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Disruption of the Golgi Apparatus by Brefeldin a Blocks Cell Polarization and Inhibits Directed Cell Migration ALEXANDER D

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Disruption of the Golgi Apparatus by Brefeldin a Blocks Cell Polarization and Inhibits Directed Cell Migration ALEXANDER D Proc. Natl. Acad. Sci. USA Vol. 91, pp. 5686-5689, June 1994 Cell Biology Disruption of the Golgi apparatus by brefeldin A blocks cell polarization and inhibits directed cell migration ALEXANDER D. BERSHADSKY*t AND ANTHONY H. FUTERMAN* Departments of *Chemical Immunology and tMembrane Research and Biophysics, Weizmann Institute of Science, Rehovot 76100, Israel Communicated by Israel Gelfand, March 14, 1994 (receivedfor review January 5, 1994) ABSTRACT The role of the Golgi apparatus in the motile kinesin antibodies results in loss of cell polarity (5, 9) activity of fibroblasts was examined with brefeldin A (BFA), supports the latter possibility. which disrupts the Golgi apparatus in a variety of cells. Upon We have examined the role of the Golgi apparatus in the incubation with BFA, Swiss mouse 3T3 fibroblasts lost their motile activity of cells. For this, we used brefeldin A (BFA) typical polarized morphology, in which the leading edge is (10-12), which disrupts the Golgi apparatus, resulting in characterized by intensive lamellipodia formation. BFA af- subsequent inhibition of vesicle transport to the cell surface fected cell asymmetry as demonstrated by a decrease in the without affecting microtubule organization (13). We report morphometric indices, dispersion, and elongation. After BFA that disruption of the Golgi apparatus leads to changes in cell treatment, cells showed little protrusional activity and did not morphology and motility that are indistinguishable from form a dense actin network at the leading edge, and conse- those caused by disruption of microtubules. These results quently the rate of cell migration into an experimental wound suggest that the generation and maintenance of fibroblast was significantly reduced. In addition, BFA prevented an asymmetry, which is a prerequisite for directed migration, increase in pseudopodial activity and prevented the formation depends on maintaining both the supply of Golgi apparatus- of long processes induced by phorbol 12-myristate 13-acetate. derived vesicles and the integrity of microtubule-based ves- The effects ofBFA on cell shape and protrusional activity were icle transport. quantitatively similar to those observed with the microtubule- disrupting agent nocodazole, although BFA had no effect on MATERIALS AND METHODS microtubule integrity. These results suggest that the integrity of both the Golgi apparatus and microtubules is necessary for Cell Culture. Swiss mouse 3T3 cells were cultured in the generation and maintenance of fibroblast asymmetry, Dulbecco's modified Eagle's medium containing 10o calf which is a prerequisite for directed cell migration. serum and maintained in a water-saturated atmosphere of7% CO2. Cells were dissociated with trypsin/EDTA and plated Directed migration of a variety of cell types depends on the at -5 x 103 per cm2 on glass coverslips. The effect of BFA polarized distribution ofprotrusions (pseudopods) at the cell on cell shape was measured 1 or 2 days after plating; BFA periphery (1). For example, in directionally migrating fibro- was prepared as a stock solution (10 mg/ml) in dimethyl blasts, most pseudopodial activity is located at the leading sulfoxide. edge ofthe cell, whereas no protrusional activity is observed Analysis of Cell Shape and Motility. Dispersion and elon- at the sides and trailing edge, resulting in the typical fan-like gation indices of cell outlines were calculated according to appearance of fibroblasts. The asymmetric distribution of Dunn and Brown (14). Cells were labeled with tetramethyl- protrusional activity is a general characteristic of directional rhodamine isothiocyanate (TRITC)-conjugated phalloidin motility and/or directional growth. Fish keratocytes, which and microscopic images were captured with a charge- are highly motile, display intensive pseudopodial activity at coupled-device camera. Cell outlines were identified and the edge of the cell which is oriented in the direction of analyzed with software provided by Z. Kam, Department of movement (2), and in neurons, protrusional activity is re- Chemical Immunology, Weizmann Institute of Science. Ap- stricted to the growth cone (3). In all of these cases, proximately 40-50 cells were analyzed for each experimental pseudopodial activity requires local polymerization and point. crosslinking of actin filaments at the leading edge (4). A confluent cell monolayer obtained 1 week after plating Polarization ofpseudopodial activity also depends on other was used for experiments on cell migration into an experi- cytoskeletal elements. Vasiliev, Gelfand, and collaborators mental wound. Experimental wounds were made in the (5-7) showed that in cultured fibroblasts, disruption of mi- middle of the coverslip by using the plunger of a disposable crotubules by drugs such as colchicine or nocodazole re- Eppendorf syringe. Video microscopy of cell migration was sulted in the loss of asymmetry of cell shape and a decrease performed with a Plan Neofluar x 100/1.3 n.a. objective of a in directed cell migration. Two potential mechanisms can Zeiss Axiophot microscope, equipped with Nomarski optics, explain the role of microtubules in maintaining cell asymme- a JVC video camera, and a Panasonic video cassette re- try. First, microtubules could play a structural role by corder. restricting the formation of pseudopods at the In experiments examining the effect of BFA pretreatment mechanically on process formation, cells were incubated with BFA for 18 sides and trailing edge ofthe cell. Second, since microtubules hr prior to addition ofphorbol 12-myristate 13-acetate (PMA, provide the framework upon which the molecular motors 100 ng/ml) for various times; BFA was present throughout kinesin and dynein vectorially transport organelles and ves- the incubation with PMA. To quantify the effect of BFA on icles (8), they may be involved in transport of the cell the formation of long-processes, cells were labeled with components necessary for formation ofthe leading edge. The observation that microinjection of fibroblasts with anti- Abbreviations: BFA, brefeldin A; PMA, phorbol 12-myristate 13- acetate; TRITC, tetramethylrhodamine isothiocyanate; C6-NBD- The publication costs of this article were defrayed in part by page charge ceramide, N-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-e-aminohex- payment. This article must therefore be hereby marked "advertisement" anoyl]-D-erythro-sphingosine. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 5686 Downloaded by guest on September 29, 2021 Cell Biology: Bershadsky and Futerman Proc. Natl. Acad. Sci. USA 91 (1994) 5687 TRITC-conjugated phalloidin to visualize cell outlines and Removal of drug with 4',6-diamidino-2-phenylindole (DAPI) to visualize the 0.6 - nucleus. A cell was considered to have a long process ("tail"; see Fig. 5C) if the length of the process was >10 times the 0.5 diameter of the nucleus. 0.4 Immunofluorescence and Fluorescence Microscopy. Micro- 0 0) tubule distribution was examined by using a monoclonal 0. 0) 0.1 0)c anti-a-tubulin antibody (clone DM1A, Sigma) and actin dis- 0.2 tribution was examined by using TRITC-conjugated phalloi- LU din. The Golgi apparatus was visualized by N-[N-(7-nitro- 0.1 2,1,3-benzoxadiazol-4-yl)- E-aminohexanoyl]-D-erythro- 0 sphingosine. (C6-NBD-ceramide) (15) after fixation in 3% 0 1 2 4 8 24 paraformaldehyde. Time (h) Time (h) FIG. 2. Quantification of the time course of the morphological RESULTS effects induced by BFA and nocodazole. Filled bars, 0.5 AM BFA; hatched bars, 10 ,M nocodazole. Values represent means ± SEM. BFA and Cell Morphology. Initial experiments demon- strated that incubation with low concentrations of BFA cell asymmetry (14). Dispersion and elongation indices de- (0.2-1 AM) resulted in dispersion of the Golgi apparatus, as creased during incubation with either 0.5 ,uM BFA or 10 tLM visualized by C6-NBD-ceramide, in Swiss 3T3 fibroblasts. nocodazole (Fig. 2), indicating loss of polarization of cell After several hours of incubation with BFA, cells lost their shape. Asymmetry was restored after removal of BFA or typical fan-like shape with prominent lamellipodial activity at nocodazole (Fig. 2), demonstrating that the effects of these the leading edge (Fig. 1A) and became discoid with a random two drugs were reversible. The loss of asymmetry was not distribution of small lamellipodia over the entire cell periph- due to "rounding" of the cells (i.e., their partial detachment ery (Fig. 1C). In control cells, the Golgi apparatus was from the substratum), since BFA-treated cells, which occu- prominently labeled with C6-NBD-ceramide (Fig. 1B) and, in pied a large area of the substrate, also had a low dispersion many cells, was positioned forward of the nucleus in the index (data not shown) and, moreover, little change was direction of the cell edge which displayed pseudopodial observed in the average projected area ofthe cell. Thus, after activity (16). Disruption of the Golgi apparatus by BFA (Fig. 8 hr, the area occupied by BFA-treated cells was 1382 ± 104 1 B and D) occurred within <1 hr of incubation; by this stage cells was 1265 ± not all cells had lost their polarized morphology (data not ,um2, that occupied by nocodazole-treated shown). However, no discernible Golgi apparatus was ob- 88 1=m2, and that occupied by control cells was 1539 ± 114 served in discoid cells after BFA treatment (Fig. 1D). These Am2. Prolonged incubation with a high concentration of BFA results indicate that disruption ofthe Golgi apparatus by BFA (5 AM) led to a more pronounced decrease in cell area and preceded changes in cell morphology. ultimately to complete rounding of the cells. The effects of BFA on cell morphology were compared BFA and the Cytoskeleton. BFA had no apparent effect on with those induced by the microtubule-disrupting agent no- the radial distribution of microtubules (Fig. 3). In contrast, codazole and quantified by using morphometric indices of significant changes in the actin cytoskeleton were observed dispersion and elongation. These indices reflect the degree of after BFA treatment (Fig.
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