Journal of Cell Science 105, 867-871 (1993) 867 Printed in Great Britain © The Company of Biologists Limited 1993

COMMENTARY Cotranslational assembly of some cytoskeletal proteins: implications and prospects

A. B. Fulton1,* and T. L’Ecuyer2 Departments of 1Biochemistry and 2Pediatrics, University of Iowa, Iowa City, IA 52242, USA *Author for correspondence

INTRODUCTION proteins synthesized during the 10 minute pulse with [35S]methionine) were fairly evenly distributed throughout The cytoskeleton is a complex three-dimensional web in the cytoplasm, both after a pulse and after a several hour the interior of eucaryotic cells. The cytoskeleton orders chase period. When the cytoskeleton was examined after a many structures in the cell and performs many kinds of pulse, autoradiographic grains were clustered over polyri- transport and motility for the cell. It is distinguished by a bosomes, rather than being evenly distributed. After release high degree of spatial differentiation and anisotropy. How from ribosomes, therefore, it appeared that a significant does the cell construct something so heterogeneous and fraction of cytoskeletal proteins remained close to their site complex? of synthesis and that their assembly into the cytoskeleton A widely used model of protein assembly postulates that was very rapid. Cytoskeletal proteins labeled during a 10 proteins are synthesized on ribosomes and released into minute pulse were found throughout the 3T3 skeletal frame- solution within the cell, before diffusing within the cell and work if protein synthesis occurred during a chase period, assembling into cytoskeletal or other structures. This model but cytoskeletal proteins remained localized near polyribo- is clearly appropriate for a variety of cytoskeletal proteins, somes if emetine inhibited protein synthesis during the including tubulin and actin (Mitchison, 1992; Theriot and chase period. This sequence of local assembly followed by Mitchison, 1992). Because these proteins assemble after redistribution was particularly striking in hemangioma cells, translation, much has been learned about their assembly by which have nearly circular profiles and are especially well- using in vitro protein chemistry and in vivo methods such spread (Fulton, 1984). These observations showed restricted as FRAP (fluorescence recovery after photobleaching). exchange of cytoskeletal proteins between a soluble pool However, a growing body of evidence supports a differ- and the cytoskeleton under these conditions. These experi- ent model of assembly for certain cytoskeletal proteins. ments suggested that many cytoskeletal proteins assembled Several observations, made with a variety of experimental onto the cytoskeleton near the time and place of synthesis. protocols, suggest that assembly of some cytoskeletal struc- To extend these observations, cytoskeletons were pre- tures is difficult to explain solely by post-translational pared from HeLa cells in an in vitro system that permitted assembly. Rather, these proteins undergo cotranslational translation to continue (Fulton and Wan, 1983). The assembly; they first associate with the cytoskeleton during cytoskeletons were diluted with varying amounts of a trans- translation, as nascent (not yet complete) peptides. The lation buffer and [35S]methionine was added to examine the range of this evidence includes in situ autoradiography of association between proteins translated in vitro and the cytoskeletal proteins, immunoprecipitation of nascent pep- cytoskeleton. If all cytoskeletal proteins enter a soluble pool tides, immunofluorescence and, most recently, fluorescent after completion and before assembly into cytoskeletal in situ hybridization. structures, then the percentage of labeled protein on the The first evidence for localized assembly came from 3T3 cytoskeleton should have decreased as the cytoskeleton was cells, in which polyribosomes, stained with acridine orange, diluted. However, the percentage of total radioactivity were examined both in intact cells and in cytoskeletons pre- observed on the cytoskeleton was independent of the extent pared by Triton extraction (Fulton et al., 1980). Polyribo- to which the cytoskeleton was diluted during translation. somes were concentrated near the nucleus; this pattern was Association with the cytoskeleton could occur as completed not affected by Triton extraction. By simultaneously stain- polypeptides or while nascent polypeptides were still on ing with acridine orange and revealing newly synthesized polyribosomes. To distinguish between these possibilities, protein with autoradiography of [35S]methionine, protein the association of cytoskeletal proteins early in translation synthesis and movement of these new proteins was observed in intact 3T3 cells and in skeletal frameworks pre- pared by Triton extraction. When intact 3T3 cells were Key words: cytoskeleton, assembly, vimentin, myosin, titin, in examined this way, autoradiographic grains (representing situ hybridization 868 A. B. Fulton and T. L’Ecuyer was examined in the presence and absence of puromycin. was detected by briefly labelling muscle cultures with Puromycin is a protein synthesis inhibitor that releases [35S]methionine, followed by a chase period. Cultures were nascent polypeptides from polyribosomes (Munro, 1967; extracted to make cytoskeletons and then crosslinked with Grollman, 1968). Most nascent polypeptides are found ethylene glycol (bis-succinimidyl succinate) (EGS). associated with the cytoskeleton early in translation because Immunoprecipitation with monoclonal antibodies to titin of their association with polyribosomes (Lenk et al., 1977). and myosin heavy chain recovered specific proteins in Only nascent polypeptides that are associated with the cross-linked complexes. Even after a pulse period as short cytoskeleton independent of the polyribosome remain on as six minutes, both titin and myosin heavy chain could be the cytoskeleton in the presence of puromycin. Nascent recovered with the antibody to the other protein, suggest- chains that are associated with the cytoskeleton solely via ing that during synthesis these proteins have a close enough polyribosomes will be released into the soluble phase upon association with each other to be crosslinked by EGS, a 15 addition of puromycin. Fulton and Wan in this experiment Å long linker. The interaction of newly synthesized titin showed persistent association of some, but not all, nascent and myosin heavy chain, both of which undergo cotransla- chains with the cytoskeleton in the presence of puromycin tional assembly though to different extents, may contribute (i.e. independent of polyribosomes). Two-dimensional gel to the remodeling of nascent myofibrils into sarcomeres. electrophoresis confirmed that the proteins that bound to Vimentin, like all intermediate filament proteins, forms the cytoskeleton during in vitro translation were represen- a coiled coil that associates with other intermediate filament tative of those that assembled in vivo. proteins to form filaments; it is another protein that has been To summarize, the first evidence for cotranslational observed to undergo cotranslational assembly to a signifi- assembly for some cytoskeletal proteins included: (1) the cant extent (Isaacs et al., 1989). By using pulse-chase exper- rapid and localized association of cytoskeletal, but not sol- iments in the presence of puromycin or RNase, or by label- uble, proteins with the cytoskeleton after a short pulse; (2) ing with [3H]puromycin, nascent chains were consistently the association of cytoskeletal proteins during in vitro trans- identified by immunoprecipitation from the cytoskeletons lation with the cytoskeleton, independent of the concentra- of chick embryonic muscle and fibroblast cells. In addition, tion of soluble proteins; and (3) the association of some, a kinetic argument for cotranslational assembly in fibrob- but not all, nascent polypeptides with the cytoskeleton lasts was made by observing that the fraction of full-length during translation, independent of polyribosomes. vimentin present on the cytoskeleton after any chase time More specific methods were developed to examine the is greater than would be expected if based solely on the cotranslational assembly of specific cytoskeletal proteins. rate of disappearance of full-length vimentin from the sol- Myosin heavy chains form coiled coils in a rod region that uble pool. No qualitative differences in 2-D electrophoretic then interacts with other myosin molecules to form thick pattern or degree of phosphorylation were noted between filaments in muscle. Nascent myosin heavy chain (MHC) the soluble and cytoskeletal fractions of vimentin. The polypeptides were found on the cytoskeleton of cultured chick embryonic skeletal muscles, consistent with cotrans- nascent vimentin chains were removed from the cytoskele- lational assembly (Isaacs and Fulton, 1987). Cultures that ton by 50 mM arginine, but not by 50 mM lysine or 1 M had been extracted with Triton were exposed to salt; thus their associations with the cytoskeleton are bio- [3H]puromycin to simultaneously label and release nascent chemically similar to those of full-length vimentin polypep- polypeptides from ribosomes. A monoclonal antibody to tides. MHC (with an epitope near the amino terminus) was used Vimentin assembly in chick embryonic erythroid cells to immunoprecipitate MHC from the cytoskeletal and sol- was also examined; the three methods used on fibroblasts uble fractions. In day 4 cultures 32% of the [3H]puromycin were all applied to the erythroid cells. Low levels of radioactivity in the MHC, representing only nascent cotranslational assembly (about 10%) were found with all polypeptides, was found on the cytoskeleton. A similar frac- three methods. These observations were consistent with ear- tion of MHC nascent polypeptides was also detected on the lier observations in erythroid cells (Blikstad and Lazarides, cytoskeleton by using [35S]methionine pulse-chase experi- 1983), where it was shown that vimentin is assembled into ments, in which puromycin or RNase released nascent intermediate filaments from a soluble precursor. Vimentin chains from polyribosomes, followed by immunoprecipita- is therefore assembled into the cytoskeleton by two differ- tion of MHC and analysis on polyacrylamide gels. ent pathways, with the relative contributions of these two Titin is a very long molecule, stretching from M line to pathways varying with cell type. Independent evidence for Z line in muscles and associating with myosin in the A the existence of cotranslational assembly of vimentin using band; it undergoes more extensive cotranslational assembly a dual isotope technique has been provided by Low et al. in cultured muscle, as suggested by the exclusive associa- (1985), in cultured human fibroblasts. tion of full length titin with the cytoskeleton after a short A recent observation consistent with cotranslational pulse, and by the observation that 74% of titin nascent assembly is the co-localization of vimentin mRNA and chains remain associated with the cytoskeleton after vimentin protein in the costameres of mature skeletal puromycin treatment (Isaacs et al., 1989). For comparison, muscle differentiating in vitro (Cripe et al., 1993). less than 20% of total cellular nascent chains were found Costameres overlie the myofibril above the Z line, with on the cytoskeleton. Therefore, nascent chains are not, as spacing the same as sarcomeres, i.e. ~1.4 mm. This partic- a class, insoluble simply because of their incomplete length. ular spacing of mRNA is too fine to generate stable pat- Titin and myosin are closely associated within minutes terns of soluble proteins, because proteins diffuse too of being synthesized (Isaacs et al., 1992). This association quickly to maintain a grid with spacing of less than 2 Cotranslational assembly of cytoskeletal proteins 869 microns. This mRNA pattern cannot, therefore, generate study of early myoblasts and myotubes showed perinuclear local gradients of soluble proteins. localization, coincident with the primary location of A parsimonious explanation of this observation is that vimentin protein. the mRNA is present at the site where vimentin protein is undergoing cotranslational assembly. Appropriate controls were performed in this experiment to exclude nonspecific IS COTRANSLATIONAL ASSEMBLY AN binding of the antibody to digoxigenin, nonspecific bind- EXPERIMENTAL ARTIFACT OR DOES IT ing of the probe to costameres or myofibril, and lack of REFLECT REAL CELLULAR PROCESSES? specificity for DNA-RNA hybrids. It is interesting that vimentin mRNA in younger muscle cells displayed several Whenever a highly novel finding is reported, it is appro- different staining patterns, being bipolar in young priate to consider whether a simpler explanation for it can myoblasts, perinuclear in elongated myoblasts, and diffuse be found in some artifact of experimental design. It seems in young myotubes. Perhaps mRNA localization will prove unlikely, however, that the whole body of observations that important for assembling and maintaining differentiated point to cotranslational assembly can be accounted for in cytoskeletal structures in the developing embryo. The rela- this way. It is worthwhile to consider in detail two partic- tionship between localized messenger RNA and cytoskele- ular possible sources of artifact, the detergent Triton X-100 tal assembly, both cotranslational and post-translational, has and the nascent chains themselves. been discussed elsewhere (Fulton, 1993). It has been recently reported that behavior of microin- Does Triton X-100 partition the cell into jected X-rhodamine-labeled vimentin, followed by fluores- representative cytoskeletal and soluble fractions? cence recovery after photobleaching (FRAP), is consistent Triton X-100 is a non-ionic detergent that came into use with a steady-state exchange of vimentin subunits along the for cytoskeletal studies during the late Seventies (Osborn length of intermediate filaments with a soluble pool and Weber, 1977; Brown et al., 1976; Lenk et al., 1977). (Vikstrom et al., 1992). Exchange of vimentin subunits In general, as is common for non-ionic detergents, Triton between the microinjected soluble pool and intermediate fil- X-100 does not denature proteins (Helenius et al., 1979) aments may at first glance seem inconsistent with cotrans- and was chosen empirically because its presence in extrac- lational assembly of vimentin. Microinjection provides the tion buffers (of the appropriate composition) permitted the cell with a sizeable pool of soluble vimentin subunits that recovery of morphologically congruous cytoskeletal struc- is not normally present, however. A concentration effect, tures. The recent observation that some soluble proteins where the likelihood of assembly of microinjected vimentin become bound to structures, especially nuclear structures, into intermediate filaments is increased because of such a in its presence (Melan and Sluder, 1992) suggests that the large soluble pool compared to nascent polypeptides avail- use of Triton X-100 should be re-examined. able for assembly, cannot be excluded. There is no reason, In the studies by Melan and Sluder, concentrations of however, to believe that assembly of vimentin (or other Triton X-100 (0.1% or 0.2%) that were below or near the cytoskeletal proteins) must exclusively occur via either critical micelle concentration (CMC), when used with post-translational assembly from a soluble pool or cotrans- buffers of moderate ionic strength, lead to artifactual redis- lationally from nascent chains on the polyribosome. The tribution of some soluble proteins, most commonly to the published data for vimentin points to about half of the nucleus but sometimes to fibrillar structures in the cyto- vimentin protein assembling post-translationally in fibrob- plasm. Higher concentrations of Triton X-100 (1%, above lasts. Perhaps incorporation of soluble subunits into the CMC) obliterated these artifactual patterns. The con- cytoskeletal structures such as intermediate filaments rep- centration of Triton X-100 used in the cotranslational resents a repair mechanism of the cell, allowing continued assembly experiments (0.5%) was not tested by Sluder, but cytoskeletal remodeling. In addition, initial assembly that this concentration is well above the CMC. In separate occurs cotranslationally does not predict whether a protein studies, 0.5% Triton X-100 produced stable cytoskeletal may disassemble and then re-assemble. preparations that did not shed protein even when the extrac- Actin is a cytoskeletal protein whose mRNA has been tion buffers contained large amounts of competing non- shown to be localized in several cell types, including muscle specific proteins (Gilbert and Fulton, 1985); with these (Lawrence and Singer, 1986). Actin mRNA appears to be buffers, 0.5% and 2% Triton X-100 gave the same results. concentrated at the periphery in young myoblasts and in In addition, if Triton X-100 were causing artifactual bind- fibroblasts, being particularly prominent in lamellipodia. ing to the cytoskeleton, it should do so independent of the High concentrations of actin mRNA were also observed in time at which a protein is synthesized. However, in most areas of cell contact. Myotubes and proliferating myoblasts of the studies discussed, either the spatial pattern or the had a much more uniform distribution of actin mRNA. The amount of material found in the cytoskeleton changed as a prominent localization of actin mRNA in peripheral regions function of time after synthesis. It is difficult to propose of motile cells closely mimics the distribution of growing models for artifacts that vary over time. actin filaments in lamellipodia. Localization of actin mes- Finally, whenever possible, comparisons were made sage may be related, therefore, to cell motility. Actin pro- between intact cells and cytoskeletons after extraction. tein appears to assemble from a soluble monomer pool. Polyribosome patterns (Fulton et al., 1980), myosin and titin Thus, co-localization of message and protein to a particu- staining (Isaacs et al., 1992) and vimentin protein and mes- lar region within a cell does not, per se, imply that cotrans- senger RNA patterns were all unchanged by this extraction lational assembly is occurring. Vimentin mRNA in this procedure. Clearly, however, a blanket assumption of such 870 A. B. Fulton and T. L’Ecuyer preservation cannot be extended automatically to all buffers et al., 1991), tryptophan synthetase b subunit (Fedorov et that contain Triton X-100; the appropriateness of a given al., 1992), and the yeast TRP3 gene product, a bifunctional buffer to a given procedure needs to be determined. protein (Crombie et al., 1992). For some proteins, peptide fragments of them can fold into the secondary structures Do nascent polypeptide chains bind non- seen in the intact molecule; a helices do so more readily specifically to the cytoskeleton? than do b pleated sheets (Dyson et al., 1992). More than 85% of all nascent peptides labeled with The hypothesis of protein folding during translational puromycin are released by 0.5% Triton X-100 (Isaacs and pauses suggests a new perspective on observations of dis- Fulton, 1987); therefore insolubility is not a general prop- crete nascent peptides of myosin, titin and vimentin (Isaacs erty of nascent peptide chains. Puromycin becomes cova- and Fulton, 1989). All three proteins displayed populations lently attached to the nascent chain as it is released from of nascent peptides that migrated as discrete bands, whether the ribosome, so this radioactivity exclusively represents the label used was methionine or puromycin. For vimentin, nascent chains. In addition, when assembly is measured one of these nascent peptides was the size that would result kinetically by examining only the full length polypeptides, from translationally pausing at a 27 bp hairpin that lies the rates of assembly agree well with measurements made within the coding sequence (Bloemendal et al., 1985). It on nascent chains (Isaacs et al., 1989). Competition with might prove informative to replace this hairpin with isocod- non-radioactive proteins did not release substantial amounts ing sequences that would not form a hairpin. of nascent peptides (Isaacs and Fulton, 1987). Nascent pep- Not all cytoskeletal proteins undergo cotranslational tides that initiated in vitro did not bind efficiently to the assembly; actin, tubulin and a -actinin are three that do not cytoskeleton (Fulton and Wan, 1983); it is difficult to see (Mitchison, 1992; Isaacs and Fulton, 1989). Do proteins how artifactual binding could differentiate between in vivo that undergo cotranslational assembly have structural fea- and in vitro initiation. For vimentin, three biochemical cri- tures in common? Vimentin and myosin heavy chain com- teria that characterize the association of full-length vimentin prise parallel coiled coils; both undergo significant cotrans- were satisfied by the nascent chains (Isaacs and Fulton, lational assembly. The globular proteins tubulin and actin, 1989). Titin and myosin nascent chains were cross-linked and the fibrillar but antiparallel dimer a -actinin are not to molecules to which the full-length molecules become cotranslationally assembled, as measured by kinetic exper- cross-linked (Isaacs et al., 1992). The cumulative weight of iments used to detect assembly during translation for these diverse observations makes it unlikely that the nascent vimentin. Thus, our methods also detect post-translational chains are displaying significant amounts of non-specific assembly. The conformation of the protein probably plays binding under these conditions. a role in permitting cotranslational assembly to occur. If so, candidates for cotranslational assembly include fibrillar, Other observations consistent with nascent self-associating proteins that assemble in parallel, not anti- peptides associating with the cytoskeleton parallel (e.g. vimentin, MHC), or elongated proteins bind- One other laboratory has reported biochemical evidence for ing to fibrillar structures (e.g. titin). As tropomyosin has a cotranslational assembly (Low et al., 1985). These studies coiled-coil fibrillar structure that undergoes parallel self- measured full-length vimentin chains, so they offer inde- assembly, it offers an interesting example to test this model pendent support for our kinetic studies. A different form of for the assembly mechanism of cotranslational assembly. evidence that is consistent with cotranslational assembly is Experiments are underway to test whether its assembly is the observation of homodimers and homopolymers of pro- cotranslational. teins that form heteropolymers if re-assembled in vitro. The Nothing is known about the factors that determine the most striking example of this is seen in the work of Gau- levels of cotranslational assembly for a particular protein. thier (1990), who detected myofibrils that were largely com- Vimentin exhibited different levels of cotranslational posed of a single isoform of myosin, although the muscles assembly in fibroblasts and erythrocytes. These cells differ were synthesizing two isoforms that co-assemble in vitro. both in the relative rates of vimentin synthesis and in Other examples, including one involving tropomyosin, have vimentin content; in addition they are different cell types. also been reported (Lin et al., 1985; Silberstein et al., 1986; The relative rate of synthesis might affect cotranslational Bandman, 1985). assembly by affecting the probability of nascent chains encountering another nascent chain. The mass of assembled vimentin might affect cotranslational assembly by affecting the probability of nascent chains encountering vimentin IMPLICATIONS AND PREDICTIONS polymerized in filaments. Cell type specific variation could result from chaperones or other accessory proteins. If one For proteins to assemble during translation, they must begin cell type had synthetic rates and a vimentin content that dif- folding as nascent peptides. Such folding appears to be pos- fered with developmental stage or growth conditions, it sible, prime facie, as many proteins re-fold in less time than might be feasible to determine whether one or both of these is required for their synthesis. Cotranslational folding of variables affects cotranslational assembly significantly. In proteins was formally presented as a hypothesis by Purvis MDCK cells, vimentin synthesis is greatest in the subcon- et al. (1987), with a particular emphasis on translational fluent cells; vimentin mass is greatest in confluent cells. pause sites that could permit folding of domains during Cotranslational assembly should be highest in the subcon- translation. Experimental support for cotranslational fold- fluent cells if relative rate is more important; it should be ing has recently been published for globin (Krasheninnikov highest in the confluent cells if vimentin mass is the sig- Cotranslational assembly of cytoskeletal proteins 871 nificant variable. These cotranslational assembly rates are the myofibrils of individual developing muscle fibers. J. Cell Biol. 110, presently being measured. 693-701. The advantage to a cell of cotranslational assembly may Gilbert, M. and Fulton, A. B. (1985). Specificity and stability of the Triton- extracted cytoskeletal framework of gerbil fibroma cells. J. Cell Sci. 73, be that organizational information is provided during pro- 335-345. tein synthesis. This could facilitate the assembly of such Grollman, A. P. (1968). Inhibitors of protein biosynthesis. J. Biol. Chem. complex cytoskeletal structures as the contractile apparatus 243, 4089-4094. in striated muscle. It may facilitate the maintenance of a Helenius, A., McCaslin, D. R., Fries, E. and Tanford, C. (1979). Properties of detergents. Meth. Enzymol. 56, 734-749. differentiated configuration of the cytoskeleton. The price Isaacs, W. B., Cook, R. K., Van Atta, J. C., Redmond, C. M. and Fulton, for this mechanism of assembly is severe restriction of time A. B. (1989). Assembly of vimentin in cultured cells varies with cell type. and location of protein assembly; these restrictions do not J. Biol. Chem. 264, 17953-17960. apply in post-translational assembly. The two processes Isaacs, W. B. and Fulton, A. B. (1987). Cotranslational assembly of may interact. Nucleation is the rate-limiting step for myosin heavy chain in developing cultured skeletal muscle. Proc. Nat. Acad. Sci. USA 84, 6174-6178. vimentin assembly and is more sensitive to protein con- Isaacs, W. B. and Fulton, A. B. (1989). Assembly of the cytoskeleton in centration than is elongation (Stewart, 1993). If cotransla- cultured muscle cells. In Cellular and Molecular Biology of Muscle tional assembly contributes a significant fraction of nuclei, Development, UCLA Symposium on Molecular and Cellular Biology, vol. it could influence the organization of the cytoskeleton out 93 (ed. L. H. Kedes and F. E. Stockdale), pp. 137-146. New York: Alan R. Liss. of proportion to its fractional contribution to assembly. Per- Isaacs, W. B., Kim, I. S., Struve, A. and Fulton, A. B. (1992). Association haps it is the balance of post-translational assembly, respon- of titin and myosin heavy chain in developing muscle. Proc. Nat. Acad. sive to the environment of the moment, and cotranslational Sci. USA 89, 7496-7500. assembly, providing structural memory, that permits the Isaacs, W. B., Kim, I. S. Struve, A. and Fulton, A. B. (1989). Biosynthesis cytoskeleton to display its wonderful variety and functional of titin in cultured skeletal muscle cells. J. Cell Biol.109, 2189-2195. Krasheninnikov, I. A., Komar, A. A. and Adzhubei, I. A. (1991). adaptations. Nonuniform size distribution of nascent globin peptides, evidence for pause localization sites, and a cotranslational protein-folding model. J. Protein Chem. 10, 445-453. Lawrence, J. B. and Singer, R. H. (1986). 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