Isolation and characterization of hamster brain polyribosome-cytomatrix complexes

Neurochem. Int. Vol. 21, No. 1, pp. 21 27, 1992 Printed in Great Britain.All rights reserved

0197-0186/9255.00+0.00 Copyright © 1992PergamonPress Ltd

ISOLATION A N D C H A R A C T E R I Z A T I O N OF H A M S T E R B R A I N P O L Y R I B O S O M E - C Y T O M A T R I X COMPLEXES PIERRE DESJARDINS, CHANTAL SAVOIE,* JOHANNE DOUCET a n d

DIDIER GAUTHIERt D6partement de Chimie et Biochimie, Universit6 de Moncton, Moncton, N.B., Canada, EIA 3E9 (Received 10 June 1991 ; accepted 20 November 1991)

Abstract--We have developed a method for the isolation of a brain subcellular fraction enriched in both highly aggregated polyribosomes and cytoskeletal proteins. This method is based on gentle dispersion of brain tissue and low speed centrifugation. This fraction is enriched in typical cytoskeletal proteins as glial fibrillary protein, neurofilament proteins and actin. Messenger RNA did not seem to be involved in the polyribosome association to the cytomatrix as shown by the effect of exposure to micrococcal nuclease. On the other hand, in vivo disruption of protein synthesis by acute experimental phenylketonuria, hypothermia or heat-shock did not cause the release of ribosomes from the cytomatrix.

It is well known that some cell polyribosomes are bound to the rough endoplasmic reticulum (Palade, 1975). Those that are not m e m b r a n e - b o u n d were thought to be "free-floating" in the cytoplasm. This view has been revised after high-voltage electron microscopy observations showing ribosomes binding to the cytoskeleton (Wolosewick and Porter, 1979). Additional biochemical as well as microscopical evidence indicates that most of actively translating ribosomes are b o u n d to the cytoskeleton framework (hereafter called cytomatrix) (Lenk et al., 1977 ; Lenk and Penman, 1979; Venrooij et al., 198l ; Howe and Hershey, 1984; Bonneau et al., 1985). Other components of the protein synthesis machinery have also been found to bind the cytomatrix in cell cultures : m R N A (Bonneau et al., 1985; Bird and Sells, 1986), aminoacyl-tRNA ligases (Mirande et al., 1985) and initiation factors (Howe and Hershey, 1984). So far, isolation of such polyribosome-cytomatrix complexes has been almost limited to cultured cells, which can be permeabilized with mild detergent, leav-

ing an intact cytomatrix easily pelletable by low-speed centrifugation. Harsher procedures, like homogenization and high-speed centrifugation, seem to disrupt polyribosome-cytomatrix interactions. Recently we have developed a method allowing the isolation, from brain tissue, of a fraction containing a significant part of polyribosomes and enriched in some cytoskeletal components (Lequang and Gauthier, 1989; Bouhtiauy et al., 1989). In this paper we report the improvement of this method, making it closer to those generally used with cell cultures. A large part of the polyribosomes were bound to this cytomatrix which was also enriched in intermediate filament proteins and actin. In order to study the binding mechanism, polyribosome-cytomatrix complexes were exposed to micrococcal nuclease. This enzyme failed to dissociate ribosomes from the cytomatrix, suggesting that m R N A was not involved in this association. On the other hand, conditions leading to a partial or total halt of the protein synthesis did not cause the release of polyribosomes from the cytomatrix.

*Present address: D6partement de Pharmacologie, Universit6 de Sherbrooke, Sherbrooke, QC, Canada, JIK 2R1. t Author to whom all correspondence should be addressed at: D+partement de chimie et biochimie, Universit6 de Moncton, Moncton, N.B., Canada, E1A 3E9. Abbreviations: DOC, sodium deoxycholate; DTT, dithiothreitol ; EGTA, ethyleneglycol-bis (fl-aminoethyl ether)-N,N,N',N'-tetraacetic acid; PMSF, phenylmet hylsulfonylfluoride.

EXPERIMENTAL PROCEDURES

Tissue fractionation

Brains of 6 1 0 day-old hamsters were removed and dispersed by gently passing them twice through a 60-mesh tissue sieve and recovered in 4.7 vol of lysis buffer (70 mM [K ÷] acetate, 5 mM MgC12, 50 mM Hepes, 20 mM KOH, 300 mM sucrose, 2.5 mM DTT, 2 mM EGTA, 1 mM PMSF). Then 0.25 vol (relative to brain weight) of 10% Triton X-100 was 21

22

PIERRE DESJARDINS el a[.

added. After gentle mixing and 5 min standing on ice, the mixture was spun at 3000 g for 3 min in a swinging bucket rotor (IEC 870). The supernatant (soluble fraction) conrained the detergent-soluble, i.e. non-cytomatrix, material. The pellet was washed once by resuspending in 4.7 vol (relalive to brain weight) of"washing" buffer (lysis buffer without EGTAI and 0.25 vol of I0% Triton X-100 was added. This resuspension was centrifuged as before. The resulting supernatant ("washing") was recovered and the pellet, containing Ihe cytomatrix, was solubilized in an appropriate buffer. Iso/alio, ~!/ hrain cj'toskeh'lal prolei,s Cytoskeletal proteins were extracted from 9 10 day-old hamster brains according to Dahl et al. (1981). Brains were homogenized in 20 vol of extraction buffer (0.1 mg/ml DNase I, I% Triton X-100, 600 mM KCI, 10 mM MgCI2, 2 mM EDTA. I mM EGTA, Na2HPO4 66.5 mM, NaHePO4 33.5 raM). After centrifugation at 12,000 g for 10 min, the pellet was rest,spended in extraction buffer without DNase and recentrifuged. The final pellet was used as a brain cytoskeletal protein standard, thereafter referred to as a standard cytoskeletal preparation, for comparative purposes. Pg~(t'rihosome sedimentation allalrsi,s' o n .s'ucrose ~]radients The cytomatrix pellet was resuspended in 4.7 vol (relative to brain weight) of dissociating buffer (DB : 300 mM [K ~] acetate, 5 mM MgCI2, 50 mM Hepes, 20 mM KOH, 2.5 mM DTT, 2 mM PMSF). Then 0.25 vol (relative to brain weight) of 10% Triton and 0.05 ml of 10% deoxycholate Isodium salt) were added. After homogenization in a Potte> Eveljhem grinder and incubation for 10 rain on ice, this mixture was clarified by centrifuging at 30,000 g for 10 rain. The supernatant contained solubilized polyribosome cytomatrix complexes components while the pellet consisted mainly of nuclei and insoluble debris. The soluble fraction and "'washing" were also clarified by deoxycholate addition and centrifugation at 30,000 g for 10 min. The resulting supernalants constituted the fractions to be analysed on sucrose gradients. Polyribosome sedimentation analyses were done on 20 47% w,,wsucrose gradients made in gradient butler (400 mM [K 1 acctatc, 5 mM MgCI,, 50 mM l-tepes, 20 mM KOH) as described (Gauthier and Murthy, 1987). The I I ml gradients were made using a gradient maker (BioComp, Fredericton, Canada) set at 80.5 and 13 rpm lbr 2.03 min and overlaid with 300 tzl of 16.3% w w sucrose in gradient buffer. This overlay contained 1% deoxycholate and 1% Triton X- 100 to dissolve structures potentially cosedimenting with the lighter ribosonral structures. The 350 ,ul samples were then layered and spun for 3.15 h (Beckman SW-40 rotor) and analysed with an ISCO UA-5 monitor and flow-cell. The sedimentation profiles wcre recorded and the area under the curve computed after baseline correction with a data reduction system (Hoefer GS-340). Eh'c~rophore.sis and immunodetection The fractions to bc electrophoresed were prepared as follows. Cytomatrix pellets were resuspended in 2.25 vol (relative to original brain weight) of dissociating buffer (300 mM [K'] acetate, 5 mM MgC12, 50 mM Hepes, 20 mM KOH, 2.5 mM DTT, 2 mM PMSF). Then 2.5 vol (relative to brain weight) of 10% sodium dodecyl sulfate (SDS) and 0.25 vol of fl-mercapto-ethanol were added. Alter thorough homogenization in a Potte~Eveljhem grinder and heating

for 5 rain in boiling water, this mixture was clarified by centrifuging at 30,000 g for l0 min. The soluble fraction and "'washing" fractions were added with 0.25 vol of SDS 10% and 0.01 vol of fl-mercapto-ethanol, heated in boiling water for 5 min and centrifuged as above. The resulting supernatants, containing solubilized proteins, were stored at 20°C until electrophoretic analyses. Electrophoreses were carried in a discontinuous buffer system (Laemmli, 1970). The gels were stained with Coomassie blue or electroeluted on nitrocellulose sheets. Electroelution was performed (Burnette, 1981) with a Bio-Rad "Trans-Blot'" system at 28 V for I h then 60 64 V overnight (Otter el al.. 1987) with cooling. The resulting blots were either stained with amidn black (Burnette, 1981) or submitted to immunodetection with alkaline phosphatase-conjugated secondary antibodies (Blake et al., 1984). Monoclonal antibodies against glial fibrillary acidic protein (clone G-A-5) (Debus et al., 1983), light neurofilament protein (clone NR 4) (Debus et al., 1983), medium-sized neurofilament protein (clone NN 18) (Debus et al., 1983), heavy neurofilament protein (clone NE 14) (Debus et al., 1983) and actin (Lin et al., 1981 ) were obtained from commercial sources. RESULTS

f ' r a c t i o n a t i o n o f brain soluble c o m p o n e n t s

into

cytomatri.v-bound

and

We have fractionated a brain extract obtained by the Triton-extraction scheme described in "'Experimental Procedures". This procedure was a modification o f that previously published (Lequang and Gauthier, 1989; Bouhtiauy et al., 1989). The most noticeable modification was the replacement o f the P o u n c e grinder homogenization by the dispersion o f the brain tissue with a 60-mesh sieve. This approach allowed us to apply to brain tissue a procedure closer to the one used for cell cultures which avoided the physical stress o f homogenization and use o f low speed centrifugation, 3000 g for 3 min. During the course o f the development o f this method many conditions were tested. For example we found no difference between 40- and 80-mesh grids. The inclusion o f protease inhibitors (PMSF, leupeptine, aprotinine, ct2-macroglobulin, e:-aminocaproic acid) did not allow the recovery o f a larger a m o u n t of polyribosomes in the cytomatrix fraction. Higher concentrations o f Triton, up to 2.5%, did not change the relative repartition of ribosomes between both fractions. Typical sedimentation profiles are shown in Fig. 1. This new method allowed us to obtain a larger fraction o f polyribosomes still associated to the cytomatrix, about 50%, compared to 2(~30% in the previous method (Bouhtiauy et al., 1989). Even if the soluble fraction and cytomatrix fractions both contained highly aggregated polyribosomes, the soluble

23

Brain polysomes---cytomatrixcomplexes •

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;EDIMENTATION - I ~ Fig. 1. Sedimentation profiles of polyribosomes. A brain homogenate was fractionated into soluble (SOL), "washing" (WSH) and cytomatrix (CMT) fractions that were analysed on a sucrose gradient as described in "Experimental Procedures". Arrows indicate monosomes.

421 fraction was enriched in ribosomal subunits (Fig. 1). Since we analysed the sedimentation profiles in gradient containing high concentrations of K ÷ (400 mM), the monosomes in each fraction resulted from RNase degradation and were not merely ribosome not associated with mRNA (Zylber and Penman, 1970) which appeared as subunits.

Identification of the cytoskeletal proteins Those fractions were electrophoresed in a 5-30% polyacrylamide gel (Fig. 2). The cytomatrix fraction shared some similitudes with a standard cytoskeletal preparation as well as with the soluble fractions. We have ascertained the identity of the major proteins by immunodetection (Fig. 3). Antibodies raised against heavy and medium-sized neurofilament protein-, and glial fibrillary acidic protein [Fig. 3(a), (b), and (c), respectively]. These intermediate filaments proteins were present practically only in the cytomatrix fraction. On the other hand actin was present in all three fractions [Fig. 3(d)]. These results unambiguously showed that the cyomatrix fraction contained most of the cell cytoskeletal proteins.

Role of mRNA in the bindin# to the cytomatrix In order to order to assess the role of mRNA in the binding of the polyribosomes on cytomatrix, we have exposed the dispersed tissue to 500 U/ml micrococcal nuclease (nuclease $7, Ca2÷-dependant) and 1.0 mM

Fig. 2. Electrophoretic analysis of protein. Fractions were obtained as described in "Experimental Procedures". About 50-75 pg of proteins electrophoresed on a 5-30% acrylamide gel (linear gradient). Lane SOL, detergent-soluble; lane WSH, "washing" ; lane CMT, cytomatrix ; lane CSK, standard cytoskeletal preparation (for comparative purposes). Migration position of relative molecular mass standards (in kDa) are shown. for 15 min before the fractionation. We have sought to eliminate the possibility that the RNA degradation could be due to continued action of the nuclease after the fractions were separated. So we have added 2.5 mM EGTA, an inhibitor of this Ca 2÷activated enzyme, to stop its effect after nuclease exposure (15 min), before the beginning of the fractionation process itself. In parallel, a fractionation was also carried out on an untreated sample (without nuclease, Ca 2+ or EGTA) as well as another one where EGTA was added at the same time as nuclease and Ca 2+, as a control. The nuclease succeeded in digesting mRNA and thus disaggregating most polyribosomes into monosomes in both fractions (Fig. 4, lower panels) compared to untreated tissue (Fig. 4, upper panels). On the other hand, EGTA succeeded in effectively stopC a 2+

PIERRE DESJARDINS et al.

24

(a)

(b)

ACTIN

SOL WSH CMT

CSK

Act

G

GFAP CSK CMT

(c)

WSH

SOL

NFP-M

SOL WSH CMT CSK

(d)

NFP-H

CSK CMT WSH SOL

Fig. 3. Immunodetection of western blots. Immunodetection was carried out as described in "Experimental Procedures" after eleetrophoresis on 7.5% (heavy and medium-sized neurofilament proteins) or 10% acrylamide gel (aetin and glial fibrillary acidic protein). Lane CMT: cytomatrix; lane SOL: soluble fraction ; lane WSH : "washing". A typical cytoskeletal preparation (lane CSK) is also shown for comparative purposes. When pure proteins were commercially available, they were used to assess the antibody specificity : lane Act, actin ; lane G, glial fibrillary acidic protein.

ping the nuclease action (Fig. 4, middle panels) thus insuring that the nuclease action occurred only during the 15 min exposure before the fractionation. The m R N A digestion failed to cause any transfer from the cytomatrix to the soluble fraction, since the relative amount of ribosomes in each fraction was not significantly changed, although polyribosomes were degraded into monosomes in both fractions.

Effect of in vivo disruption o f protein synthesis In order to get insights on the importance of polyribosmes-cytomatrix association we have studied the effect various conditions known to bring about polyribosome disruption. For example, experimental hyperphenylalaninaemia, an experimental acute phenylketonuria, can inhibit protein synthesis and cause polyribosome disruption (Aoki and Siegel, 1970; Binek et al., 1981). We have studied the effect of this condition, induced by phenylalanine injection, on the association of polyribosome to the cytomatrix (Fig. 5). The treatment itself caused a partial polyribosome disassembly (corn-

pare left to right panels). However no fraction was more sensitive than the other although a small amount of material did appear in the "washing" fraction (middle right panel). Hypothermia is also known to slow down brain protein synthesis and cause polyribosome disaggregation. Anaesthetized animals were submitted to hypothermia (4~'C for 20 min) as another means to depolymerize brain polyribosomes (Fig. 6). The same observations were made: the slight polyribosome disaggregation was not associated with a shift from one fraction to the other not to a different response from soluble or cytomatrix ribosomes. Finally we used a treatment allowing the disaggregation of most brain polyribosomes, hyperthermia (heat-shock) (Fig. 7). Anaesthetized animals were exposed at 4 Y C for 20 min. This led to a massive disruption of the in vivo protein synthesis as shown by the large increase of monosomes at the expense of polyribosomes (Fig. 7, lower panels). This disruption was not accompanied by an extensive transfer of these monosomes from one fraction to the other.

25

Brain polysomes---cytomatrix complexes

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SEDIMENTATION

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~

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- -

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Fig. 4. Effect of micrococcal nuclease on the association of polyribosomes to the cytoskeletal fraction. A cell extract was divided into three aliquots that were independently fractionated into soluble (SOL, left panels), "washing" (not shown) and cytomatrix (CMT, right panels) fractions. One of those aliquot was treated with micrococeal nuclease and CaC12 and incubated for 15 min at 21°C, after which EGTA was added (TR, lower panels). As a "positive control" (CTR +, middle panels), the second aliquot was added with EGTA immediately followed by microccal nuclease and CaC12 were added. A "negative control" ( C T R - , upper panels), the third aliquot, did not receive any added chemicals. Final concentrations of micrococcal nuclease, CaC12and EGTA were 500 U/ml, 1 mM and 2.5 mM, respectively. Each aliquot was then fractionated and analysed for polyribosome sedimentation as described in "Experimental Procedures". Arrows indicate monosomes. DISCUSSION

We have isolated a cell fraction enriched in both polyribosomes and cytoskeletal elements. We have modified our previous procedure: replacement of homogenization by tissue dispersion, reduced centrifugation speed to separate the cytomatrix material from the soluble fraction, etc. This new method was closer to the one generally used to prepare polyribosome-cytomatrix complexes from cell cultures, which consist in exposing monocellular layers to a mild anionic detergent that will solubilize cell and organelle membranes, leaving intact the cytomatrix. We have improved the method to obtain a fraction enriched in both polyribosomes and cytoskeletal components from brain tissue. Similarly we have exposed dispersed brain fragments, obtained by passing through 60-mesh screens to Triton X-100. So this anionic detergent could quickly attack large surfaces of cells and rapidly dissolve the membranes. This has

m

SEDIMENTATION

-I~

Fig. 5. Effect of hyperphenylalaninaemia on the association of polyribosomes to the cytomatrix. Animals were intraperitoneally injected with phenylalanine (1 mg/g body weight) (pku, right panels) and controls were injected with and equivalent volume of saline (ctr, left panels). Brains were removed 60 min later and fractionated as described in "Experimental Procedures" into soluble (SOL, upper panels), "washing" (WSH, middle panels) and cytomatrix (CMT, lower panels) fractions. Arrows indicate monosomes. replaced the previous homogenization that destroyed, or at least broke into smaller pieces, most of the whole cytomatrix. The cytomatrix nature of the obtained material was unambiguously demonstrated by electrophoresis and immunodetection. The presence of polyribosomes in this cytomatrix fraction suggests an association between the cytomatrix and the protein synthesis apparatus. We believe such an association genuinely exists in brain cells. Indeed the presence of polyribosomes in a cytomatrix fraction can hardly be considered as a mere contamination. Such a possibility has been already proposed. For instance it has been shown that some aminoacyl-tRNA ligases may bind to some "reticulum endoplasmic remnants" co-isolated with cytoskeletal structures (Dang et al., 1983). However such a contamination does not happen in our case. Firstly, up to 2 washes failed to remove polyribosomes from the fraction in which they are found (data not shown). Secondly, the polyribosomes that pelleted at 10,000 g when the cytomatrix was intact did not do so at 30,000 g after its disruption with DOC. Thirdly, moderate salt conditions, which disrupted cytomatrix, succeeded in transferring polyribosomes from the cytomatrix to the soluble fraction (data not shown),

26

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D E S J A R D I N S el al.

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Fig. 6. Efl'ect of hypothermia on the association of polyribosomes to the cytomatrix. Anaesthetized animals were exposed to 4'C for 20 min to simulate hypothermia (hypo, lower panels). "'Positive controls" (ctr+, middle panels), were anaesthetized, injected with saline, returned to the dam and left for 15 rain. "'Negative control" (ctr - , upper panels) were not subjected to any treatment. Brains from each groups were then fractionated into soluble (SOL, left panels), "washing" (not shown) and cytomatrix (CMT, right panels) fractions that were analysed on a sucrose gradient as described in "Experimental Procedures". Arrows indicate monosomes.

such a condition not being k n o w n to destroy endoplasmic structures. On the other h a n d artifactual e n t r a p m e n t of polyribosomes within the cytomatrix networks, to which smaller m o n o s o m e s a n d subunits should be less sensitive, can also be dismissed. Indeed such m o n o s o m e s and subunits produced in vitro (e.g. exposure to micrococcal nuclease) or in vivo (e.g. heat shock) remained b o u n d to the cytomatrix (Figs 4 and 5). The electrophoretic pattern o f our cytomatrix fraction shared similarities with that obtained by A d a m s et al. (Fig. 6). Their fraction also contained polyribosomes a n d was enriched in actin. O u r results extend to brain cells as with the previous reports, mainly dealing with cultured cells, showing that polyribosomes are b o u n d to the cellular framework. The binding m e c h a n i s m is far from being understood. A direct binding has been suggested in most papers supporting this polyribosomes--zytomatrix association (Lenk a n d P e n m a n , 1979: Lenk et al., 1979; Wolosewick and Porter, 1979; Cervera et al., 1981, Venroij et al., 1981 ; Howe and Hershey, 1984: B o n n e a u et al., 1985; Lequang and Gauthier, 1989). However a "'restricted diffusion" scheme may

0

(/)

~,

SOL

HS

CMT HS

SEDIMENTATION -I~ Fig. 7. Effect of heat-shock on the association of polyribosomes to the cytomatrix. Anaesthetized animals were exposed to a heat shock at 43 C for 15 min (HS, lower panels). "'Positive controls" (ctr+, middle panels), were anaesthetized, injected with saline, returned to the dam and left for 15 min. "Negative control" (ctr - , upper panels) were not subjected to any treatment. Brains from each groups were then fractionated as described and analysed as in Fig. 6. Arrows indicate monosomes.

be envisioned. In such a mechanism we can hypothesize that polyribosomes (or a subset of them) as well as other organelles are kept within boundaries defined by cytomatrix elements. The nature of this restricted diffusion could be due to mere e n t r a p m e n t in structures with pores smaller than the organelles, or to other mechanisms. A direct binding mechanism, a l t h o u g h much easier to understand a n d visualize, is not easily reconciled with results presented in this paper and elsewhere. F o r instance E D T A treatment fails to release at least one ribosomal subunit (Howe and Hershey, 1984). Indeed if the binding occurs t h r o u g h ribosomes themselves, we should expect that either the small or the large one is directly involved while the other one is not directly bound. A binding of both subunit at the same time is harder to envision. The nature of the cytomatrix or cytoskeletal component(s) involved is also u n k n o w n . Indirect evidence rule out the possibility that intermediate filaments are instrumental in the binding process. Indeed intermediate filaments are not dissociated by conditions (deoxycholate a n d 350 m M K +) that free polyribosomes from the cytomatrix. M u c h harsher conditions (5"/0 SDS and 0.5% fi'-mercaptoethanol) are required to significantly dissociate intermediate ilia-

Brain polysomes--eytomatrix complexes ments in o u r p r e p a r a t i o n s (C. Savoie, u n p u b l i s h e d observations). In s u m m a r y , we have i m p r o v e d o u r m e t h o d o f preparing cytomatrix fraction c o n t a i n i n g actively translating polyribosomes. Various in vivo a n d in vitro treatments, otherwise k n o w n to effectively block protein synthesis, did n o t cause a release of those polyribosomes from the cytomatrix, indicating t h a t this association was not only due to m R N A binding. O n the other h a n d intermediate filaments did not seem to be the cytoskeletal structure involved in this association. Acknowledgements--This work has been supported by grants from the Natural Sciences and Engineering Research Council of Canada and from the Facult6 des Etudes Sup6rieures et de la Recherche de l'Universit6 de Moncton.

REFERENCES

Aoki K. and Siegel F. L. (1970) Hyperphenylalaninaemia. Disaggregation of brain polyribosomes in young rats. Science 168, 129-130 Binek P. A., Johnson T. C. and Kelly C. J. (1981) Effect of 7-methylphenylalanine and phenylalanine on brain polyribosomes. J. Neurochem. 36, 147(~1484. Bird R. C. and Sells B. H. (1986) Cytoskeleton involvement in the distribution of mRNP complexes and small cytoplasmic RNAs. Biochim. Biophys. Acla 868, 215-225. Blake M. S., Johnston K. H., Russel-Jones G. J. and Gotschlich E. C. (1984) A rapid, sensitive method for the detection of alkaline phosphatase-conjugated anti-antibody on western blots. Analyt. Biochern. 136, 175 179. Bonneau A.-M., Darveau A. and Sonenberg N. (1985) Effect of viral infection on host protein synthesis and mRNA association with the cytoplasmic cytoskeletal structure. J. CellBiol. 100, 1209 1218. Bouhtiauy I., Choukri Y., Turpin C. and Gauthier D. (1989) Characterization of hamster brain polyribosome-cytomatrix complexes. Neurochem. Res. 14, 635~640. Burnette W. N. (1981) Western blotting : electrophoretic transfer of protein from sodium dodecyl sulfate gels to unmodified nitrocelulose and radiographic detection with antibody and radioiodinated protein A. Analyt. Biochem. 112, 195 203. 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. Cell 23,113 120. Dahl D., Ruegger D. C., Bignami A., Osborne M. and Weber K. (1981) Vimentin, the 57,000 molecular weight protein of fibroblast filament is the major cytoskeletal component of immature glia. Eur. J. Cell Biol. 24, 191 196.

27

Dang C. V., Yang D. C. H. and Pollard T. D. (1983) Association of methionyl-tRNA synthetase with detergent insoluble components of the rough endoplasmic reticulum. J. CellBiol. 96, 1138 1146. Debus E., Weber K. and Osborn M. (1983) Monoclonal antibodies specific for glial fibrillary acidic (GFA) protein and for each of the neurofilament triplet polypeptides. Differentiation 25, 193 203. Gauthier D. and Murthy M. R. V. (1986) Polysomes during early postnatal development of brain in the rat. Neurochern. Res. 11, 1373-1378. Gauthier D. and Murthy M. R. V. (1987) Efficacy of RNAse inhibitors during brain polysome isolation. Neurochem. Res. 12, 335 339. Howe J. G. and Hershey J. W. B. (1984) Translational initiation factor and ribosome association with the cytoskeletal framework fraction from HeLa cells. Cell 37, 85 93. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 685. Lenk R. and Penman S. (1979) The cytoskeletal framework and poliovirus metabolism. Cell 16, 289 301. Lenk R., Ransom L. and Penman S. (1977) A cytoskeletal structure with associated polyribosomes obtained from HeLa cells. Cell 10, 67 78. Lequang H. and Gauthier D. (1989) Isolation of polyribosomes-~cytoskeleton complexes from hamster brain. Neurochem. Res. 14, 239 243. Lin J. J-C. (1981) Monoclonal antibodies against myofibrillar components of rat skeletal muscle decorates the intermediate filaments of cultured cells. Proc. natn. Acad. Sci. U.S.A. 78, 2335 2339. Mirande M., Le Corre D., Louvard D., Regio H., Pailliez J.-P. and Waller J.-P. (1985) Association of an aminoacyltRNA synthetase complex and of phenylalanyl-tRNA synthetase with the cytoskeletal framework fraction from mammalian cells. Expl. Cell Res. 156, 91 102. Otter T., King K. M. and Witman G. B. (1987) A twostep procedure for efficient electrotransfer of both highmolecular-weight (>400,000) and low-molecular-weight ( < 20,000) proteins. Analyt. Biochem. 162, 371~377. Palade G. E. (1975) Intracellular aspects of the process of protein synthesis. Science 189, 347 358. Ramsay J. C. and Steele W. J. (1979). Quantitative isolation and properties of nearly homogeneous populations of undegraded free and bound ribosomes from rat brain. J. Neurochem. 28, 517 527. Venrooij W. J. v., Sillekens P. T. G., Eekelen C. A. G. v. and Reinders R. J. (1981) On the association of mRNA with the cytoskeleton in uninfected and adenovirus-infected human KB cells. Expl. Cell Res. 135, 79 91. Wolosewick J. J. and Porter K. R. (1979) Microtrabeclar lattice of the cytoplasmic ground substance. Artifact or reality? J. Cell Biol. 82, 114-139. Zylber E. A. and Penman S. (1970) The effect of high ionic strength on monomers, polyribosomes and puromycintreated polyribosomes. Biochim. Biophys. Acta 204, 221 229.