CELL STRUCTURE AND FUNCTION 22: 325-334 (1997) © 1997 by Japan Society for Cell Biology Process of Dispersion and Fragmentation of Golgi Complexby Microtubule Bundles Formed in Taxol Treated HeLa Cells Harumi Hoshino, Atsushi Tamaki, and Tatsuo Yagura* Laboratory of Life Science, Department of Chemistry, Faculty of Science, Kwansei Gakuin University, 1-1- 155, Uegahara, Nishinomiya-shi, Hyogo-ken 652, Japan Keywords: Golgi complex/microtubule/taxol/immunoelectronmicroscopy ABSTRACT.By means of a monoclonal antibody (mAbG3A5)against Golgi membrane glycoprotein, we have visualized the relative position of cytoplasmic polymerized microtubule bundles to Golgi stack cisternae in taxol treated HeLa cells, and found extensive fragmentation of the Golgi stack cisternae brought about by micro- tubule bundles. Within a 1 h period of taxol treatment, polymerization of cytoplasmic microtubules increased rapidly to form microtubule bundles, while the Golgi complex dispersed slightly along with the polymerized mi- crotubule bundles. After 2 to 3 h taxol treatment the dispersal of the Golgi complex from the microtubule-organ- izing center (MTOC)to cytoplasmic periphery rapidly progressed in the direction which the microtubules ran. At early dispersal, the microtubule bundles were oriented apart from the stretched end of the Golgi stack cister- nae and exhibited little direct contact with the Golgi stack cisternae membrane. The Golgi stack cisternae then began to wind around the microtubule bundles, followed by the beginning of fragmentation of the Golgi stack cisternae. At this step, some of the microtubules seemed to attach to a part of the Golgi stack cisternae. After prolonged exposure of cells to taxol (25 h) the microtubule bundles were highly developed throughout the cells and most of the Golgi fragments were trapped at their termini. In these cells, extensive fragmentation of Golgi stack cisternae occurred, resulting in small Golgi vesicles bound to the microtubule bundle. The Golgi complex is composed of several stacks ar- membraneand the reassembly of fragment vesicles into ranged in a circumscribed juxtanuclear region to which Golgi stacks. The transport of newly synthesized pro- the microtubule-organizing center (MTOC) localizes teins through the Golgi cisternae is maintained in cells (19). The association of the Golgi complex and microtu- in which microtubules are depolymerized and the Golgi bules has been demonstrated often in several systems (5, stacks are dispersed (6). These observations have sug- 14, 15), with microtubules being instrumental in the gested that someunknownmechanismis at work to orderly membrane traffic through Golgi elements (12). maintain the integrity, location and function of the The candidate proteins for mediating the interaction be- Golgi complex in addition to or independent of the func- tween the Golgi complex and microtubules have also tion of microtubules. been identified, and have been shownto be associated Wemade a highly specific monoclonal antibody with the cytoplasmic face of Golgi membranes (1, 3). (mAbG3A5) to Golgi membrane glycoprotein (pi38), On the basis of these and several other observations (9, which we used previously to reveal immunocytochemi- 19), microtubules are thought to play a role in maintain- cally the ultrastructure of Golgi vesicles in mitotic cells ing the integrity and location of the Golgi complex in in- (2, 24). In this study, we present a detailed immunocyto- terphase cells. However,although microtubules run in chemical analysis of the mechanismfor the microtubule the vicinity of the Golgi complex (23), ultrastructural bundle mediated intracellular disposition of elements of studies have not revealed the binding of the Golgi cister- the Golgi complex in taxol treated cells using the mAb nae to microtubules (18, 23), such as that shown for mi- G3A5antibody. Taxol, which stabilizes microtubule as- crotubules and endoplasmic reticulum in taxol treated sembly (17), causes the Golgi complex to move toward cells (ll, 17). Recently, Veit et al (21) have shown the minus end of the microtubule bundle (15). Weexam- the microtubule-independent breakdown of the Golgi ined the dispersal event of the Golgi complex through stabilization of microtubule assembly and pursued the fate of the Golgi stack cisternal membrane in taxol * To whomcorrespondence should be addressed. Tel: 81-798-54-6406, Fax: 81-798-51-0914 treated HeLa cells by immunoelectron microscopy us- e-mail: [email protected] ing the mAbG3A5antibody. Our results extend those Abbreviation: MTOC;microtubule-organizing center. of previous studies by showing that the dispersal of the 325 H. Hoshino et al. Golgi complex is initiated later than the formation of microtubule bundles and that prolonged treatment of taxol breaks the Golgi stack cisternae into small vesicles that bind to the microtubule bundle. Moreover, we showthat microtubule bundles run in the vicinity of the Golgi stack cisternae but that they have only slight con- tact with the Golgi stack cisternae during early taxol treatment. The Golgi stack cisternae, however, are be- ing stretched by microtubule bundles, suggesting some unknownstructure mediatingthese twostructures. MATERIALS AND METHODS Reagents. Taxol was obtained from WakoChemical Co.. Stock solution of taxol was prepared at 10 mMin dimethyl sul- foxide and stored in small aliquots at -40°C. Cell Culture. HeLa cells were grown in Eagle's minimum essential medium supplemented with 5%fetal calf serum. In experiments using taxol, the concentration of dimethyl sulfox- ide added along with the drug as a solvent to the culture was 0. 1%(final concentration). Cells were cultivated with the drug at 37°C before preparation as indicated. Antibodies. Hybridoma cell line (mAbG3A5) that se- creted monoclonalantibody against pi38 of humanorigin was prepared as described previously (24) and purified by chro- matography on columns of affi-Gel protein A. Rabbit anti- body to tubulin was obtained from ICN Biomedicals, Inc.; FITC-conjugated rabbit anti-mouse IgG antibody was from DAKOA/S; Rhodamine-conjugated donkey anti-rabbit IgG antibody was from CHEMICONInternational Inc. Immunofluorescencemicroscopy and immunoperoxidase microscopy. The preparations were madeby the methods de- scribed previously (2). Specimens were observed and photo- graphed with an Olympusfluorescence microscope for immu- nofluorescence microscopy, and ultrathin sections were exam- ined with a JEOL JEM-100S electron microscope. Western immunoblotting of tubulins. Quantification of microtubule polymer in taxol treated HeLa cells was carried out principally by the method described by Liao et al. (10). HeLa cells were extracted with PEM(100 mMPipes, 10 mM EGTAand 2 mMMgCl2, pH 7.0) containing 0.5 mg/ml sapo- nin for 1 minute after drug treatment, harvested with a scraper, washed 2 times by centrifugation, and then prepared for SDS-PAGE.Blots were performed as described previous- Fig. 1. Patterns of distribution of the Golgi complex in taxol treated cells. The distribution of the Golgi complex in cells treated with 10 ^Mtaxol was examined with indirect immunofluorescence mi- croscopy after labeling with mAbG3A5antibody. The distribution of the Golgi complex was classified according to their fluorescence pat- terns: (A) pattern 1, no change, similar to those observed in untreated control cells; (B) pattern 2, a portion of the Golgi stack cisternae are being pulled out and fragmenting; (C) pattern 3, entire Golgi stack cis- ternae are extended and fragmented; (D) pattern 4, fragmented Golgi Fig. 1. stack cisternae are dispersed to cell periphery. The bar indicates 10 ftm. 326 Dispersion and Fragmentation of Golgi by Taxol ly (8) using anti-tubulin antibody and alkaline phosphatase conjugated-second antibody. RESULTS S80 / Microtubule polymerization precedes dispersal of \560- / Golgi complex during taxol treatment. Treatment of f\ cells with taxol for 3 h results both in polymerization of !O / / microtubule bundles in the cell periphery and dispersal 2 40- / of the Golgi complex (16). In HeLacells treated with taxol we could classify several patterns of distribution I£20-/ / of the Golgi complex within the cytoplasm. Fig. 1 sum- marizes the patterns revealed by immunofluorescence 1 / procedure using mAbG3A5antibody and, in Fig. 2, the 0« . , å , . change of patterns over time during taxol treatment. 0 2 4 6 Pattern 1 is the same as the Golgi complex in non- Incubation Time After Addition of Taxol (h) treated control cells, and the Golgi complex in pattern 2 is not dispersed from its MTOCregion but a part of it is Fig. 3. Formation of microtubule bundles in cells during taxol treat- being pulled towards the outer cytoplasm. The total ment. Cells exhibiting apparent microtubule bundles in their cyto- number of cells exhibiting patterns 3 and 4, in which the plasm were counted during the course of taxol treatment (10 fiM) us- Golgi complexes are obviously dispersed in the cyto- ing immunofluorescence microscopy. plasm from the MTOC,gradually increased with time tion of tubulin over time during taxol treatment. First, anti-tubulinwe employed antibody.immunofluorescentA plot of themicroscopyratio of theusingcellsan antibodywith microtubulevs. time bundlesperiod ofvisualizedtaxol treatmentwith anti-tubulinis shown in period of taxol treatment. The ratio of cells exhibiting 70%Fig. of3.theMicrotubulecells at 1bundlesh after weretaxol observedaddition, inwithmorealmostthan withall oftaxol.the cellsNext,exhibitingthe changebundlesin theafteramountlongerof