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Localization of the PP2A B56γ Regulatory Subunit at the Golgi Complex

2003; Elsevier BV; Volume: 162; Issue: 2 Linguagem: Inglês

10.1016/s0002-9440(10)63842-4

1525-2191

Akihiko Ito, Yu‐ichiro Koma, Miwa Sohda, Kenji Watabe, Teruaki Nagano, Yoshio Misumi, Hiroshi Nojima, Yukihiko Kitamura,

Lipid Membrane Structure and Behavior

The BL6 subline was derived from the F10 line, which was derived from the B16 mouse melanoma cell line. BL6 cells are more invasive than F10 cells and differ genetically from F10 cells by an alteration of the gene encoding the B56γ regulatory subunit of protein phosphatase 2A (PP2A). This alteration results in the transcription of mRNA encoding a truncated variant of the B56γ1 isoform (Δγ1). When F10 cells were stained with a polyclonal antibody that recognizes three B56γ isoforms, B56γ1, B56γ2, and B56γ3, the immunofluorescent signals co-localized well with the cis-Golgi marker proteins. When BL6 cells were fractionated in a sucrose gradient, B56γ1 and B56γ2, but not B56γ3, were present in the Golgi-enriched fraction. This fraction also contained the catalytic subunit of PP2A. FLAG-tagged Δγ1 preferentially localized to the trans-Golgi area rather than the cis-Golgi. This localization was the same as that of FLAG-tagged B56γ1. NIH3T3 cells stably expressing Δγ1 transported a mutant viral protein from the endoplasmic reticulum to the plasma membrane much faster than wild-type cells. Their directional migration, as assessed by the advance of cells into a cell-free area, was also elevated. As Δγ1 reduces the activity of the B56γ-containing PP2A holoenzymes, these results suggest that the normal holoenzymes suppress vesicle transport and that Δγ1 might increase the invasive ability of BL6 cells by activating Golgi function. The BL6 subline was derived from the F10 line, which was derived from the B16 mouse melanoma cell line. BL6 cells are more invasive than F10 cells and differ genetically from F10 cells by an alteration of the gene encoding the B56γ regulatory subunit of protein phosphatase 2A (PP2A). This alteration results in the transcription of mRNA encoding a truncated variant of the B56γ1 isoform (Δγ1). When F10 cells were stained with a polyclonal antibody that recognizes three B56γ isoforms, B56γ1, B56γ2, and B56γ3, the immunofluorescent signals co-localized well with the cis-Golgi marker proteins. When BL6 cells were fractionated in a sucrose gradient, B56γ1 and B56γ2, but not B56γ3, were present in the Golgi-enriched fraction. This fraction also contained the catalytic subunit of PP2A. FLAG-tagged Δγ1 preferentially localized to the trans-Golgi area rather than the cis-Golgi. This localization was the same as that of FLAG-tagged B56γ1. NIH3T3 cells stably expressing Δγ1 transported a mutant viral protein from the endoplasmic reticulum to the plasma membrane much faster than wild-type cells. Their directional migration, as assessed by the advance of cells into a cell-free area, was also elevated. As Δγ1 reduces the activity of the B56γ-containing PP2A holoenzymes, these results suggest that the normal holoenzymes suppress vesicle transport and that Δγ1 might increase the invasive ability of BL6 cells by activating Golgi function. B16 mouse melanoma cells were originally established from a de novo melanoma tumor.1Fidler IJ Biological behavior of malignant melanoma cells correlated to their survival in vivo.Cancer Res. 1975; 35: 218-224PubMed Google Scholar These cells underwent 10 rounds of in vivo selection to yield the F10 subline, which in turn went through six rounds of in vitro selection to yield the BL6 subline.2Hart IR The selection and characterization of an invasive variant of the B16 melanoma.Am J Pathol. 1979; 97: 587-600PubMed Google Scholar, 3Poste G Doll J Hart IR Fidler IJ In vitro selection of murine B16 melanoma variants with enhanced tissue-invasive properties.Cancer Res. 1980; 40: 1636-1644PubMed Google Scholar Every additional round of selection increased the metastatic potential of the cells. Consequently, although both cells metastasize to the lungs after being injected intravenously into mice, BL6 cells can metastasize to the lungs even after being injected subcutaneously.2Hart IR The selection and characterization of an invasive variant of the B16 melanoma.Am J Pathol. 1979; 97: 587-600PubMed Google Scholar, 3Poste G Doll J Hart IR Fidler IJ In vitro selection of murine B16 melanoma variants with enhanced tissue-invasive properties.Cancer Res. 1980; 40: 1636-1644PubMed Google Scholar When we analyzed the gene expression of the two sublines to identify the differences responsible for the heightened metastatic potential of BL6,4Nakaji T Kataoka TR Watabe K Nishiyama K Nojima H Shimada Y Sato F Matsushima H Endo Y Kuroda Y Kitamura Y Ito A Maeda S A new member of the GTPase superfamily that is upregulated in highly metastatic cells.Cancer Lett. 1999; 147: 139-147Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 5Kataoka TR Ito A Asada H Watabe K Nishiyama K Nakamoto K Itami S Yoshikawa K Ito M Nojima H Kitamura Y Annexin VII as a novel marker for invasive phenotype of malignant melanoma.Jpn J Cancer Res. 2000; 91: 75-83Crossref PubMed Scopus (35) Google Scholar, 6Ito A Kataoka TR Watanabe M Nishiyama K Mazaki Y Sabe H Kitamura Y Nojima H A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation.EMBO J. 2000; 19: 562-571Crossref PubMed Scopus (144) Google Scholar, 7Ito A Katoh F Kataoka TR Okada M Tsubota N Asada H Yoshikawa K Maeda S Kitamura Y Yamasaki H Nojima H A role for heterologous gap junctions between melanoma and endothelial cells in metastasis.J Clin Invest. 2000; 105: 1189-1197Crossref PubMed Scopus (170) Google Scholar, 8Watabe K Ito A Asada H Endo Y Kobayashi T Nakamoto K Itami S Takao S Shinomura Y Aikou T Yoshikawa K Matsuzawa Y Kitamura Y Nojima H Structure, expression and chromosome mapping of MLZE, a novel gene which is preferentially expressed in metastatic melanoma cells.Jpn J Cancer Res. 2001; 92: 140-151Crossref PubMed Scopus (92) Google Scholar, 9Nakamoto K Ito A Watabe K Koma Y Asada H Yoshikawa K Shinomura Y Matsuzawa Y Nojima H Kitamura Y Increased expression of a nucleolar Nop5/Sik family member in metastatic melanoma cells: evidence for its role in nucleolar sizing and function.Am J Pathol. 2001; 159: 1363-1374Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar we found that in BL6 cells, a retrotransposon had been inserted into an intronic region of one allele of the gene encoding the B56γ regulatory subunit of protein phosphatase type 2A (PP2A).6Ito A Kataoka TR Watanabe M Nishiyama K Mazaki Y Sabe H Kitamura Y Nojima H A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation.EMBO J. 2000; 19: 562-571Crossref PubMed Scopus (144) Google Scholar PP2A consists of a series of serine/threonine phosphatase holoenzymes that are composed of a common dimeric core of invariable catalytic (C) and structural (A) subunits associated with a variable regulatory (B) subunit.10Usui H Imazu M Maeta K Tsukamoto H Azuma K Takeda M Three distinct forms of type 2A protein phosphatase in human erythrocyte cytosol.J Biol Chem. 1988; 263: 3752-3761Abstract Full Text PDF PubMed Google Scholar The regulatory subunit is extremely diverse because it is constituted by members from at least three unrelated families, namely, PR55 (or simply B), B56 (B′), and PR72 (B″).11Virshup DM Protein phosphatase 2A: a panoply of enzymes.Curr Opin Cell Biol. 2000; 12: 180-185Crossref PubMed Scopus (294) Google Scholar Each of these families in turn consists of several subfamilies, each of which contains several proteins resembling each other structurally. B56 seems to be the most complex of the three families as its members are encoded by five distinct mammalian genes that produce at least 13 splicing isoforms. One of these genes is B56γ, which is spliced in three different ways to produce the three isoforms belonging to the B56γ subfamily, namely, B56γ1, B56γ2, and B56γ3. In BL6 cells, the 5′ part of the original B56γ gene is replaced with the retrotransposon sequence, which results in the abundant expression of a chimeric mRNA species that encodes a mutant protein, termed Δγ1, that lacks the N-terminal 65 amino acid residues of the B56γ1 isoform.6Ito A Kataoka TR Watanabe M Nishiyama K Mazaki Y Sabe H Kitamura Y Nojima H A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation.EMBO J. 2000; 19: 562-571Crossref PubMed Scopus (144) Google Scholar Okadaic acid (OA) has been used to examine the function of PP2A both in vitro and in vivo. It is a complex fatty acid polyketal12Takai A Bialojan C Troschka M Ruegg JC Smooth muscle myosin phosphatase inhibition and force enhancement by black sponge toxin.FEBS Lett. 1987; 217: 81-84Crossref PubMed Scopus (244) Google Scholar, 13Bialojan C Ruegg JC Takai A Effects of okadaic acid on isometric tension and myosin phosphorylation of chemically skinned guinea-pig taenia coli.J Physiol. 1988; 398: 81-95Crossref PubMed Scopus (61) Google Scholar and inhibits several types of protein phosphatases, including PP1, PP2A, PP4, and PP5.12Takai A Bialojan C Troschka M Ruegg JC Smooth muscle myosin phosphatase inhibition and force enhancement by black sponge toxin.FEBS Lett. 1987; 217: 81-84Crossref PubMed Scopus (244) Google Scholar, 13Bialojan C Ruegg JC Takai A Effects of okadaic acid on isometric tension and myosin phosphorylation of chemically skinned guinea-pig taenia coli.J Physiol. 1988; 398: 81-95Crossref PubMed Scopus (61) Google Scholar, 14Huang X Cheng A Honkanen RE Genomic organization of the human PP4 gene encoding a serine/threonine protein phosphatase (PP4) suggests a common ancestry with PP2A.Genomics. 1997; 44: 336-343Crossref PubMed Scopus (29) Google Scholar, 15Dobson S Kar B Kumar R Adams B Barik S A novel tetratricopeptide repeat (TPR) containing PP5 serine/threonine protein phosphatase in the malaria parasite, plasmodium falciparum.BMC Microbiol. 2001; 1: 31Crossref PubMed Scopus (45) Google Scholar When an intact cell is treated with 0.5 μmol/L of OA, the Golgi complex fragments into numerous clusters of Golgi-derived vesicles and tubules that then disperse in the cytoplasm.16Dinter A Berger EG Golgi-disturbing agents.Histochem Cell Biol. 1998; 109: 571-590Crossref PubMed Scopus (324) Google Scholar, 17Thyberg J Moskalewski S Disorganization of the Golgi complex and the cytoplasmic microtubule system in CHO cells exposed to okadaic acid.J Cell Sci. 1992; 103: 1167-1175PubMed Google Scholar, 18Lucocq J Berger E Hug C The pathway of Golgi cluster formation in okadaic acid-treated cells.J Struct Biol. 1995; 115: 318-330Crossref PubMed Scopus (22) Google Scholar During the Golgi fragmentation, intracellular vesicle transport is arrested.16Dinter A Berger EG Golgi-disturbing agents.Histochem Cell Biol. 1998; 109: 571-590Crossref PubMed Scopus (324) Google Scholar, 19Chou CF Omary MB Mitotic arrest with anti-microtubule agents or okadaic acid is associated with increased glycoprotein terminal GlcNAc's.J Cell Sci. 1994; 107: 1833-1843PubMed Google Scholar Because PP1 and PP2A are the major components of the serine/threonine phosphatase activity in the mammalian cell,20Cohen P Classification of protein-serine/threonine phosphatases: identification and quantification in cell extracts.Methods Enzymol. 1991; 201: 389-398Crossref PubMed Scopus (222) Google Scholar and because OA completely inhibits PP2A and PP1 at 1 nmol/L and 5 μmol/L, respectively,20Cohen P Classification of protein-serine/threonine phosphatases: identification and quantification in cell extracts.Methods Enzymol. 1991; 201: 389-398Crossref PubMed Scopus (222) Google Scholar it is possible that the Golgi fragmentation caused by the OA treatment is because of the OA-induced inhibition of PP2A. In other words, PP2A may be involved in maintaining the morphology of the Golgi complex and in regulating Golgi complex-mediated vesicle transport. However, this possibility has never been closely examined. It is believed that the regulatory subunits of PP2A control PP2A functions by directing particular trimeric PP2A holoenzymes into specific subcellular compartments as well as by enhancing the PP2A phosphatase activity on specific substrates.6Ito A Kataoka TR Watanabe M Nishiyama K Mazaki Y Sabe H Kitamura Y Nojima H A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation.EMBO J. 2000; 19: 562-571Crossref PubMed Scopus (144) Google Scholar, 11Virshup DM Protein phosphatase 2A: a panoply of enzymes.Curr Opin Cell Biol. 2000; 12: 180-185Crossref PubMed Scopus (294) Google Scholar, 21Kamibayashi C Estes R Slaughter C Mumby MC Subunit interactions control protein phosphatase 2A: effects of limited proteolysis, N-ethylmaleimide, and heparin on the interaction of the B subunit.J Biol Chem. 1991; 266: 13251-13260Abstract Full Text PDF PubMed Google Scholar, 22Kamibayashi C Estes R Lickteig RL Yang S-I Craft C Mumby MC Comparison of heterotrimeric protein phosphatase 2A containing different B subunits.J Biol Chem. 1994; 269: 20139-20148Abstract Full Text PDF PubMed Google Scholar Our previous work showed that both B56γ1 and Δγ1 localize in the perinuclear region when they are expressed exogenously as epitope-tagged proteins.6Ito A Kataoka TR Watanabe M Nishiyama K Mazaki Y Sabe H Kitamura Y Nojima H A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation.EMBO J. 2000; 19: 562-571Crossref PubMed Scopus (144) Google Scholar This work also showed that Δγ1 is incapable of promoting the dephosphorylation of specific substrates that is normally mediated by the B56γ subunit-containing PP2A holoenzyme.6Ito A Kataoka TR Watanabe M Nishiyama K Mazaki Y Sabe H Kitamura Y Nojima H A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation.EMBO J. 2000; 19: 562-571Crossref PubMed Scopus (144) Google Scholar Thus, although Δγ1 seems capable of acting as a targeting subunit, it lacks the ability to enhance PP2A activity in a substrate-specific manner. Given that PP2A may be crucially involved in Golgi function, we asked whether B56γ1 and Δγ1 actually localize to the Golgi complex, thereby serving to target the PP2A holoenzymes to the Golgi complex. If so, the expression of Δγ1 instead of B56γ1 may interfere with the normal regulation of Golgi function. This possibility may also explain how the expression of Δγ1 could enhance the metastatic potential of BL6 cells. It may be that B56γ1-containing PP2A holoenzymes normally down-regulate Golgi functions such as the transport of various molecules to their destined sites. Efficient vesicular transport is required for establishment of cell polarity and directional cell migration.23Bershadsky AD Futerman AH Disruption of the Golgi apparatus by brefeldin A blocks cell polarization and inhibits directed cell migration.Proc Natl Acad Sci USA. 1994; 91: 5686-5689Crossref PubMed Scopus (106) Google Scholar, 24Kulkarni SV Gish G van der Geer P Henkemeyer M Pawson T Role of p120 Ras-GAP in directed cell movement.J Cell Biol. 2000; 149: 457-470Crossref PubMed Scopus (135) Google Scholar By interfering with the B56γ1-containing PP2A holoenzymes, Δγ1 may accelerate Golgi-mediated vesicle transport, thereby enhancing the migratory properties of BL6 cells. In the present study, we show that an antibody (Ab) recognizing the endogenous B56γ subunits localizes to the cis-Golgi complex in the interphase. During Golgi fragmentation in mitosis or because of treatment with a microtubule-depolymerizing drug, the B56γ subunit remained associated with the Golgi complex fragments. Of the three isoforms of the B56γ subunit, B56γ1 and B56γ2 were detected in the Golgi-enriched fraction of the cell lysate. When B56γ1 and Δγ1 were transiently expressed as FLAG-tagged proteins, both isoforms localized mainly to the trans-Golgi area rather than the cis-Golgi area. We found that NIH3T3 cells stably expressing Δγ1 transported a viral protein via the Golgi to the plasma membrane faster than wild-type cells and that they were also more proficient in directional migration. These observations suggest that Δγ1 expression might increase the migratory abilities of BL6 cells by disrupting normal Golgi function. B16 melanoma sublines F10 and BL6 were kindly provided by Dr. I. J. Fidler (University of Texas, Houston, TX). NIH3T3 mouse fibroblastic cells, NRK rat kidney cells, and COS-7 monkey kidney cells were purchased from the American Type Culture Collection (Manassas, VA). All cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum. The pCX4bsr vector is derived from the pCXbsr vector25Akagi T Shishido T Murata K Hanafusa H v-Crk activates the phosphoinositide 3-kinase/AKT pathway in transformation.Proc Natl Acad Sci USA. 2000; 97: 7290-7295Crossref PubMed Scopus (126) Google Scholar with a minor modification, and was kindly provided by Dr. T. Akagi (Osaka Bioscience Institute, Osaka, Japan). The AscI-NotI cDNA fragments of Δγ1 or B56γ1 were described previously.6Ito A Kataoka TR Watanabe M Nishiyama K Mazaki Y Sabe H Kitamura Y Nojima H A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation.EMBO J. 2000; 19: 562-571Crossref PubMed Scopus (144) Google Scholar The pCX4bsr vector was digested with EcoRI, blunted with T4 DNA polymerase, digested with NotI, and ligated with the AscI-NotI cDNA fragment of Δγ1 or B56γ1 that had been blunted with T4 DNA polymerase at its 5′ end (pCX4bsr-Δγ1 or pCX4bsr-B56γ1). Using the AscI-NotI cDNA fragment of Δγ1 or B56γ1 as a template, polymerase chain reaction was performed using the following primers: sense, 5′-GGCCTGCGTGCTTACATCAGGAAACAGA-3′; anti-sense, 5′-ACGGTACCCCGCAACACTCCCGAGTTACTCTCTTTTTTAG-3′. The anti-sense primer contains a mutation at the third nucleotide of the stop codon of Δγ1 or B56γ1 (TGA to TGC) and a KpnI site at its 3′ end. The 3′ part of the polymerase chain reaction-amplified cDNA fragment was ligated with the 5′ part of the AscI-NotI cDNA fragment at the EcoNI site, and then digested with KpnI. The resulting AscI-KpnI cDNA fragment was inserted into the multiple cloning site of the p3XFLAG-CMV-14 expression vector (Sigma Chemical Co., St Louis, MO) in which a synthetic linker carrying an AscI site (AGCTTGGCGCGCCA) had been inserted into the HindIII site. The plasmid constructs allowed expression of Δγ1 or B56γ1 tagged with FLAG at their C-termini. NIH3T3 cells were transfected with pCX4bsr-Δγ1 or empty pCX4bsr vector by using the Fugene 6 transfection regent (Roche Diagnostics Co. Ltd., Indianapolis, IN) according to the manufacturer's instructions. After transfection, cells were selected by resistance to blasticidin (3 μg/ml; Invitrogen, Carlsbad, CA) for 3 weeks to obtain single colonies. For transient expression of exogenous FLAG-tagged proteins, similar procedures were performed using p3XFLAG-CMV-14-Δγ1 or p3XFLAG-CMV-14-B56γ1 vector. After culturing for 24 hours, cells were processed for immunofluorescence. Two Abs specific for the B56γ subunits were used. Their production has been described previously.6Ito A Kataoka TR Watanabe M Nishiyama K Mazaki Y Sabe H Kitamura Y Nojima H A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation.EMBO J. 2000; 19: 562-571Crossref PubMed Scopus (144) Google Scholar Briefly, Δγ1 and the B56γ isoforms B56γ1 and B56γ2 were produced as recombinant proteins as described previously,6Ito A Kataoka TR Watanabe M Nishiyama K Mazaki Y Sabe H Kitamura Y Nojima H A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation.EMBO J. 2000; 19: 562-571Crossref PubMed Scopus (144) Google Scholar and were used to immunize rabbits. Affinity-purified IgG fractions from the rabbit sera were examined for their specificity by Western blot analysis as described below. Anti-pan-B56γ Ab was derived from the rabbit immunized with recombinant B56γ1, whereas anti-B56γ1/2 Ab was from the rabbit immunized with a mixture of recombinant B56γ1, B56γ2, and Δγ1. Other primary Abs used are specific for giantin (polyclonal Ab26Misumi Y Sohda M Tashiro A Sato H Ikehara Y An essential cytoplasmic domain for the Golgi localization of coiled-coil proteins with a COOH-terminal membrane anchor.J Biol Chem. 2001; 276: 6867-6873Crossref PubMed Scopus (42) Google Scholar), GM130 (clone 35; Transduction Laboratories, Lexington, KY), Rab8 (clone 4, Transduction Laboratories), p115 (clone 46, Transduction Laboratories), Golgi 58K (58K-8, Sigma), the PP2A C subunit (clone 46, Transduction Laboratories), syntaxin-6 (clone 30, Transduction Laboratories), calnexin (clone 37, Transduction Laboratories), lamin B (M-20; Santa Cruz Biotechnology, Santa Cruz, CA), α-tubulin (DM 1A, Sigma), and FLAG (polyclonal Ab, Sigma). Secondary Abs used are the peroxidase-labeled anti-rabbit or anti-mouse IgG Abs (MBL Co. Ltd., Nagoya, Japan), Cy2- and Cy5-labeled anti-rabbit IgG Abs (Jackson ImmunoResearch, West Grove, PA), and Cy2-, Cy3-, and Cy5-labeled anti-mouse IgG Abs (Jackson ImmunoResearch). Cells were grown on coverslips in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. In some cases, cells were incubated with nocodazole (5 μg/ml) for 2 hours at 37°C before fixation. After washing with phosphate-buffered saline (PBS), cells were fixed and mildly permeabilized with methanol at −20°C for 10 minutes. No further permeabilization of cells was performed with disruptive agents such as Triton X-100. Cells were blocked with 2% bovine serum albumin (fraction V, Sigma) in PBS and then incubated in the blocking solution with anti-B56γ1/2 Ab at 1:400 dilution (the approximate protein concentration is 0.6 μg/ml), another Ab as indicated at 1:500 dilution, or both. Control experiments included two procedures. One was that after blocking, cells were incubated with preimmune rabbit serum at 1:20 dilution instead of the anti-B56γ1/2 Ab. The second involved diluting the anti-B56γ1/2 Ab at 1:400 in the blocking solution and adding this to a tube containing the recombinant B56γ2 protein at a concentration of 0.1 to 6.0 μg/ml. After leaving the tube for 1 hour at room temperature, the mixture was poured onto the coverslips bearing the cells that had just been blocked with 2% bovine serum albumin. The reaction with the primary Abs or preimmune serum was continued for 2 hours at 4°C. After washing with PBS, the cells were stained with a mixture of Cy5-labeled anti-rabbit IgG Ab and Cy2-labeled anti-mouse IgG Ab. To visualize DNA, after the cells had been incubated with the primary Abs, they were incubated with 0.5 mg/ml of DNase-free RNase for 1 hour in the blocking solution. The cells were then stained with a mixture of Cy5-labeled anti-rabbit IgG Ab, Cy3-labeled anti-mouse IgG Ab, and the nucleic acid stain SYTO13 (500 nmol/L; Molecular Probes, Eugene, OR). Cells were visualized using a confocal laser-scanning microscope (LSM510; Carl Zeiss, OberKochen, Germany). NIH3T3 cells were plated onto coverslips and the following day, cells growing in log phase were incubated in the presence of 2.5 mmol/L of thymidine for 20 hours to arrest the cells at the G1/S phase. Cells were then incubated for 10 hours in fresh medium without thymidine to promote the growth of unarrested cells. Subsequently, 5 μg/ml of aphidicolin was added to the medium and the incubation was continued for 15 hours. Cells were washed several times to remove the drug and incubated in fresh medium for 6 to 9 hours to allow progression into mitosis. Cells on coverslips were stained as described above. BL6 cells were homogenized in 0.25 mol/L of sucrose and protease inhibitor mixture with a nitrogen bombardment apparatus (Parr Instrument Co. Ltd., Moline, IL.), followed by centrifugation at 1000 × g for 10 minutes. The supernatant was separated by the method of Balch and colleagues27Balch WE Dunphy WG Braell WA Rothman JE Reconstitution of the transport of protein between successive compartments of the Golgi measured by the coupled incorporation of N-acetylglucosamine.Cell. 1984; 39: 405-416Abstract Full Text PDF PubMed Scopus (553) Google Scholar to obtain the Golgi fraction. Briefly, the postnuclear supernatant was adjusted to 1.4 mol/L of sucrose by the addition of ice-cold 2.3-mol/L sucrose containing 10 mmol/L Tris-HCl and 1 mmol/L ethylenediaminetetraacetic acid. It was then loaded into a SW28 tube and overlaid with 1.2 mol/L and 0.8 mol/L of sucrose containing 10 mmol/L of Tris-HCl. The gradients were centrifuged at 90,000 × g for 2.5 hours in the SW28 rotor. The turbid band at the 0.8/12-mol/L sucrose interface was harvested by syringe puncture. NIH3T3 cells were grown in one 150-mm dish to 90% confluency. The cells were washed four times with a prechilled solution containing 10 mmol/L Tris (pH 7.5) and 0.25 mol/L sucrose and then scraped from the plates. The cells in an ∼1-ml solution were supplemented with 10 μl of protease inhibitor cocktail (Sigma) and passed through a 25G3/4 needle 15 times on ice. The cell lysate was centrifuged at 1500 × g for 10 minutes at 4°C. The resulting postnuclear supernatant was mixed with 2 mol/L of sucrose to a final concentration of 1.6 mol/L of sucrose. The mixture (∼2 ml) was loaded at the bottom of a centrifuge tube and overlaid with 1.3, 1.0, and 0.8 sucrose in 10 mmol/L of Tris (pH 7.5) (2.5 ml of each sucrose density), and 0.5 mol/L of sucrose (1.5 ml). Equilibrium centrifugation was performed with a RPS40T rotor (Hitachi, Tokyo, Japan) at 135,000 × g for 14 hours at 4°C. Fractions (∼1 ml per fraction) were collected from the top. Cells were lysed in buffer containing 10 mmol/L Tris-HCl (pH 8.0), 1 mmol/L ethylenediaminetetraacetic acid, 0.5% Nonidet P-40, and 1 mmol/L phenylmethylsulfonyl fluoride. Equal quantities of cell lysates or fractionated cell extracts were denatured in sodium dodecyl sulfate gel loading buffer, separated on 10% sodium dodecyl sulfate-acrylamide gels, transferred to Immobilon (Millipore, Bedford, MA), and reacted with the primary Ab indicated. After washing, the blots were incubated with peroxidase-labeled secondary Ab and then reacted with Renaissance reagents (NEN, Boston, MA) before exposure. The expression plasmid pCDM8.1 vector (Invitrogen) encoding VSVG from the temperature-sensitive (ts) 045 mutant strain fused with the enhanced GFP (Clontech, Palo Alto, CA) at its C-terminus (VSVG-GFP)28Presley JF Cole NB Schroer TA Hirschberg K Zaal KJ Lippincott-Schwartz J ER-to-Golgi transport visualized in living cells.Nature. 1997; 389: 81-85Crossref PubMed Scopus (3) Google Scholar was kindly provided by Dr. J. Lippincott-Schwartz (National Institutes of Health, Bethesda, MD). Cells were transiently transfected with pCDM8.1-VSVG-GFP using the Fugene 6 transfection reagent (Roche Diagnostics). After transfection, the cells were cultured at 40°C for 16 hours. Thirty minutes before shifting the temperature from 40°C to 32°C, 100 μg/ml of cycloheximide was added to each dish according to the methods of Toomre and colleagues.29Toomre D Keller P White J Olivo JC Simons K Dual-color visualization of trans-Golgi network to plasma membrane traffic along microtubules in living cells.J Cell Sci. 1999; 112: 21-33Crossref PubMed Google Scholar The cells were subsequently fixed at the indicated times after the temperature shift and then processed for fluorescence microscopy and quantification as follows. Methanol-fixed cells were incubated with the anti-p115 Ab and stained with Cy5-labeled anti-mouse IgG Ab to define the Golgi area. Images were created on the computer monitor of a confocal laser-scanning microscope (LSM510) through a filter set suitable for detecting fluorescein and Cy5 signals. The fluorescence intensity of VSVG-GFP in the Golgi area was measured with an image processing system of LSM510. Using the histogram tools in this system, the Cy5-labeled Golgi area and the whole cell area were measured in a unit of μm2 by delineating the margins of both areas with a digital cursor. The mean GFP fluorescence intensities in both areas were also measured in arbitrary units and the mean background fluorescence intensity was subtracted from both values. The corrected mean intensities multiplied by the delineated areas yielded the total GFP fluorescence intensities in the Golgi area and in the whole cell area. Proportions of the former to the latter were then calculated. The proportion at time 0 was expressed as 1. Proportions at other time points were converted into values relative to the proportion at time 0. We examined 25 cells at each time point and calculated the mean and SD. Experiments were repeated three times with similar results. The wound closure assay was performed according to the methods of Goodman and colleagues30Goodman SL Vollmers HP Birchmeier W Control of cell locomotion: perturbation with an antibody directed against specific glycoproteins.Cell. 1985; 41: 1029-1038Abstract Full Text PDF PubMed Scopus (47) Google Scholar Cells were cultured in 12-well plates in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. The initial plating was adjusted after 24 hours to yield subconfluent monolayers at the same cell density. The monolayers were wounded by scratching the bottom of the culture plates with a plastic scraper so that a cross with 1-mm-long lines was made. Phase contrast images of the wounded monolayers were photographed with a camera of a Diaphot microscope (Nikon, Tokyo, Japan). After washing, the monolayers were incubated for 10 hours in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and then photographed again. By using the center point of the wound cross as a guide, two photos taken before and after the 10-hour incubation were compared. A Coolscan III film scanner (Nikon) was used to create photo images on a monitor of a Macintosh computer (Power Mac G4) equipped with an image processing software (Adobe Photoshop Ver 5.0; Adobe System Inc., Mountain View, CA) and a digital cursor. On the monitor, the width of the initial wounds was ∼5 cm.