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Home , Extracellular space, Golgi apparatus

Proc. Nati Acad. Sci. USA Vol. 80, pp. 4286-4290, July 1983

Role of in the distribution of the : Effect of taxol and microinjected anti-a-tubulin ( organization/) JURGEN WEHLAND*, MARYANNA HENKARTt, RICHARD KLAUSNERt, AND IGNACIO V. SANDOVALt§ *Laboratory of Molecular and tImmunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205; and *Laboratory of Biochemistry and Metabolism, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20205 Communicated by Gilbert Ashwell, April 21, 1983 ABSTRACT Immunofluorescence microscopy reveals that both organization and distribution of both microtubules and the Gol- organizing center (MTOC) and Golgi apparatus are gi apparatus were studied by dual indirect immunofluores- contained in the same perinuclear area of A549 cells in inter- cence microscopy as described (6). Cells were permeabilized by phase. The cells display long microtubules stretching radially from immersion in cold methanol (-20°C) for 2 min. The Golgi ap- the MTOC to the plasma membrane. Treatment of cells with taxol paratus was studied by using a rabbit monospecific results in polymerization of microtubules without relation to the was MTOC and formation of microtubule bundles predominantly lo- raised against the Golgi ,B-galactosyltransferase and calized in the periphery. After incubation with taxol, the Golgi a gift of E. G. Berger (7). Microtubules were studied by using apparatus is fragmented and is conspicuously present in areas of the rat monoclonal antibody YL 1/2. Rhodamine-conjugated the enriched in microtubules. Incubation of cells with goat-anti-rabbit IgG and fluorescein-conjugated goat-anti-rat Colcemid results in complete depolymerization of microtubules IgG were purified by affinity chromatography with, respec- and fragmentation of the Golgi into elements randomly distrib- tively, columns of rabbit IgG and rat IgG coupled to Sepharose uted throughout the cytoplasm. Cells treated with taxol before being 4B. These antibodies were used as second antibodies in indirect incubated with-Colcemid contain large numbers of Golgi-derived immunofluorescence microscopy studies. Microinjection of cells elements in close association with Colcemid-resistant microtu- was performed as described (6, 8) with solutions of 20 mg of bules. Microtubule depolymerization by vinblastine also is fol- antibody per ml of phosphate-buffered saline (pH 7.0). The lowed by fragmentation of the Golgi apparatus. These Golgi-de- YOL rived elements show no association with the atypical polymers of two rat monoclonal anti-a-tubulin antibodies, YL 1/2 and tubulin induced by vinblastine. The codistribution of Golgi-de- 1/34, used in the microinjection experiments were provided by rived elements with taxol-induced microtubule bundles can be re- J. V. Kilmartin (9). Both antibodies, which were characterized versed by microinjection of a monoclonal (YL 1/2) antibody re- as IgGs, were conjugated to fluorescein to study microtubules acting specifically with the tyrosylated form of a-tubulin. by direct immunofluorescence. YL 1/2 reacted specifically with the tyrosylated form of a-tubulin as shown by (i) immunoau- The processing of that are synthesized in the endo- toradiography of tyrosylated and detyrosylated a-tubulin re- plasmic reticulum before being secreted or sorted to the plasma solved from ,B-tubulin by NaDodSO4/polyacrylamide gel elec- membrane and cytoplasmic takes place in the Golgi trophoresis and (ii) its ability to bind specifically to the synthetic apparatus (for a review, see ref. 1). The Golgi apparatus consists peptide Gly-Glu3-Gly-Glu2-Tyr, which corresponds to the car- of vesicles and several functionally related cysternae stacked in boxyl-terminal amino acid sequence of tyrosylated a-tubulin. a defined order (1) and found in the vicinity of the microtubule YOL 1/34 reacted with both tyrosylated and detyrosylated a- organizing center (MTOC) (2). The MTOC initiates the poly- tubulin as shown by immunoautoradiography. The volume of merization and organizes the distribution of microtubules of antibody solution injected into the cells was approximately 10% cells in interphase (3). The proximity of the Golgi apparatus to of the cell volume (8). Approximately 1.2 amol of either an- the MTOC and the ability of Colcemid both to depolymerize tibody (IgG Mr) 160,000) was injected per cell. This antibody microtubules and to induce the fragmentation of the Golgi ap- concentration was shown to have no effect on either the poly- paratus (4, 5) have suggested that microtubules may play a role merization or depolymerization of microtubules both in vitro in maintaining the integrity and location of the Golgi apparatus and in vivo (unpublished results). For electron microscopy, cell in cells in interphase. Here we present our studies on (i) the in 0.125 M ca- effects of the antimitotic drug taxol on the integrity and location monolayers were fixed in 2.5% glutaraldehyde of the Golgi apparatus in the cell, (ii) the codistribution of mi- codylate buffer (pH 7.4) at room temperature. They were post- crotubules and Golgi-derived elements in the cytoplasm of taxol- fixed in OS04, stained in situ with uranyl acetate, dehydrated treated cells, and (iii) the ability of a monoclonal antibody (YL in ethanol, and embedded in Epon. After polymerization of the 1/2) reacting with the tyrosylated form of a-tubulin to reverse Epon, representative areas were selected for sectioning. Serial the association of the Golgi-derived elements with microtu- sections parallel to the plane of the monolayer were cut and bules. examined so that the distribution of microtubules and Golgi elements could be evaluated thoroughly. Stock solutions of taxol MATERIALS AND METHODS and vinblas- A549 (human lung carcinoma) cells were grown in Dulbecco's (National Cancer Institute), Colcemid (GIBCO), modified medium containing 10% fetal calf serum. The tine (Sigma) were prepared in dimethyl sulfoxide and diluted Eagle's 1: 1,000 when added to the cell culture medium. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviation: MTOC, microtubule organizing center. ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. § To whom reprint requests should be addressed. 4286 Downloaded by guest on September 25, 2021 Biochemistry: Wehland et al. Proc. Nati Acad. Sci. USA 80 (1983) 4287

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FIG. 1. (Legend appears at the bottom of the next page.) Downloaded by guest on September 25, 2021 4288 Biochemistry: Wehland et al. Proc. Natl. Acad. Sci. USA 80 (1983) RESULTS whereas YOL 1/34 reacted with both tyrosylated and detyro- Studies on the polymerization of microtubules of cells in in- sylated a-tubulin. We observed that injection of approximately terphase revealed that microtubules were initiated in the amor- 1.2 amol of YL 1/2 antibody into taxol-treated cells produced phous material of the MTOC and grew radially to the plasma a marked rearrangement of the microtubules in the cytoplasm membrane (3). The high density of microtubules in the prox- (in Fig. 1 compare H with E and F). Fluorescent staining of the imity of the MTOC produced a strong fluorescent spot in cells Golgi- apparatus in these cells revealed large numbers of Golgi studied by immunofluorescence microscopy with anti-tubulin elements uniformly distributed throughout the cytoplasm with- antibodies (10, 11). The MTOC of A549 cells in interphase out relation to microtubules (Fig. 1H'). In cells not treated with growing in normal medium was located in the vicinity of the taxol, injection with YL 1/2 antibody displayed normal micro- nucleus (Fig. lA; see the strong fluorescent spots near the nu- tubules and numerous Golgi fragments randomly scattered clei). Immunofluorescence microscopy studies with the anti-f- throughout the cytoplasm (data not shown). Taxol-treated cells galactosyltransferase antibody revealed that. the Golgi appara- injected with YOL 1/34 antibody continued to display a cor- tus was located in the perinuclear area containing the MTOC relation between the distribution of Golgi elements and mi- (Fig. IA'). Treatment of cells with 10 ,/M taxol for 3 hr resulted crotubule bundles accumulated in the cell periphery (data not in polymerization of free microtubules showing no connection shown). Injection of YOL 1/34 antibody into cells not treated with the MTOC (an effect already shown in other cell lines; refs. with taxol had no effect on either microtubules or the Golgi ele- 12 and 13). The free microtubules bundled and accumulated in ment distribution (data not shown). the periphery of the cell, leaving large areas of the cytoplasm The ability of Golgi elements to associate with polymers of impoverished in microtubules (Fig. 1 E and F; see also ref. 12). tubulin other than microtubules was studied by incubating cells The immunofluorescence showed the Golgi apparatus to be with vinblastine. Treatment of cells with 50 ,uM vinblastine for fragmented in these taxol-treated cells (Fig. 1 E' and F'). Al- 15 min resulted in complete disassembly of microtubules and though the Golgi marker f&galactosyltransferase moved from formation of a limited number of paracrystals (Fig. 1C) con- the vicinity of the nucleus to areas of the cytoplasm enriched sisting of short, curled ribbons of tubulin (14, 15). The disas- in microtubules, the question arose as to whether the Golgi ap- sembly of microtubules induced by vinblastine was complete paratus maintained its distinctive structure after taxol treat- before any significant changes in the integrity and position of ment. Electron microscopy of serial thin sections cut from taxol- the Golgi apparatus could be detected (Fig. 1 C and C'). How- treated cells revealed morphologically identifiable Golgi struc- ever the depolymerization of microtubules and formation of tures including vesicles and typical, stackedcisternae in the cell tubulin paracrystals was followed within 90 min by the frag- periphery (Fig. 2). The Golgi elements were found inter- mentation of the Golgi apparatus into elements that were uni- spersed with microtubules and in the vicinity of well-defined formly distributed throughout the cytoplasm, with no apparent bundles of microtubules. Control cells never exhibited such codistribution with the tubulin paracrystals (in Fig. 1 compare multiple peripheral localizations of the Golgi structures. Im- D with D'). munofluorescence microscopy showed that incubation of un- treated cells with 1 A.M Colcemid for 2 hr resulted in complete DISCUSSION depolymerization of microtubules (Fig. 1B) and fragmentation Previous studies have demonstrated that the Golgi apparatus of the Golgi apparatus into elements randomly distributed and the MTOC are found in the same perinuclear area in rat throughout the cytoplasm (Fig. 1B'). Pretreatment of cells with kidney (NRK) cells in interphase (2). Furthermore, these two 10 ttM taxol before incubation with Colcemid stabilized the mi- structures simultaneously move together during cell translo- crotubules against Colcemid-induced depolymerization (Fig. cation to the perinuclear region facing the leading edge of the 1G). Moreover, in these cells, the fragments of the Golgi were cell (2). Treatment of cells with Colcemid has been shown both conspicuously found in areas of the cytoplasm containing Col- to depolymerize microtubules and to induce fragmentation of cemid-resistant microtubules (Fig. 1G'). the Golgi elements into elements that are randomly distributed The role of tubulin in the interaction of microtubules with throughout the cytoplasm (4, 5, 16, 17). Results such as these the Golgi apparatus was studied by injecting the YL 1/2 and have prompted speculation-that either the MTOC or the mi- YOL 1/34 monoclonal anti-a-tubulin antibodies into cells. YL crotubules (or both) play a role in preserving the normal struc- 1/2 reacted specifically with the tyrosylated form of a-tubulin, ture and localization of the Golgi apparatus. We tested further

FIG. 1 (on preceding page). (A and A') Microtubules and Golgi apparatus as displayed by A549 cells growing in normal medium. Observe the long microtubules stretching radially from the vicinity of the nucleus to the plasma membrane (A) and the perinuclear location of the Golgi ap- paratus in the area of intense fluorescence containing the MTOC (A'). (B andB') Microtubule depolymerization and fragmentation anddispersion of the Golgi apparatus in A549 cells treated with Colcemid. Cells were incubated with 1 ,uM Colcemid for 2 hr. Note the complete disappearance ofmicrotubules (B) and the fragmentation ofthe Golgi apparatus into elements distributed uniformly throughout the cytoplasm (B'). (C, C',D, and D') Microtubule depolymerization, formation of tubulin paracrystals, and fragmentation and dispersion of the Golgi apparatus in A549 cells in- cubated with 50 AM vinblastine for 15 min (C and C') and 90 min (D and D'). Observe the fast substitution oftubulin paracrystals for microtubules (C) and the slower fragmentation of the Golgi apparatus (C') after 15 min of incubation with vinblastine. Note the different distribution of tubulin paracrystals and Golgi-derived elements in the cytoplasm after the 90-min incubation of the cells with vinblastine (compare D and D'). (E, E', F, and F') Effects of taxol on the integrity of the Golgi apparatus and the distribution of Golgi-derived elements and microtubules in the cytoplasm of A549 cells. Cells were incubated with 10 ,uM taxol for 3 hr. Observe the predominant location of microtubules in the periphery of the cells (E andF) and the fragmentation ofthe Golgi apparatus (E' andF') into elements that were located in areas ofthe cytoplasm enriched in microtubules. (G and G') Codistribution of Golgi-derived elements with Colcemid-resistant microtubules in the cytoplasm of taxol-treated A549 cells. Cells were treated with 10 AM taxol for 3 hr and then incubated with 1 ,LM Colcemid for 2 hr. Observe the conspicuous accumulation of Golgi-derived elements (G') in areas of the cytoplasm containing Colcemid-resistant microtubules. (H and H') Effect of the monoclonal YL 1/2 anti-tyrosylated a-tubulin antibody on the distribution ofmicrotubules and Golgi-derived elements in the cytoplasm ofA549 taxol-treated cells. Cells treated with 10 A±M taxol for 3 hr were injected with approximately 1.2 amol of fluorescein-conjugated YL 1/2 antibody per cell; 2 hr later the cells were fixed. Microtubules were studied by direct immunofluorescence microscopy, and the Golgi elements were studied by indirect immunofluorescence microscopy. Observe the changes in microtubule organization (compare H with E and F) and distribution of Golgi elements (compare H' with E' and F') produced by YL 1/2 in taxol-treated cells. The patches of fluorescence shown in the right side ofH' correspond to the Golgi-derived elements of a taxol-treated cell not injected with antibody. (Bars = 20 ,um.) Downloaded by guest on September 25, 2021 Biochemistry: Wehland et al. Proc. Nati Acad. Sci. USA 80 (1983) 4289

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_ a -s s ; .--eso ho >' Aid _ an and Sl; s tab. 4, >? j- ;'^'ant;tS. a_ He .I- A are lE5S-_|| .t. .. An .. Zeus r. Ad Am $ s - -. .:-i Z )0*. % ., , en A.> In;.. . ; ; w X -- w , . 1} - w Z i; S o i, . .: t| .10, s - a. *ib FIG. 2. Electron micrograph of a taxol-treated cell. This example from a set of serial sections shows one of a number of well-organized Golgi structures (G) interspersed among microtubules in the periphery ofthe cell. The segments of microtubules above the Golgi apparatus in this plane of section are part of a large band of microtubules passing beneath the Golgi apparatus in deeper planes of section. Microtubules are marked with arrows. The light area in the upper right is . The Golgi apparatus was notfound distributed in these locations, and the microtubule bundles were absent in untreated cells. (Bar = 1 Am.) A-....'e these hypotheses by examining the effects of the antimitotic as organizing sites for the Golgi apparatus. In other experi- drug taxol on the organization of microtubules and Golgi ap- ments along these lines, we examined the effect on the distri- paratus in A549 cells in interphase. bution of the Golgi elements of two monoclonal anti-tubulin Treatment of interphase cells with taxol results in polymer- antibodies reacting with a-tubulin. We reasoned that, if mi- ization of microtubules with no relationship to the MTOC (12, crotubules were involved in determining the location and or- 13). These microtubules accumulate as bundles in the cell pe- ganization of the Golgi apparatus within the cell, we might find riphery (12, 13). The MTOC most likely remains intact after anti-tubulin antibodies that would interfere with this function such treatment (12). Despite this, our results show that the Golgi when injected into cells. It is interesting that, of the two anti- apparatus is no longer preserved as a single perinuclear struc- a-tubulin antibodies tested, only the one reacting with the ty- ture near the MTOC but is fragmented. The cellular localiza- rosylated carboxyl terminus of a-tubulin reversed the apparent tion of the Golgi fragments appears to coincide with-that of the association between the Golgi apparatus and microtubules ob- peripheral microtubule bundles. The Golgi apparatus also frag- served in both normal and taxol-treated cells. This result in- ments in cells whose microtubules have been depolymerized dicates that the carboxyl terminus of tyrosylated a-tubulin plays with colchicine (4, 5, 16, 17) or during (16, 18). In these a role in the interaction of the microtubules with the Golgi ap- cases, however, there are no cytoplasmic microtubules, and the paratus. We do not know whether the carboxyl end of dety- fragmented Golgi apparatus is randomly distributed through- rosylated a-tubulin plays a similar role. Further studies will be out the cytoplasm. The relocation of the Golgi-derived ele- required to determine if the Golgi apparatus interacts with mi- ments among the peripheral microtubule bundles in taxol-treated crotubules directly through tubulin or by means of the proteins cells suggests that microtubules play an important role in the attached to them. organization and location of the Golgi apparatus in the cell. This Electron microscopy of serial thin sections of taxol-treated is also emphasized by the failure of Colcemid to disperse the cells reveals the presence of some characteristic Golgi cyster- Golgi-derived elements in cells whose taxol-treated microtu- nae and vesicles interspersed with microtubules in the periph- bules are resistant to Colcemid-induced depolymerization. This ery of the cell. However, we have not seen the extensive in- result also indicates that it is unlikely that Colcemid, in addition timate relationship between microtubules and recognizable Golgi to binding to tubulin, has some other direct effect on the Golgi apparatus that has been observed between microtubules and membranes that leads independently to both Golgi apparatus rough in cultures of dorsal root gan- fragmentation and microtubule depolymerization. glion-spinal cord treated with taxol (19). It is possible that dur- We do not know the nature of the interaction of the Golgi ing the reorganization of the Golgi apparatus, many of the Golgi- apparatus with microtubules. The experiments with vinblastine derived elements would become dissociated from typical stacks illustrate that tubulin paracrystals (14, 15) do not appear to serve of cysternae and clusters of vesicles. This would make their Downloaded by guest on September 25, 2021 4290 Biochemistry: Wehland et al. Proc. Natl. Acad. Sci. USA 80 (1983) identification on morphologic grounds alone impossible. Iden- 6. Wehland, J., Osborn, M. & Weber, K. (1977) Proc. Nati Acad. Sci. tification of these elements will require the use of immuno- USA 74, 5613-5617. cytochemical techniques at the electron microscopic level. 7. Berger, E. G., Mandel, T. & Schilt, U. (1981)J. Histochem. Cy- tochem. 29, 364-370. These studies add to the list of observations that suggests 8. Graessmann, M. & Graessmann, A. (1976) Proc. Natl Acad. Sci. that microtubules play a role in determining the intracellular USA 73, 366-370. localization of the Golgi apparatus (4, 5, 16-18, 20). There are 9. Kilmartin, J. V., Wright, B. & Milstein, C. (1982)J. Cell Biol 93, two striking details that emerge from this study. First, specific 576-582. rearrangement of intact microtubules leads to a similar rear- 10. Brinldey, B. R., Fuller, G. M. & Highfield, D. P. (1975) Proc. Natl rangement of the Golgi apparatus. Second, a monoclonal an- Acad. Sci. USA 72, 4981-4985. 11. Osborn, M. &'Weber, K. (1976) Proc. Nati Acad. Sci. USA 73, 867- tibody against tubulin, which shows no direct interaction with 871. the Golgi apparatus, leads to a dramatic dispersal of the Golgi 12. DeBrabander, M., Gevens, G., Nuydens, R., Willebrords, R. & apparatus when microinjected into cells. The latter point opens DeMey, J. (1981) Proc. Natl. Acad. Sci. USA 78, 5608-5612. a potentially fruitful approach for the study of organelle-cy- 13. Schiff, P. B. & Horwik, S. B. (1980) Proc. Nati Acad. Sci. USA 77, toskeletal interaction. Unfortunately, the studies reported here 1561-1565. do not elucidate the molecular nature of the interaction, be it 14. Erickson, H. P. (1975) Ann. N.Y. Acad. Sci. 253, 51-52. 15. Fujiwava, K. & Tilney, L. G. (1975) Ann. N.Y. Acad. Sci. 253, 27- direct or indirect, between microtubules and the Golgi appa- 50. ratus. 16. Hiller, G. & Weber, K. (1982) Exp. Cell Res. 142, 85-94. 1. Farquhar, M. G. & Palade, G. E. (1981)J. Cell Biol 91, 77s-103s. 17. Lin, J. J.-C. & Queally, S. A. (1982)J. Cell Biol. 92, 108-112. 2. Kupfer, A., Louvard, D. & Singer, S. J. (1982) Proc. Nat. Acad. 18. Zeligs, J. D. & Wollman, S. H. (1979)J. Ultrastr. Res. 66, 53-77. Sci. USA 79, 2603-2607. 19. Masurovsky, E. B., Peterson, E. R., Crain, S. M. & Horwitz, S. 3. Picket-Heaps, J. (1969) Cytobios 1, 257-280. B. (1981) Brain Res. 217, 392-398. 4. Robbins, E. & Gonatas, N. K. (1964)J. Histochem. Cytochem. 12, 20. Wehland, J. & Sandovap, I. V. (1983) Proc. Natl Acad. Sci. USA 704-711. 80, 1938-1941. 5. Moskalewski, S., Jhyberg, J., Lohmander, S. & Friberg, V. (1975) Exp. Cell Res. 95, 440-454. Downloaded by guest on September 25, 2021