Proc. Natl. Acad. Sci. USAVol. 88, pp. 6318-6322, July 1991Cell Biology

Post-translational modification of microtubules is a component ofsynergic alterations of cytoskeleton leading to formation ofcytoplasmic processes in fibroblasts

(tumor promoter/detyrosination of tubulin/intermediate fflaments/vimentin)

I. S. TINT*, A. D. BERSHADSKYt, I. M. GELFAND*t§, AND J. M. VASILIEV*ttDepartment of Biological Sciences, Rutgers University, Newark, NJ 07102; institute of Systems Research, Academy of Sciences, Moscow 117234, U.S.S.R.;*Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, Moscow 119899, U.S.S.R.; and tCancer Research Center,Academy of Medical Sciences of the U.S.S.R., Kashirskoje shosse 24, Moscow 115478, U.S.S.R.

Contributed by I. M. Gelfand, March 11, 1991

ABSTRACT The protein kinase C activator phorbol 12-myristate 13-acetate (PMA) induces rapid and reversible shapechanges in cultured fibrobasts: extension of motile lamellas isfollowed by transformation of these lameilas into nonmoilestalklke processes. This "lamefla-to-stalk" tranformation wasfound to be associated with the formation of microtubules en-riched in detyrosinated a-tubulin. This change was local: micro-tubules in motile lameflas at the distal ends of the processes andin the cell bodies were not enriched in detyrosinated a-tubulin.Detyrosinated microtubules in the processes were more resistantto Colcemid treatment than other microtubules of PMA-treatedand control cells. The effects ofPMA were reversible and couldbe abolished by sphingosi e, a specific inhibitor of protein kinaseC. Besides modification of microtubules, lamella-to-stalk trans-formation is associated with the ingrowth of intermediate fia-ments into the extensions. Earlier it was found that this trans-formation is also associated with the profound reorganization ofthe system of actin microfilaments. Thus, all three cytoskeletalsystems are altered simultaneously during PMA-induced forma-tion of processes. Similar "cytoskeletal synergies" may playessential roles in many morphogenetic processe-e.g., in thegrowth of neurites.

Transformation of motile lamellas into stalk-like processes isan essential component of many important morphogeneticreorganizations (1). For instance, extension of neurites byneurons involves transformation of proximal parts of motilelamellar growth cones at the ends of these neurites intononmotile shafts ("stalks"; see, e.g., ref. 2). Cultured fibro-blasts treated with the protein kinase C (PKC) activatorphorbol 12-myristate 13-acetate (PMA) provide a convenientsystem for the investigation of mechanisms of lamella-to-stalk transformations: PMA rapidly induces in these cellsextensions of lamellas and their further transformation intonarrow nonmotile processes (3). Our previous studies haveshown that the microfilament system undergoes drasticchanges during reorganization of lamellas into stalks inPMA-treated cells: the well-developed microfilament net-work collapses and loses its contractility (4). The aim ofexperiments described in this paper was to investigate thealterations of other cytoskeletal systems-i.e., microtubulesand intermediate filaments-in the course of lamella-to-stalktransformation in PMA-treated fibroblasts. In particular, wehad previously investigated the alterations in the degree ofdetyrosination of a-tubulin in the microtubules. Detyrosina-tion is a characteristic post-translational modification ofa-tubulin in polymerized microtubules performed by a spe-cial enzyme (for a review, see ref. 5). Microtubules enriched

in detyrosinated a-tubulin were found to characterize severaltypes of stable cytoplasmic extensions, especially neurites ofcultured neurons (6, 7). Results presented in this paper showthat PMA-induced lamella-to-stalk transformation is accom-panied by a considerable increase of detyrosinated tubulinwithin the transforming extensions; this modification is alsocorrelated with the growth ofvimentin intermediate filamentsinto the extensions.

MATERIALS AND METHODSThe 152/15 subline (8) of spontaneously transformed mousefibroblast line CAK-7 (9) was used. The cells were grown onglass coverslips in a 1:1 (vol/vol) mixture of Eagle's basalmedium and 0.5% lactalbumin hydrolysate supplementedwith lo bovine serum for 48 hr. Cell density was approx-imately 104 cells per cm2. PMA (Sigma) was used at a finalconcentration of 10 ng/ml. The cells were incubated withPMA for 1-2 hr. D-Sphingosine sulfate (Sigma) was added ata final concentration of 6 gM together with PMA or sepa-rately for 1 hr.

Cell permeabilization with Triton X-100 was performed asdescribed earlier (10). After permeabilization the cells werefixed with 0.1-0.2% glutaraldehyde (Serva). Free aldehydegroups were blocked by sodium borohydride (Sigma; 2mg/ml in phosphate-buffered saline) for 10 min and withlysine (Sigma; 2% solution in phosphate-buffered saline) for1 hr. For indirect immunofluorescence staining we used thefollowing antibodies.

(i) Mouse monoclonal antibody to tubulin, TU-01, waskindly donated by V. Viklicky (Institute of Molecular Ge-netics, Prague) (11). This antibody reacts with a-tubulin andmay recognize both tyrosinated and detyrosinated microtu-bules (which contain Tyr-tubulin and Glu-tubulin, respec-tively).

(ii) Rabbit polyclonal antibody to tubulin from bovine brainwas affinity purified according to ref. 12. It reacts with a- and13-tubulin and also recognizes tyrosinated and detyrosinatedmicrotubules.

(iii) Rabbit polyclonal antibody to detyrosinated a-tubulin(Glu-tubulin) was a generous gift of J. C. Bulinski (Universityof California, Los Angeles). It reacts with detyrosinateda-tubulin only (13).

(iv) Monoclonal mouse antibody to tyrosinated a-tubulin(YL1/2) was a generous gift of J. V. Kilmartin (Cambridge,England). It reacts with tyrosinated a-tubulin (14).

(v) Monoclonal mouse antibody to vimentin (IIID3), whichstains intermediate filaments of fibroblasts (10) was obtained

Abbreviations: PMA, phorbol 12-myristate 13-acetate; PKC, proteinkinase C.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 88 (1991) 6319

in the laboratory of 0. Rokhlin (All-Union Cardiology Sci-entific Center, Moscow).These antibodies were revealed by fluorescein-conjugated

goat anti-mouse IgG (1:50 dilution) and rhodamine-con-jugated goat anti-rabbit IgG (1:100 dilution) (both obtainedfrom Sigma).

RESULTSControl cells of the 152/15 line had polygonal, fusiform, orfanlike shapes (Fig. 1A). Antibodies to total tubulin (TU-01 orpolyclonal antibody) revealed a dense radial network ofmicrotubules filling the whole cell body, including its periph-eral parts and processes (Fig. 2A). Antibody to Glu-tubulinstained only few short microtubules radiating from microtu-bule-organizing centers (Fig. 2B); in some cells detyrosinatedmicrotubules were absent. Antibody to vimentin revealednumerous intermediate filaments surrounding the nucleus.The peripheral parts of lamellas were often free from theintermediate filaments.PMA induced an alteration of cell shape within 20-40 min.

As described before (3), most cells extended large semicir-cular lamellas from various parts of their edges. Proximalparts of these lamellas often underwent progressive narrow-ing, leading to their transformation into stalk-like cylindricalprocesses with lamella leaflike parts at their distal ends (Fig.1B). After 2-3 hr most cells acquired several long narrowprocesses formed from lamellas.The cells fixed after 1-2 hr of incubation with PMA had

numerous microtubules revealed by antibodies to total tubu-lin. Microtubules formed rather tight bundles in the narrowstalklike parts of the extensions and had more loose radiatingpatterns in the lamellar parts (Fig. 3 A and C). Glu-tubulinrevealed by corresponding antibody was abundant in themicrotubules localized in stalklike parts of the cells (Fig. 3 Band D). In contrast, Glu-tubulin staining of microtubuleslocalized in the cell body was absent or weak, except for a fewmicrotubules radiating from the organizing centers. Intensestaining of microtubule bundles with Glu-tubulin antibody in

FIG. 1. Effect of PMA treatment on cell shape. (A) Control152/15 cell. (B) The 152/15 cell after PMA treatment for 1 hr; noteseveral processes with lamellas formed at distal ends. (Bars, 20 ,um.)

FIG. 2. Double-label immunofluorescence of microtubules andintermediate filaments in control 152/15 cells. (A) Staining withantibody TU-01, revealing all microtubules of the cell. (B) Stainingwith antibody to Glu-tubulin, revealing detyrosinated microtubules.Short detyrosinated microtubules radiate from the microtubule-organizing center. (Bar, 10 ,um.)

the stalklike processes disappeared when microtubules en-tered cell bodies at the proximal ends ofthese processes (Fig.3D); this staining also disappeared when microtubules en-tered the lamellar parts of extensions at their distal ends (Fig.3B). In the domains enriched with Glu-tubulin, staining fornonmodified Tyr-tubulin with corresponding antibody wasalso positive; obviously, only a part of a-tubulin moleculesundergoes modification in these domains.

Staining for vimentin revealed the abundance of interme-diate filaments in the cell bodies and in the stalklike pro-cesses; intermediate filaments were absent from the lamellas(Fig. 4B). Combined staining for Glu-tubulin and vimentindemonstrated the colocalization of microtubules rich in dety-rosinated tubulin and bundles of vimentin filaments. Inparticular, at the intermediate stages oflamella-to-stalk trans-formation both types of structures were present in thestalklike parts but absent from lamellar parts of the exten-sions (Figs. 3B and 4B), which contained only tyrosinatedmicrotubules (Fig. 4A).The cells incubated with PMA for 2 hr and then returned

into PMA-free medium for another 4 hr restored their usualshape. Their staining for total tubulin, Glu-tubulin, andintermediate filaments was similar to controls.

Incubation of the cells with PMA in combination with aspecific inhibitor of PKC, sphingosine (15, 16), did not leadto apparent changes in cell morphology. Staining both forGlu-microtubules and for total microtubules also remainedidentical to controls.

Incubation for 15 min with demecolcin (0.5 ,ug/ml) led tothe disappearance of microtubules from control cells. PMA-treated cells also lost their microtubules, except for Glu-microtubules, which were often seen in the cell processes.

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Proc. Natl. Acad. Sci. USA 88 (1991)

FIG. 3. Double-label immunofluorescence of microtubules in PMA-treated cells. Staining was with antibody TU-01 (A and C) or withantibody to Glu-tubulin (B and D). Detyrosinated microtubules are localized in stalklike processes (B and D). Proximal lamellas (B) and cell bodies(D) are free from detyrosinated microtubules. (Bars, 20 jAm.)

Longer incubation (30-45 min) with Colcemid led to disap-pearance of all microtubules.

DISCUSSIONMicrotubules Rich in Detyrosinated Tubulin Characterize

the Cytoplasmic Processes Induced by Phorbol Ester. Exper-iments described above show that the phorbol ester PMAgreatly increases the number of microtubules rich in dety-rosinated a-tubulin in cultured fibroblasts. The characteristicand most spectacular feature of PMA-induced modulation ofmicrotubules is its close association in time and place with

morphologic reorganizations and, more specifically, with oneparticular stage ofthese reorganizations, formation ofnarrowstalklike processes. Microtubules growing into extendedperipheral lamellas are not yet enriched in Glu-tubulin;modulation occurs only when and where these lamellas havebeen collapsed into stalks. Probably microtubule modulationspreads in the same direction.

In other cell systems modified microtubules were found tobe increased in stability and resistance to disruption bymicrotubule-depolymerizing drugs (17-22). Modified micro-tubules in PMA-treated cells also have an increased resis-tance to Colcemid. Evidence obtained in other systems

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Proc. Natl. Acad. Sci. USA 88 (1991) 6321

FIG. 4. Double-label immunofluorescence of PMA-induced cellprocesses with polyclonal antibody to tubulin (A) and 11ID3 antibodyto vimentin (B). Distal lamella does not contain intermediate fila-ments (B), but it has many tyrosinated microtubules (A). (Bar, 10/Im.)

suggests that detyrosination and other post-translationalmodifications of microtubules are a consequence of theirincreased stability rather than its cause (22, 23).Most microtubules in cultured interphase cells have a

characteristic radial pattern, and it is usually accepted thatthey all are associated with the centrosome. Microtubuledomains containing detyrosinated tubulin are usually local-ized in the stalklike extensions of PMA-treated cells, whilemore central parts in corresponding sectors of the cell bodyas well as more peripheral lamellar parts are not enriched inmodified tubulin. It is not likely that the detyrosinateddomain can be localized in the middle portion of a continuouscentrosome-associated microtubule. It can be suggested thatmodified peripheral microtubules in PMA-treated cells arenot associated with centers, as was shown earlier for axonalmicrotubules (24) and for stable microtubules in epithelialcells (25).The most characteristic effect of PMA is strong activation

of many subtypes of PKC (26). In our experimental systemmodulation of microtubules is rapidly induced by low con-centrations ofPMA, is easily reversed by removal of this drugfrom the medium, and is suppressed by an inhibitor of PKC,sphingosine. Thus, it seems reasonable to regard modulationsof microtubules as one ofthe manifestations of the pleiotropic

effects of PKC activation. In different experimental systemsPKC phosphorylates a great number of molecular species,including microtubule-associated proteins MAP-2 (27) and r(28) and intermediate filament protein vimentin (29). It re-mains unknown which of these cytoskeletal targets of PKC,if any, is essential for the modification of microtubules.PMA Induces Synergic Cytoskeletal Reorganization. Be-

sides microtubule modification, lamella-to-stalk transforma-tion also involves the profound reorganization of anothercytoskeletal system, actin microfilaments; the network ofmicrofilaments present in the lamellas is dismantled and actincortex loses its contractility (3, 4, 30, 31).

This transformation is also correlated with -the growth ofintermediate filaments into peripheral extensions: vimentinantibody reveals these filaments in the stalklike parts ofextensions but not in the lamellas.Thus, PMA-induced morphological alterations are associ-

ated with reorganizations of all the three cytoskeletal systemsoccurring at the same time and at the same places. Complexreorganizations of this type can be named "cytoskeletalsynergies." One cannot exclude that each cytoskeletal sys-tem is changed independently in the course of these reorga-nizations. However, it seems more probable that variouscytoskeletal systems interact with one another during thereorganization. What is the nature of these possible interac-tions? The presence of microtubules is not essential fordisorganization of actin accompanying lamella-to-stalk trans-formation: these transformations can be induced by PMA inthe cells with microtubules depolymerized by Colcemid (31).Intermediate filaments are the only type of cytoskeletalelement that is always found in cytoplasmic stalklike pro-cesses induced by PMA in all types of experiments; asalready indicated, intermediate filaments are usually absentfrom lamellas before their transformation into these pro-cesses. We suggest that the presence of intermediate fila-ments is essential for morphological transformation and foraccompanying alterations of actin cortex and microtubules.This possibility needs further experimental testing.

Cytoskeletal reorganizations occurring at the boundarybetween the lamellar growth cone and the stem ofthe growingneurites of neurons are rather similar to those between thelamellar and stalklike parts of the processes formed byPMA-treated fibroblasts. More specifically, intermediate fil-aments (neurofilaments) are present only in the shafts, not inthe growth cone. The shafts contains microtubules rich indetyrosinated a-tubulin, while the microtubules in the lamel-lar growth cone do not contain stainable amounts of thismodified tubulin (6, 7). Probably, lamella-to-stalk transfor-mations in various cell types have common mechanisms.

We are very grateful to Dr. J. C. Bulinski for antibody againstGlu-tubulin, to Dr. V. Viklicky for TU-01 antibody, and to Dr. J. V.Kilmartin for YL1/2 antibody. We highly appreciate help andencouragement provided to us by Dr. Harvey H. Feder. This workwas partially supported by the Rutgers University Exchange Pro-gram under the direction of I.M.G.

1. Vasiliev, J. M. & Gelfand, I. M. (1988) Cell Diff. 24, 75-82.2. Bray, D. & Chapman, K. (1985) J. Neurosci. 5, 3204-3213.3. Dugina, V. B., Svitkina, T. M., Vasiliev, J. M. & Gelfand,

I. M. (1987) Proc. Natl. Acad. Sci. USA 84, 4122-4126.4. Lyass, L. A., Bershadsky, A. D., Vasiliev, J. M. & Gelfand,

I. M. (1988) Proc. Natl. Acad. Sci. USA 85, 9538-9541.5. Barra, H. S., Arce, C. A. & Argarana, C. E. (1988) Mol.

Neurobiol. 2, 133-153.6. Robson, S. & Burgoyne, R. D. (1989) Cell Motil. Cytoskeleton

12, 273-282.7. Bass, P. W. & Black, M. M. (1990) J. Cell Bin. 111, 495-507.8. Bershadsky, A. D., Brodskaya, R. M., Mansurov, P. G.,

Stavrovskaya, A. A. & Stromskaya, T. B. (1984) Eksp. Onkol.6, 27-32 (in Russian).

Cell Biology: Tint et al.

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 28

, 202

1

6322 Cell Biology: Tint et al.

9. Farber, R. A. & Liskay, R. M. (1974) Cytogenet. Cell Genet.13, 384-396.

10. Bershadsky, A. D., Tint, I. S. & Svitkina, T. M. (1987) CellMotil. Cytoskeleton 8, 274-283.

11. Viklicky, V., Draber, P., Hasek, J. & Bartek, J. (1982) CellBiol. Int. Rep. 6, 725-731.

12. Fuller, G. M., Brinkley, B. R. & Boughter, J. M. (1975) Sci-ence 187, 948-950.

13. Gundersen, G. G., Kalnoski, M. H. & Bulinski, J. C. (1984)Cell 38, 779-789.

14. Kilmartin, J. V., Wright, B. & Milstein, C. (1982) J. Cell Biol.93, 576-582.

15. Hannun, Y. A. & Bell, R. M. (1989) Science 243, 500-507.16. Merrill, A. H. & Stevens, V. L. (1989) Biochim. Biophys. Acta

1010, 131-139.17. Schulze, E., Asai, D. J., Bulinski, J. C. & Kirschner, M. (1987)

J. Cell Biol. 105, 2167-2177.18. Webster, D. R., Gundersen, G. G., Bulinski, J. C. & Borisy,

G. G. (1987) Proc. Natl. Acad. Sci. USA 84, 9040-9044.19. Kreis, T. E. (1987) EMBO J. 6, 2597-2606.20. Cambray-Deakin, M. A. & Burgoyne, R. D. (1987) Cell Motil.

Cytoskeleton 8, 284-291.

Proc. Nati. Acad. Sci. USA 88 (1991)

21. Prescott, A. R., Vestberg, M. & Warn, R. M. (1989) J. Cell Sci.94, 227-236.

22. Khawaja, S., Gundersen, G. G. & Bulinski, J. C. (1988) J. CellBiol. 106, 141-149.

23. Webster, D. R., Wehland, J., Weber, K. & Borisy, G. G. (1990)J. Cell Biol. 111, 113-122.

24. Bray, D. & Bunge, M. B. (1981) J. Neurocytol. 10, 589-605.25. Bre, M.-H., Kreis, T. E. & Karsenti, E. (1987) J. Cell Biol. 105,

1283-12%.26. Nishizuka, Y. (1984) Nature (London) 308, 693-698.27. Tsuyama, S., Bramblett, G. T., Huang, K.-P. & Flavin, M.

(1986) J. Biol. Chem. 261, 4110-4116.28. Hoshi, M., Nishida, E., Miyata, Y., Sakai, H., Miyoshi, T.,

Ogawara, H. & Akiyama, T. (1987) FEBS Lett. 217, 237-241.29. Huang, C. K., Devanney, J. F. & Kennedy, S. P. (1988) Bio-

chem. Biophys. Res. Commun. 150, 1006-1011.30. Danowsky, B. A. & Harris, A. K. (1988) Exp. Cell. Res. 177,

47-59.31. Bershadsky, A. D., Ivanova, 0. Y., Lyass, L. A., Pletyush-

kina, 0. Y., Vasiliev, J. M. & Gelfand, I. M. (1990) Proc. Natl.Acad. Sci. USA 87, 1884-1888.

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