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The role of the cytoskeleton in eukaryotic protein synthesis
Cell Biology
International
zRepotTs, Vol. 7, No. 4, April
1983
245
THE ROLE OF THE CYTOSKELETON IN EUKARYOTIC PROTEIN SYNTHESIS (A minireview) Peter * Biocenter, + Switzerland Friedrich land
Nielsen*, University Miescher
Susan
Goelz
of Institute,
+
Basel,
and Hans Trachsel* CH-4056 CH-4002
Basel, Basel,
Switzer-
immunological and morphological investigaBiochemical, tions demonstrate that within the cytoplasm of eukaryotic cells there is an intricate architecture of filaments called the cytoskeleton (for a review, see Brinkley, B.R. 1981, Goldman, R.D. et al., 1976). The cytoskeleton includes three major filamentous systems, namely microfilaments (Clarke, M. and Spudich, J-A., 1977, Lazarides, intermediate filaments (Lazarides, E., 1980), E ., 1976), microtubules (Goldman, R.D. et al., 1976, Olmsted, J.B. and Borisy, G.G., 1973, Tucker, J.B., 1979) and a large number of filament-associated proteins. In addition, a considerable portion of the cytoplasm may be organized into a irregular meshwork termed the microtrabecular lattice (Wolosewick, J.J. and Porter, K.R., 1976, Wolosewick, J.J. and Porter, K.R., 1979). A variety of functions have been ascribed to the cytoThey include influence on cell motility, cell skeleton. shape, intracellular transport and spatial distribution of cellular organelles (Goldman, R.D. et al., 1976, Clarke, M. and Spudich, J.A., 1977). In this review we examine the relatively recent hypothesis that the cytoskeleton is involved in the regulation of protein synthesis. We do this by reviewing evidence which bears on three questions: 1. Are components involved in protein synthesis associated with the of the cytocytoskeleton? 2. If so, with which elements skeleton are they associated? 3. Does this association play a role in the regulation of protein synthesis? 0309-16511831040245-10/$03.00/O
@ 1983 Academic
Press Inc. (London)
Ltd.
246
Cell Biology
International
1. Are components involved ted with the cytoskeleton? Several lines somes engaged skeleton.
of in
evidence protein
in
Reports,
protein
suggest synthesis
that are
Vol. 7, No. 4, April
synthesis
1983
associa-
mRNA and ribobound to the cyto-
a) Electronmicroscopical evidence: Examination by high voltage electron microscopy of whole cell mounts from tissue culture cells reveals microtubules, intermediate filaments, microfilaments and the microtrabecular lattice (Wolosewick, J.J. and Porter, K.R., 1976). Embedded in this lattice which extends throughout the cytoplasm are the cytoplasmic organelles including the endoplasmic reticulum. The only translational component directly visible by electron microscopy, the ribosome, is frequently seen at junctions of the microtrabecular lattice, on filaments, or attached to membranes (Wolosewick, J.J. and Porter, K.R., 1976). Often, ribosomes are seen in clusters suggestive of polyribosomes. Gentle extraction of cells with low concentrations of non-ionic detergents removes 50-70% of the cellular protein (termed the soluble fraction), and leaves behind the cytoskeletal fraction (Lenk, R. et al., 1977). Although it corresponds fairly well to the cytoskeleton seen in unextracted cells, the cytoskeleton remaining after detergent extraction is usually less complex. Membranes and membraneous organelles are absent, as is much of the delicate microtrabecular lattice (Schliwa, M. et 1981, Schliwa, M. and van Blerkom, J., 1981, Cerveal., 1981). Ribosomes can still be seen, howra, M. et al., ever, apparently associated with either amourphous material or with surviving filaments (Lenk, R. et al., 1977, Schliwa, M. et al., 1981). b) Biochemical evidence: In most biochemical experiments, the term cytoskeleton is used to refer to the cytoplasmic material which remains after cells have been extracted with a mild nonionic detergent (Lenk, R. et al., 1977, Brown, S. et al., Sucrose gradient analysis of the ribosomes in the 1976). soluble and cytoskeletal fractions shows that most of the polyribosomes remain in the cytoskeletal fraction while monomeric ribosomes are found in the soluble fraction (van Venrooij, W.J. et al., 1981). This is also true when cytoskeletons are prepared under conditions where microtubules are extracted (Lenk, R. et al., 1977). Reduction of the amount of pGlyribosomes by cell starvation (Lee, 1971) I poliovirusand vesicular stomatit:s S.Y. et al.,
Cell Biology
International
Reports,
Vol. 7, No. 4, April
1983
247
virus (VSV)-infection (Lenk, R. and Penman, S., 1979, or by the addition of protein M. et a1.,1981), Cervera, synthesis inhibitors (Lenk, R. et al., 1977, van Ven1981) results in the extraction of rooij, W.J. et al., nearly all the ribosomes as 80s monomers in the soluble fraction. These findings suggest that ribosomes are primarily associated with the cytoskeleton when they are translating mRNA and that this association is either much weaker or non-existant when ribosomes are not synthesizing protein. Consistent with the idea that ribosomes are associated with some stationary component of the cytoplasm are reports of a non-uniform distribution of ribosomes within the cell as studied by nucleic acid staining of 3T3 1980) or cellular microcells (Fulton, A.B. et al., dissection of insect salivary gland cells (Edstrbm, J.-E. and Lonn, U., 1976). The association of polyribosomes with the cytoskeleton is apparently through mRNA. In HeLa cells (Lenk, R. et al., 1977), ascidian follicle cells (Jeffery, W.R., 1982), human KB cells (van Venrooij, W.J. et al., 1981) and Ehrlich ascites tumor cells+(Lemieux, R. and Beaud, G 1982) 70-80% of the poly(A) mRNA as measured by hybridization with oligo(dT) or poly(U) is found in the cytoskeletal fraction. Treatment of the cells with fluoride ions (Lenk, R. et al., 1977, Lemieux, R. and Beaud, G., 1982) or pactamycin (Lenk, R. and Penman, S., 1979) which cause the dissociation of ribosomes from mRNA, results in the loss of ribosomes but not of mRNA from the cytoskeletal fraction. On the other hand, ribonuclease treatment of the cytoskeleton releases ribosomes and mRNA (Lenk, R. et al., 1977). The association of mRNA with the cytoskeleton is also indirectly suggested by the finding that mRNA in oocytes, eggs and embryos of several organisms is not uniformly distributed (Ernst, S.G. et al., 1980, Capco, D.G. and Jackie, H * I 1982, Capco, D.G. and Jeffery, W.R., 1982). Following infection with many kinds of viruses, there is a shift in translation from cellular to viral mRNA (Bablanian, R., 1975). Although this shut-off of host mRNA translation is not yet understood, in two cases it has been reported to correlate temporally with a shift of host mRNA's from the cytoskeletal to the soluble fraction (van Venrooij, W.J. et al., 1981, Lenk, R. and Penman, S., 1979). As is true for cellular mRNA in uninfected cells, viral mRNA in infected cells is found in polyribosomes associated with the cytoskeleton (Cervera, 1981, van Venrooij, W.J. et al., 1981, Lenk, M. et al.,
248
R. and Penman,
Cell Biology
S.,
International
1979,
Reports,
Ben-Ze'ev,
A.
Vol. 7, No. 4, April
et
al.,
1983
1981).
In addition to ribosomes and mRNA, the association of other translational components with the cytoskeleton has It is estimated from two reports been investigated. (Cervera, M. et al., 1981, Lemieux, R. and Beaud, G., tRNA is found in the 1982) that 80-85% of the cellular The remaining 15-20% which is found in soluble fraction. the cytoskeletal fraction may be tRNA bound to polyribosomes. 1982) studied the locaZumbe et al. (Zumbe, A. et al., tion in baby hamster kidney (BHK) cells of proteins involved in the binding of the 5'-terminal m7G(5')-cap structure of mRNA (cap binding proteins, CBP). They used a monoclonal antibody which reacted with several proteins (Sonenberg, N. and Trachsel, H., 1982), one of which (a 50K-CBP) shared tryptic peptides Mr 50K polypeptide, with a Mr 24K polypeptide (24K-CBP) previously identified as a CBP (Sonenberg, N. et al., 1978). Analysis of the cytoskeletal proteins reacting with the antibody on protein blots revealed the presence of the 50K-CBP and a When this antibody was used to stain Mr 93X polypeptide. the cytoskeletal preparations of BHK cells by indirect immunofluorescence, an intermediate filament-like netthese observations show that 50Kwork was seen. Although CBP is associated with the cytoskeletal fraction, the possibility cannot be ruled out that the filamentous staining is due to reaction with the Mr 93K polypeptide or other cross-reacting proteins which were not seen on the protein blots of the cytoskeletal fraction. This is a general problem in immunofluorescence microscopy experiments. Using polyclonal antibodies directed against initiation factor eIF-2 and monoclonal antibodies against initiation factor eIF-4A, we find 90% of eIF-2 and 90-95% of eIF-4A in the soluble fraction in CV-1 (african green monkey kidney), mouse 3T3 and HeLa cells (our unpublished results). These measurements were made by spotting serial dilutions of soluble and cytoskeletal fraction on nitrocellulose and then comparing the highest dilution where staining was still visible following incubation, first with the specific antiserum, and then with peroxidaseImmunofluorescence microscopy coupled second antibody. experiments with the antibodies directed against eIF-2 and eIF-4A show no staining of the cytoskeleton. Concerning association or lack of association of translational components with the cytoskeleton, we should keep
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1983
in mind that the preparation of the cytoskeleton involves extraction procedures during which rearrangements of cellular components could occur. This is indicated by the finding of Schliwa et al. (Schliwa, M. et al., 1981, Schliwa, M. and van Blerkom, J., 1981) that the species of proteins solubilized by the detergent extraction of cells depend upon the extraction conditions used. It is therefore difficult to exclude the possibility that translational components could artifactually associate or dissociate from the cytoskeleton during preparation. The results presented above are made more credible, however, because different fractionation conditions have been used, all leading essentially to the same conclusion. In summary, based on electronmicroscopical, biochemical we believe that a signifiand immunological evidence, cant portion of cytoplasmic ribosomes (namely those bound to mRNA in polyribosomes), mRNA and 5OK-CBP are associated with the cytoskeleton, while tRNA and the initiation factors eIF-2 and eIF-4A are not. 2. With which elements ribosomes associated?
of
the
cytoskeleton
are
mRNA and
The cytoskeletal components involved in binding of mRNA and ribosomes (through their attachement to mRNA) are not known. The salient features of the association of mRNA with the cytoskeleton are a) the association survives detergent extraction, even under conditions which disrupt microtubules and much of the microtrabecular lattice (Lenk, R. et al., 1977), and b) the association does not survive detergent extraction of cells which have been incubated with the microfilament-disrupting drug cytochalasin B before extraction (Lenk, R. et al., 1977). In addition, Feramisco and coworkers have mentioned (without presenting the data), that the disruption of the vimentin filament network in fibroblast cells changes neither the two-dimensional gel pattern of proteins synthesized nor the distribution of sites in the cell where proteins are made (Thomas, G.P. et al., 1981, Lin, J. -C. and Feramisco, J-R., 1981), indicating that translational components are not associated with intermediate filaments. Taking these observations at face one is tempted to conclude that mRNA and ribovalue, somes are associated with microfilaments. However, evidence from several laboratories suggests that the cytoskeleton in intact cells is a highly interconnected net.work rather than a collection of isolated filament systems: First, disruption of microtubules in living
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1983
cells using colchicine results in the collapse of the vimentin filament system around the nucleus (Goldman, the disruption of actin filaments R.D., 1971). Second, following cytochalasin B treatment results in substantial disruption of the cytoskeleton obtained following detergent extraction (Schliwa, M. and van Blerkom, J., 1981). Third, high molecular weight proteins related to microtubule-associated proteins were found to copurify with intermediate filaments in several tissue culture cell lines (Pytela, R. and Wiche, G., 1980). Furthermore, ultrastructural examination of cells reveals networks of actin filaments and strand-like elements of the microtrabecular lattice which form extensive bridges between all types of filaments (Wolosewick, J.J. and Porter, K.R., 1979, Webster, R.E. et al., 1978, Schliwa, M. and van Blerkom, J., 1981). In this light, the above conclusion must be modified to say that mRNA and ribosomes appear to be associated with microfilaments directly or with a structure whose cytoskeletal attachement is dependent on the integrity of the microfilaments. Such a structure could be the microtrabecular lattice. This would fit with the finding in whole mount electron microscopy experiments that ribosomes are found in close proximity to the microtrabecular lattice (Wolosewick, J.J. and Porter, K-R., 1976, Wolosewick, J.J. and Porter, K.R. Substantiation of such a possibility awaits bio1979). chemical and functional characterization of the microtrabecular lattice. 3. Does the cytoskeleton synthesis?
association of mRNA and ribosomes play a role in the regulation of
with the protein
So far, the evidence for a regulatory role of the association of mRNA and ribosomes with the cytoskeleton is Arguing for a regulatory role are only correlative. those experiments described earlier which demonstrate an association of ribosomes with the cytoskeleton when translating mRNA and a loss of this association when protein synthesis is reduced (Lenk, R. et al., 1977, Cervera, M. et al., 1981, van Venrooij, W.J. et al., 1981, Lee, S.Y. et al., 1971, Lenk, R. and Penman, S., 1979). Several groups have studied the possibility that the cell maintains a large portion of its mRNA, and perhaps its active mRNA exclusively in a cytoskeletal-bound form. Cervera et al. (Cervera, M. et al., 1981) isolated mRNA from the soluble and cytoskeletal fractions of HeLa cells, translated it in a mRNA-dependent in vitro translation
Cell Biology
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1983
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system and analyzed the proteins by two-dimensional gel The gel patterns corresponding to the electrophoresis. two mRNA fractions were different, suggesting the existence of a mechanism whereby a specific population of mRNA's is preferentially bound to the cytoskeleton. Similar results were obtained by van Venrooij et al. (van Venrooij, W.J. et al., 1981) in human KB cells. Furthermore, they were able to demonstrate that changes in the cytoskeletal mRNA population correlated with changes in protein synthesis. Pulse-labeling experiments show that newly synthesized mRNA appears first in the cytoplasm associated with the cytoskeleton and later is found in the soluble fraction (Jeffery, W.R., 1982). A similar flow of viral mRNA first to the cytoskeleton and later to the soluble fraction is seen in VSV-infected HeLa cells (Cervera, M. et al., 1981) and SV40-infected BSC-1 cells (african green monkey kidney, Ben-Ze'ev, A. et al., 1981). That its association with the cytoskeleton may play an important role in determining the activity of mRNA is suggested by the observations from poliovirus-infected HeLa cells (Cervera, M. et a1.,1981), adenovirus-infected human KB cells (van Venrooij, W.J. et al., 1981), and SV40-infected BSC-1 cells (Ben-Ze'ev, A. et al., 1981) that the time of appearance of viral mRNA roughly correlates with a) the synthesis of viral protein, b) the reduction in the amount of host mRNA in and c) the reduction in the the cytoskeletal fraction, synthesis of host proteins. Vaccinia virus may be an exception since it has been recently reported that early after infection, a portion of the polyribosomes containing viral mRNA can be found in the soluble fraction and no significant release of host mRNA from the cytoskeleton is observed (Lemieux, R. and Beaud, G., 1982). The general correlation of mRNA association and activity is relatively crude and therefore the question whether the association of mRNA with the cytoskeleton plays a regulatory role still awaits definitive proof. We suggest that future work in this field should include: 1) further investigation of the association of protein synthesis initiation, elongation and termination factors with the cytoskeleton, taking into account the possibility of artifactual association/dissociation during examination. Possible experiments include cross-linking or direct in situ labeling of unextracted cells with anti.bodies or other specific probes. 2) characterization of a) the elements of the cytoskeleton involved in mRNA binding, b) the region of the
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1983
mRNA molecule which interacts with the cytoskeleton, c) possible accessory proteins involved in mRNA binding, and d) alterations in these components following cell viral infection, hormone treatment, etc. stress, 3) demonstration that specific mRNA's (viral or cellular) shuttle between the soluble and cytoskeletal fractions and that these movements correlate with the synthesis of the corresponding proteins. A suitable system for such a demonstration could, for example, be the hormonally regulated synthesis of a protein known to be under translational control. 4) development of in vitro systems in which the interaction between cytoskeletal and translational components can be studied. REFERENCES Structural and functional alteraBablanian,R. (1975). tions in cultured cells infected with cytocidal Progress in Medical Virology,19,40-83. viruses. Ben-Ze'ev,A.,Horowitz,M.,Skolnik,H.,Abula~a,R.,Laub,O. The metabolism of SV40 RNA is and Aloni,Y.(1981). associated with the cytoskeletal framework. Virology,111,475-487. of the cytoBrinkley,B.R.(1981). Summary: Organization plasm.Cold Spring Harbor Symposia on Quantitative Biology,XLVI,1029-1040. Levinson,W.and Spudich,J.A.(1976). Cytoskeletal Brown,S., elements of chick embryo fibroblasts revealed by detergent extraction.Journal of Supramolecular Structure,l,ll9-130. Capc0,D.G. and Jackle,H.(1982). Localized protein synthesis during oogenesis of Xenopus laevis: Analysis by in situ translation.Developmental Biology,z,41-50. Capc0,D.G. and Jeffery,W.R.(1982). Transient localizations of messenger RNA in Xenopus laevis oocytes. Developmental Biology,89,1-12. Cervera,M.,Dreyfuss,G. andPenman,S.(1981). Messenger RNA is translated when associated with the cytoskeletal framework in normal and VSV-infected HeLa cells. Cell,z,113-120. Clarke,M. and Spudich,J.A.(1977). Nonmuscle contractile proteins: The role of actin and myosin in cell motility and shape determination.Annual Review of Biochemistry,46,797-822. Edstrom,J.-E. and LBnn,U.(1976). Cytoplasmic zone analysis. The Journal of Cell Biology,s,562-572.
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Hough-Evans,B.R.,Britten,R.J. and Davidson, Ernst,S.G., Limited complexity of the RNA in microE.H.(1980). meres of sixteen-cell sea urchin embryo. Developmental Biology,E,119-127. 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.Ce11,2,849-857. Goldman,R.D.(1971). The role of three cytoplasmic fibres in BHK-21 cell motility.The Journal of Cell Biology, z,752-762. Goldman,R.D.,Pollard,T. and Rosenbaum,J.(1976).Cell motility.Cold Spring Harbor Conferences on Cell Proliferation. Messenger RNA in the cytoskeletal Jeffery,W.R.(1982). framework:Analysis by in situ hybridization. The Journal of Cell Biology,E,l-7. Lazarides,E.(1976). Two general classes of cytoplasmic actin filaments in tissue culture cells: The role of tropomyosin.Journal of Supramolecular Structure,?, 531-563. Lazarides,E.(1980). Intermediate filaments as mechanical integrators of cellular space.Nature,283,249-256. Lee,S.Y., Krsmanovic,V. and Brawerman,G.(1971). Attachement of ribosomes to membranes during polysome formation in mouse sarcoma 180 cells.The Journal of Cell Biology,49,683-691. Lemieux,R. and BGud,G.(1982). Expression of vaccinia virus early mRNA in Ehrlich ascites tumor cells. European Journal of Biochemistry,l29,273-279. Lenk,R., Ransom,L., Kaufmann,Y. and Penman,S.(1977). A cytoskeletal structure with associated polyribosomes obtained from HeLa cells.Ce11,%,67-78. Lenk,R. The cytoskeletal framework and Penman,S. (1979). and poliovirus metabolism.Cell,l6,289-301. and Feramisco,J.R.(1981).Disruption of the in Lin,J.-C. vivo distribution of the intermediate filaments in fibroblasts through the microinjection of a specific monoclonal antibody.Ce11,24,185-193. and Borisy,G.G.(1973). Microtubules.Annual Olmsted,J.B. Review of Biochemistry,42,507-540. High molecular weight polyPytela,R. and Wiche,G.(1980). peptides (270,000-340,OOO)from cultured cells are related to hog brain microtubule-associated proteins but copurify with the intermediate filaments. Proceedings of the National Academy of Sciences(USA), -77,4808-4812.
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Schliwa,M. and van Blerkom,J.(1981). Structural interaction of cytoskeletal components.The Journal of Cell Biology,90,222-235. and Porter,K.R.(1981). Stavan Blerkom,J. Schliwa,M., bilization of the cytoplasmic ground substance in detergent-opened cells and a structural and biochemical analysis of its composition.Proceedings of the National Academy of Sciences(USA),78,4329-4333. Sonenberg,N. and Trachsel,H.(1982). Probing the function of the eukaryotic 5 '-cap structure using monoclonal antibodies to cap binding proteins. Current Topics in Cellular Regulation,z,65-88. Sonenberg,N.,Morgan,M.A.,Testa,D.,Colonna,R.J. and Shatkin,A.J.(1978). A polypeptide in eukaryotic initiation factors that crosslinks specifically to the 5'-terminal cap in mRNA.Proceedings of the National Academy of Sciences(USA),G,4843-4847. Thomas,G.P.,Welch,W.J.,Mathews,M.B. and Feramisc0,J.R. (1981). Molecular and cellular effects of heat-shock and related treatments of mammalian tissue-culture cells.Cold Spring Harbor Symposia on Quantitative Biology,XLVI, 985-996. Tucker,J.B.(1979). Spatial organization of microtubules. Microtubules(eds.Roberts,K. and Hyams,J.S.). Academic Press,London,314-357. van Venrooij,W.J.,Sillekens,P.T.G.,Ekelen,C.A.G. and On the association of mRNA with Reinders,R.J.(1981). the cytoskeleton in uninfected and adenovirus-infected human KB cells.Experimental Cell Research,135,79-91. Webster,R.E.,Osborn,M. and Weber,K.(1978). Visualization of the same PtK2 cytoskeleton by both immunofluorescence and low power electron microscopy. Experimental Cell Research,117,47-61. Wolosewick,J.J. and Porter,K.R.(1976). Stereo highvoltage electron microscopy of whole cells of the human diploid line WI-38.American Journal of Anatomy, 147,303-324. Wolosewick,J.J. and Porter,K.R.(1979). Microtrabecular lattice of the cytoplasmic ground substance. The Journal of Cell Biology,82,114-139. Zumbe,A.,Stahli,C. and Trachselz.(1982). Association of a Mr 50,000 cap binding protein with the cytoskeleton in baby hamster kidney cells.Proceedings of the National Academy of Sciences(USA),E,2927-2931.
Received:
10th February
1983
Accepted:
14th February
1983
International
zRepotTs, Vol. 7, No. 4, April
1983
245
THE ROLE OF THE CYTOSKELETON IN EUKARYOTIC PROTEIN SYNTHESIS (A minireview) Peter * Biocenter, + Switzerland Friedrich land
Nielsen*, University Miescher
Susan
Goelz
of Institute,
+
Basel,
and Hans Trachsel* CH-4056 CH-4002
Basel, Basel,
Switzer-
immunological and morphological investigaBiochemical, tions demonstrate that within the cytoplasm of eukaryotic cells there is an intricate architecture of filaments called the cytoskeleton (for a review, see Brinkley, B.R. 1981, Goldman, R.D. et al., 1976). The cytoskeleton includes three major filamentous systems, namely microfilaments (Clarke, M. and Spudich, J-A., 1977, Lazarides, intermediate filaments (Lazarides, E., 1980), E ., 1976), microtubules (Goldman, R.D. et al., 1976, Olmsted, J.B. and Borisy, G.G., 1973, Tucker, J.B., 1979) and a large number of filament-associated proteins. In addition, a considerable portion of the cytoplasm may be organized into a irregular meshwork termed the microtrabecular lattice (Wolosewick, J.J. and Porter, K.R., 1976, Wolosewick, J.J. and Porter, K.R., 1979). A variety of functions have been ascribed to the cytoThey include influence on cell motility, cell skeleton. shape, intracellular transport and spatial distribution of cellular organelles (Goldman, R.D. et al., 1976, Clarke, M. and Spudich, J.A., 1977). In this review we examine the relatively recent hypothesis that the cytoskeleton is involved in the regulation of protein synthesis. We do this by reviewing evidence which bears on three questions: 1. Are components involved in protein synthesis associated with the of the cytocytoskeleton? 2. If so, with which elements skeleton are they associated? 3. Does this association play a role in the regulation of protein synthesis? 0309-16511831040245-10/$03.00/O
@ 1983 Academic
Press Inc. (London)
Ltd.
246
Cell Biology
International
1. Are components involved ted with the cytoskeleton? Several lines somes engaged skeleton.
of in
evidence protein
in
Reports,
protein
suggest synthesis
that are
Vol. 7, No. 4, April
synthesis
1983
associa-
mRNA and ribobound to the cyto-
a) Electronmicroscopical evidence: Examination by high voltage electron microscopy of whole cell mounts from tissue culture cells reveals microtubules, intermediate filaments, microfilaments and the microtrabecular lattice (Wolosewick, J.J. and Porter, K.R., 1976). Embedded in this lattice which extends throughout the cytoplasm are the cytoplasmic organelles including the endoplasmic reticulum. The only translational component directly visible by electron microscopy, the ribosome, is frequently seen at junctions of the microtrabecular lattice, on filaments, or attached to membranes (Wolosewick, J.J. and Porter, K.R., 1976). Often, ribosomes are seen in clusters suggestive of polyribosomes. Gentle extraction of cells with low concentrations of non-ionic detergents removes 50-70% of the cellular protein (termed the soluble fraction), and leaves behind the cytoskeletal fraction (Lenk, R. et al., 1977). Although it corresponds fairly well to the cytoskeleton seen in unextracted cells, the cytoskeleton remaining after detergent extraction is usually less complex. Membranes and membraneous organelles are absent, as is much of the delicate microtrabecular lattice (Schliwa, M. et 1981, Schliwa, M. and van Blerkom, J., 1981, Cerveal., 1981). Ribosomes can still be seen, howra, M. et al., ever, apparently associated with either amourphous material or with surviving filaments (Lenk, R. et al., 1977, Schliwa, M. et al., 1981). b) Biochemical evidence: In most biochemical experiments, the term cytoskeleton is used to refer to the cytoplasmic material which remains after cells have been extracted with a mild nonionic detergent (Lenk, R. et al., 1977, Brown, S. et al., Sucrose gradient analysis of the ribosomes in the 1976). soluble and cytoskeletal fractions shows that most of the polyribosomes remain in the cytoskeletal fraction while monomeric ribosomes are found in the soluble fraction (van Venrooij, W.J. et al., 1981). This is also true when cytoskeletons are prepared under conditions where microtubules are extracted (Lenk, R. et al., 1977). Reduction of the amount of pGlyribosomes by cell starvation (Lee, 1971) I poliovirusand vesicular stomatit:s S.Y. et al.,
Cell Biology
International
Reports,
Vol. 7, No. 4, April
1983
247
virus (VSV)-infection (Lenk, R. and Penman, S., 1979, or by the addition of protein M. et a1.,1981), Cervera, synthesis inhibitors (Lenk, R. et al., 1977, van Ven1981) results in the extraction of rooij, W.J. et al., nearly all the ribosomes as 80s monomers in the soluble fraction. These findings suggest that ribosomes are primarily associated with the cytoskeleton when they are translating mRNA and that this association is either much weaker or non-existant when ribosomes are not synthesizing protein. Consistent with the idea that ribosomes are associated with some stationary component of the cytoplasm are reports of a non-uniform distribution of ribosomes within the cell as studied by nucleic acid staining of 3T3 1980) or cellular microcells (Fulton, A.B. et al., dissection of insect salivary gland cells (Edstrbm, J.-E. and Lonn, U., 1976). The association of polyribosomes with the cytoskeleton is apparently through mRNA. In HeLa cells (Lenk, R. et al., 1977), ascidian follicle cells (Jeffery, W.R., 1982), human KB cells (van Venrooij, W.J. et al., 1981) and Ehrlich ascites tumor cells+(Lemieux, R. and Beaud, G 1982) 70-80% of the poly(A) mRNA as measured by hybridization with oligo(dT) or poly(U) is found in the cytoskeletal fraction. Treatment of the cells with fluoride ions (Lenk, R. et al., 1977, Lemieux, R. and Beaud, G., 1982) or pactamycin (Lenk, R. and Penman, S., 1979) which cause the dissociation of ribosomes from mRNA, results in the loss of ribosomes but not of mRNA from the cytoskeletal fraction. On the other hand, ribonuclease treatment of the cytoskeleton releases ribosomes and mRNA (Lenk, R. et al., 1977). The association of mRNA with the cytoskeleton is also indirectly suggested by the finding that mRNA in oocytes, eggs and embryos of several organisms is not uniformly distributed (Ernst, S.G. et al., 1980, Capco, D.G. and Jackie, H * I 1982, Capco, D.G. and Jeffery, W.R., 1982). Following infection with many kinds of viruses, there is a shift in translation from cellular to viral mRNA (Bablanian, R., 1975). Although this shut-off of host mRNA translation is not yet understood, in two cases it has been reported to correlate temporally with a shift of host mRNA's from the cytoskeletal to the soluble fraction (van Venrooij, W.J. et al., 1981, Lenk, R. and Penman, S., 1979). As is true for cellular mRNA in uninfected cells, viral mRNA in infected cells is found in polyribosomes associated with the cytoskeleton (Cervera, 1981, van Venrooij, W.J. et al., 1981, Lenk, M. et al.,
248
R. and Penman,
Cell Biology
S.,
International
1979,
Reports,
Ben-Ze'ev,
A.
Vol. 7, No. 4, April
et
al.,
1983
1981).
In addition to ribosomes and mRNA, the association of other translational components with the cytoskeleton has It is estimated from two reports been investigated. (Cervera, M. et al., 1981, Lemieux, R. and Beaud, G., tRNA is found in the 1982) that 80-85% of the cellular The remaining 15-20% which is found in soluble fraction. the cytoskeletal fraction may be tRNA bound to polyribosomes. 1982) studied the locaZumbe et al. (Zumbe, A. et al., tion in baby hamster kidney (BHK) cells of proteins involved in the binding of the 5'-terminal m7G(5')-cap structure of mRNA (cap binding proteins, CBP). They used a monoclonal antibody which reacted with several proteins (Sonenberg, N. and Trachsel, H., 1982), one of which (a 50K-CBP) shared tryptic peptides Mr 50K polypeptide, with a Mr 24K polypeptide (24K-CBP) previously identified as a CBP (Sonenberg, N. et al., 1978). Analysis of the cytoskeletal proteins reacting with the antibody on protein blots revealed the presence of the 50K-CBP and a When this antibody was used to stain Mr 93X polypeptide. the cytoskeletal preparations of BHK cells by indirect immunofluorescence, an intermediate filament-like netthese observations show that 50Kwork was seen. Although CBP is associated with the cytoskeletal fraction, the possibility cannot be ruled out that the filamentous staining is due to reaction with the Mr 93K polypeptide or other cross-reacting proteins which were not seen on the protein blots of the cytoskeletal fraction. This is a general problem in immunofluorescence microscopy experiments. Using polyclonal antibodies directed against initiation factor eIF-2 and monoclonal antibodies against initiation factor eIF-4A, we find 90% of eIF-2 and 90-95% of eIF-4A in the soluble fraction in CV-1 (african green monkey kidney), mouse 3T3 and HeLa cells (our unpublished results). These measurements were made by spotting serial dilutions of soluble and cytoskeletal fraction on nitrocellulose and then comparing the highest dilution where staining was still visible following incubation, first with the specific antiserum, and then with peroxidaseImmunofluorescence microscopy coupled second antibody. experiments with the antibodies directed against eIF-2 and eIF-4A show no staining of the cytoskeleton. Concerning association or lack of association of translational components with the cytoskeleton, we should keep
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in mind that the preparation of the cytoskeleton involves extraction procedures during which rearrangements of cellular components could occur. This is indicated by the finding of Schliwa et al. (Schliwa, M. et al., 1981, Schliwa, M. and van Blerkom, J., 1981) that the species of proteins solubilized by the detergent extraction of cells depend upon the extraction conditions used. It is therefore difficult to exclude the possibility that translational components could artifactually associate or dissociate from the cytoskeleton during preparation. The results presented above are made more credible, however, because different fractionation conditions have been used, all leading essentially to the same conclusion. In summary, based on electronmicroscopical, biochemical we believe that a signifiand immunological evidence, cant portion of cytoplasmic ribosomes (namely those bound to mRNA in polyribosomes), mRNA and 5OK-CBP are associated with the cytoskeleton, while tRNA and the initiation factors eIF-2 and eIF-4A are not. 2. With which elements ribosomes associated?
of
the
cytoskeleton
are
mRNA and
The cytoskeletal components involved in binding of mRNA and ribosomes (through their attachement to mRNA) are not known. The salient features of the association of mRNA with the cytoskeleton are a) the association survives detergent extraction, even under conditions which disrupt microtubules and much of the microtrabecular lattice (Lenk, R. et al., 1977), and b) the association does not survive detergent extraction of cells which have been incubated with the microfilament-disrupting drug cytochalasin B before extraction (Lenk, R. et al., 1977). In addition, Feramisco and coworkers have mentioned (without presenting the data), that the disruption of the vimentin filament network in fibroblast cells changes neither the two-dimensional gel pattern of proteins synthesized nor the distribution of sites in the cell where proteins are made (Thomas, G.P. et al., 1981, Lin, J. -C. and Feramisco, J-R., 1981), indicating that translational components are not associated with intermediate filaments. Taking these observations at face one is tempted to conclude that mRNA and ribovalue, somes are associated with microfilaments. However, evidence from several laboratories suggests that the cytoskeleton in intact cells is a highly interconnected net.work rather than a collection of isolated filament systems: First, disruption of microtubules in living
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cells using colchicine results in the collapse of the vimentin filament system around the nucleus (Goldman, the disruption of actin filaments R.D., 1971). Second, following cytochalasin B treatment results in substantial disruption of the cytoskeleton obtained following detergent extraction (Schliwa, M. and van Blerkom, J., 1981). Third, high molecular weight proteins related to microtubule-associated proteins were found to copurify with intermediate filaments in several tissue culture cell lines (Pytela, R. and Wiche, G., 1980). Furthermore, ultrastructural examination of cells reveals networks of actin filaments and strand-like elements of the microtrabecular lattice which form extensive bridges between all types of filaments (Wolosewick, J.J. and Porter, K.R., 1979, Webster, R.E. et al., 1978, Schliwa, M. and van Blerkom, J., 1981). In this light, the above conclusion must be modified to say that mRNA and ribosomes appear to be associated with microfilaments directly or with a structure whose cytoskeletal attachement is dependent on the integrity of the microfilaments. Such a structure could be the microtrabecular lattice. This would fit with the finding in whole mount electron microscopy experiments that ribosomes are found in close proximity to the microtrabecular lattice (Wolosewick, J.J. and Porter, K-R., 1976, Wolosewick, J.J. and Porter, K.R. Substantiation of such a possibility awaits bio1979). chemical and functional characterization of the microtrabecular lattice. 3. Does the cytoskeleton synthesis?
association of mRNA and ribosomes play a role in the regulation of
with the protein
So far, the evidence for a regulatory role of the association of mRNA and ribosomes with the cytoskeleton is Arguing for a regulatory role are only correlative. those experiments described earlier which demonstrate an association of ribosomes with the cytoskeleton when translating mRNA and a loss of this association when protein synthesis is reduced (Lenk, R. et al., 1977, Cervera, M. et al., 1981, van Venrooij, W.J. et al., 1981, Lee, S.Y. et al., 1971, Lenk, R. and Penman, S., 1979). Several groups have studied the possibility that the cell maintains a large portion of its mRNA, and perhaps its active mRNA exclusively in a cytoskeletal-bound form. Cervera et al. (Cervera, M. et al., 1981) isolated mRNA from the soluble and cytoskeletal fractions of HeLa cells, translated it in a mRNA-dependent in vitro translation
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system and analyzed the proteins by two-dimensional gel The gel patterns corresponding to the electrophoresis. two mRNA fractions were different, suggesting the existence of a mechanism whereby a specific population of mRNA's is preferentially bound to the cytoskeleton. Similar results were obtained by van Venrooij et al. (van Venrooij, W.J. et al., 1981) in human KB cells. Furthermore, they were able to demonstrate that changes in the cytoskeletal mRNA population correlated with changes in protein synthesis. Pulse-labeling experiments show that newly synthesized mRNA appears first in the cytoplasm associated with the cytoskeleton and later is found in the soluble fraction (Jeffery, W.R., 1982). A similar flow of viral mRNA first to the cytoskeleton and later to the soluble fraction is seen in VSV-infected HeLa cells (Cervera, M. et al., 1981) and SV40-infected BSC-1 cells (african green monkey kidney, Ben-Ze'ev, A. et al., 1981). That its association with the cytoskeleton may play an important role in determining the activity of mRNA is suggested by the observations from poliovirus-infected HeLa cells (Cervera, M. et a1.,1981), adenovirus-infected human KB cells (van Venrooij, W.J. et al., 1981), and SV40-infected BSC-1 cells (Ben-Ze'ev, A. et al., 1981) that the time of appearance of viral mRNA roughly correlates with a) the synthesis of viral protein, b) the reduction in the amount of host mRNA in and c) the reduction in the the cytoskeletal fraction, synthesis of host proteins. Vaccinia virus may be an exception since it has been recently reported that early after infection, a portion of the polyribosomes containing viral mRNA can be found in the soluble fraction and no significant release of host mRNA from the cytoskeleton is observed (Lemieux, R. and Beaud, G., 1982). The general correlation of mRNA association and activity is relatively crude and therefore the question whether the association of mRNA with the cytoskeleton plays a regulatory role still awaits definitive proof. We suggest that future work in this field should include: 1) further investigation of the association of protein synthesis initiation, elongation and termination factors with the cytoskeleton, taking into account the possibility of artifactual association/dissociation during examination. Possible experiments include cross-linking or direct in situ labeling of unextracted cells with anti.bodies or other specific probes. 2) characterization of a) the elements of the cytoskeleton involved in mRNA binding, b) the region of the
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1983
mRNA molecule which interacts with the cytoskeleton, c) possible accessory proteins involved in mRNA binding, and d) alterations in these components following cell viral infection, hormone treatment, etc. stress, 3) demonstration that specific mRNA's (viral or cellular) shuttle between the soluble and cytoskeletal fractions and that these movements correlate with the synthesis of the corresponding proteins. A suitable system for such a demonstration could, for example, be the hormonally regulated synthesis of a protein known to be under translational control. 4) development of in vitro systems in which the interaction between cytoskeletal and translational components can be studied. REFERENCES Structural and functional alteraBablanian,R. (1975). tions in cultured cells infected with cytocidal Progress in Medical Virology,19,40-83. viruses. Ben-Ze'ev,A.,Horowitz,M.,Skolnik,H.,Abula~a,R.,Laub,O. The metabolism of SV40 RNA is and Aloni,Y.(1981). associated with the cytoskeletal framework. Virology,111,475-487. of the cytoBrinkley,B.R.(1981). Summary: Organization plasm.Cold Spring Harbor Symposia on Quantitative Biology,XLVI,1029-1040. Levinson,W.and Spudich,J.A.(1976). Cytoskeletal Brown,S., elements of chick embryo fibroblasts revealed by detergent extraction.Journal of Supramolecular Structure,l,ll9-130. Capc0,D.G. and Jackle,H.(1982). Localized protein synthesis during oogenesis of Xenopus laevis: Analysis by in situ translation.Developmental Biology,z,41-50. Capc0,D.G. and Jeffery,W.R.(1982). Transient localizations of messenger RNA in Xenopus laevis oocytes. Developmental Biology,89,1-12. Cervera,M.,Dreyfuss,G. andPenman,S.(1981). Messenger RNA is translated when associated with the cytoskeletal framework in normal and VSV-infected HeLa cells. Cell,z,113-120. Clarke,M. and Spudich,J.A.(1977). Nonmuscle contractile proteins: The role of actin and myosin in cell motility and shape determination.Annual Review of Biochemistry,46,797-822. Edstrom,J.-E. and LBnn,U.(1976). Cytoplasmic zone analysis. The Journal of Cell Biology,s,562-572.
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Received:
10th February
1983
Accepted:
14th February
1983