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Role of Microtubules in the Distribution of the Golgi Apparatus

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Proc. Nati Acad. Sci. USA Vol. 80, pp. 4286-4290, July 1983 Biochemistry Role of microtubules in the distribution of the Golgi apparatus: Effect of taxol and microinjected anti-a-tubulin antibodies (organelle organization/cytoskeleton) JURGEN WEHLAND*, MARYANNA HENKARTt, RICHARD KLAUSNERt, AND IGNACIO V. SANDOVALt§ *Laboratory of Molecular Biology 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- microtubule 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 antibody results in polymerization of microtubules without relation to the was MTOC and formation of microtubule bundles predominantly lo- raised against the Golgi enzyme ,B-galactosyltransferase and calized in the cell 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 cytoplasm 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 proteins 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 organelles 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 l 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.
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