucrose gradient (Fig. 8A, fractions 1 and 2). The polyribosomal nature of the RNA in fractions 1 and 2 of Fig. 8A was suggested by the displacement with EDTA of the polyribosomal mRNA to the region of the gradient known to contain free mRNPs (fractions 5-7 of Fig. SC)1(Jenkins et al., 1978; Moon et al., 1980). Little translational activity was detected in any of the fractions of the blastula SOL gradient (Fig. 8B), although after treat.ment of the SOL with EDTA translationally active mRNA was detected in fractions 5-7 (Fig. 8D). This result. suggests that small amounts of translationa~ly competent SOL polyribosomal mRNA were distributed between many gradient fractions, and the diluted mRNA was only detectable after treatment with EDTA shifted the mRNA to primarily one fraction (Fig. 8D). The detection of mRNA in the SOL gradient does not, however, definitively establish that these messageswere present in polyribosomes, or that these messageswere free in the cytoplasm. These results support the analyses based upon the absorbance of the gradients at 260 nm and, taken together, indicate that more polyribosomes were present in the CSK than in the SOL of the blastula. TranslationaIly active mRNA was, however, present in both fractions. In contrast to the results obtained with blastulae, most of the translational activity of eggs (Fig. 9) was present in the SOL gradient and divided between fraction 1 (the polyribosome region) and fractions 4-7 (the small polyribosome and free mRNP region) (Fig. 9B). Fraction 6 probably contains the highest concentration of the nonpolyribosomal mRNP complexes of eggs. These mRNPs sediment with a broad distribution averaging about 60 S (Kaumeyer et al., 1978; Moon et al., 1980). TABLE 2 MONORIBOSOME AND POLYRIBOSOME DISTRIBUTION IN THE CYTOSKELETAL FRAMEWORK OF A. punctulata EGGS AND HATCHED BLASTULAE Percentage in cytoskeleton” Developmental Unfertilized
stage
eggs
Hatched blastulae
Monoribosomes 30 (1) 16 (2) 19
Polyribosomes undetectable 90
a The percentage of monoribosomes and polyribosomes in the CSK was calculated from the area of the peaks shown in Fig. ‘7.The baseline was established by comparison of the absorbance tracing of EDTAtreated samples and blank gradients.
3. Blaslula
d A
2
“1,
4
6
8 Fraction
,
2
,
,
4
)
,
6
,
8
Number
FIG. 7. Segregation of monoribosomes and polyribosomes between the SOL and the CSK of (A) eggs and (B) hatched blastula embryos. The absorbance at 254 nm is shown for SOL and CSK centrifuged on lo-40% sucrose gradients and pumped through an absorbance monitor with the top of the gradient (right) as the leading edge f-). Identical samples were pretreated with 30 mM EDTA before centrifugation (- - -). In panel A the dashed line merges with the solid line. For clarity, the dashed line in panel B is shown in the postmonoribosome region only. The discontinuity in the absorbance in panel B is due to a twofold change of scale. Fractions similar to those used for RNA purification and in vitro translation are indicated.
After treatment with EDTA, all of the translatable mRNA of the egg SOL was detected in fractions 5-7 of the gradient (Fig. 9C). The sensitivity to EDTA of the mRNA present in gradient fractions 1 and 4 of Fig. 9A implies that the mRNA in these fractions was associated with detergent-extracted polyribosomes which were not part of the CSK. The small amount of translational activity present in the egg CSK was located in fraction 6, suggesting that it represented nonpolyribosomal mRNP (Fig. 9A). Our results show that most of the mRNA in eggs sediments in sucrose gradients as free mRNP and is present in the SOL whereas most of the mRNA in blastulae occurs as polyribosomes in viva and is present in the CSK. The retention of polyribosomes in the CSK of embryos was not species specific or dependent upon the use of the BEB extraction buffer. When A. punctulatahatched blastulae were extracted with SEMEB (without EGTA or EDTA), most polyribosomes were retained in the CSK. The SOL and CSK of S. purpurutus eggs and hatched blastulae were also examined for translationally active mRNA across sucrose gradients (data not shown). Consistent with the data presented here for A. punctuhta, most of the translatable mRNA in the eggs of 5’. purpwatus was present in the nonpolyribosomal mRNP region of the SOL, and most of the translatable
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not allow precise quantitation, a rough estimate is that very little translatable mRNA is retained in the CSK of the unfertilized egg, and that approximately 50% or more of the translatable mRNA is present in the CSK of blastulae. The association of egg mRNA with the CSK could occur shortly after fertilization at the same time as the recruitment of stored messages into polyribosomes, though this awaits further analysis. Our finding that mRNA-~ytoskeletal interactions change during development is consistent with the observation that viral polyribosomes, but not host messages, are associated with the CSK in infected HeLa cells (Lenk and Penman, 1979). The correlation noted here between the low level of
FIG. 8. Translation across sucrose gradients of blastula CSK and SOL. A. ~~ct~~~~ blastulae (6 hr, 2O“C after fertilization) CSK (panels A and C) and SOL (panels B and D) were centrifuged on lo-40% sucrose gradients similar to those in Fig. 7 with (panels C and D) or without (panels A and B) pretreatment with 30 mM EDTA. Gradient fractions corresponding approximately with those in Fig. 7 (top of the gradients on the right), and pelleted material (lane B), were extracted and the RNA was translated in the rabbit reticulocyte lysate. The fluorograph of the 10% ~olyacrylam~de gel localized sea urchin messages in the sucrose gradients.
mRNA in hatched blastulae was present in the polyribosome region of the CSK after extraction of the cells with S~MEB containing 5 mfl/l EGTA. Since the retention of polyribosomes in the CSK was not species specific, the next step is to determine whether or not the apparent association of polyribosomes with the CSK involves specific sites on both the polyribosomes and the CSK. In summary, the results of the present investigation show that a marked increase occurs in the percentage of translatable mRNA which is retained in the CSK during development. Although the in vitro translation method used to detect CSK-associated messages does
FIG. 9. Translation across sucrose gradients of egg CSK and SOL. A&a& pun&da CSK (panels A and C) and SOL (panels B and D) were centrifuged on lo-40% sucrose gradients similar to those in Fig. 7 with (panels C and D) or without (panels A and B) pretreatment with 30 mM EDTA. The direction of centrifugation was from right to left. Gradient fractions correspond approximately with those in Fig. 7, and fraction B contained pelleted material. Fraction 8 of panel C was lost. RNA extracted from the sucrose gradient fractions was translated in the rabbit reticulocyte lysate and the products directed by sea urchin or endogenous (E) messages were separated on a 10% polyacrylamide gel followed by fluorography.
MOON
ET AL.
Cytoskeletal
mRNA-CYK interactions and the relative translational inactivity of the egg raiseis the possibility that the postfertilization recruitment of mRNAs for translation may be dependent upon association of the message with cytoskeletal elements. In eggs and blastulae, however, a small yet detectable amount of translatable mRNA, some of which may have been polyribosomal, was present in the SOL. Thus, at present it must be concluded that although there appears to be a correlation between activation of protein synthesis and the retention of mRNAs in the CSK, this apparent association may not be absolutely required for translation. Alternatively, it is possible that the association of mRNAs with the CSK enhances the efficiency of translation after fertilization, or that this association is a mechanism which promotes a nonrandom spatial distribution of maternal mRNA during early development.
mRNA
in
Sea. Urchins
457
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We thank E. Rosenthal for provision of rabbit reticulocyte lysate and hefpful suggestions; L. Hyman, Rr. 3. Ruderman, and Dr. T. Hunt, for supplies and equipment; and Dr. M. A. Rankin, for performing the lipid analysis. S. Houghton provided technical assistance in eleetron microscopy. This work was supported by NIH Grants HD-13970 (W.R.J.), HD-11070 (M.B.H.), NRSA HD-07183 (R.T.M.), and 323HD07098 (Embryology course, Marine Biological Laboratory, Woods Hole, Mass.) and American Cancer Society Institutional Grant IN 26U (R.T.M.). REFERENCES BEN ZE’EV, A., DUERR, A., SOLOMON, F., and PENMAN, S. (1979). The outer boundary of the cytoskeleton: A lamina derived from plasma membrane proteins. C& 17,853-865. BRADFORD, M. (1376). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Bz’mhem. 72.248-254. BROWN, S., LEVINSON, W., and SPUDICH, J. A. (1976). Cytoskeletal elements of duck embryo fibroblasts revealed by detergent extraction. J. supramd .%%ct. 5,113-130. CERVERA, M., DREYFUSS, G., and PENMAN, S. (1981). Messenger RNA is translated when associated with the cytoskeletal framework in normal and VSV-infected HeLa cells. CeEE 23, 113-120. EPEL, D. (1970). Methods for removal of the vitelline layer of sea urchin eggs. II. Controlled exposure to trypsin Lo eliminate postfertilization clumping of embryos. Exp. Cell Res. 61, 69-70. EPEL, D., WEAVER, A. M., and MAZIA, D. (1970). Methods for removal of the vitelline layer of sea urchin eggs. I. Use of dithiothreitol (Cleland reagent). Exp. CeZl Res. 61, 64-68. FULTON, A. B., WAN, K. M., and PENMAN, S. (1980). The spatial distribution of polyribosomes in 3T3 cells and the associated assembly of proteins into the skeletal framework. Cell 20, 849-857. GLISIN, V., CRKVENJAKOV, R., and BYUS, C. (1974). Ribonueleic acid isolation by cesium chloride centrifugation. Biochemistry 13,26332637.
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