Advertisement

Histochemistry and Cell Biology

, Volume 122, Issue 2, pp 95–109 | Cite as

The hypolipidemic compound cetaben induces changes in Golgi morphology and vesicle movement

  • Werner J. Kovacs
  • Michael Schrader
  • Ingrid Walter
  • Herbert StanglEmail author
Original Paper

Abstract

The human hepatoma cell line HepG2 was used to study the effect of cetaben, a non-fibrate hypolipidemic drug, on cell morphology and vesicle distribution. Cetaben treatment correlated with a fragmentation and/or condensation of Golgi cisternae and the appearance of large electron-lucent vesicles. The Golgi apparatus, demonstrated, for example, by fluorescence-lectin histochemistry, was fragmented after cetaben treatment. The lectin-positive remnants were dispersed throughout the cytoplasm, but with a preference for being transported to tips of cells. However, microtubules and the intermediate filaments as well as the actin microfilaments were unchanged after cetaben treatment indicating that changes in Golgi morphology are not caused by alterations in the cytoskeleton. Cetaben decreases the cholesterol content due to inhibition of cholesterol biosynthesis. Changes in the intracellular cholesterol content are known to influence the intracellular vesicle distribution and are most likely responsible for cetaben-induced Golgi alterations, as depletion of cellular cholesterol by starvation or lovastatin and/or cyclodextrin treatment resulted in a similar redistribution of Golgi-derived wheat germ agglutinin vesicles. These lectin-stained vesicles colocalized with lysosomal marker proteins such as Limp-1 and Lamp-2, but not with the early endosomal markers Rab5 and EEA1. Upon removal of cetaben the lectin- and Limp-1/Lamp-2-costained vesicles dissociated and were transported back to the perinuclear region. Thus, cetaben-induced changes such as fragmentation of the Golgi apparatus and the dispersion of lysosomes away from their juxtanuclear location were reversible.

Keywords

Cetaben Golgi apparatus Peroxisomes Lysosomes HepG2 Cholesterol 

Notes

Acknowledgements

We are grateful to W. Tschulenk, N. Kielhausen, and B. Machac for technical assistance. Antibodies to catalase, PMP70, ER HMG-CoA reductase, IPP isomerase, and squalene synthase kindly provided by Drs. A. Voelkl, P. Edwards, S. Krisans, and. I. Shechter. This project was supported in part by OENB project number 8198 and Anton Dreher Gedächtnisschenkung number 318.

Supplementary material

supp.pdf (401 kb)
(PDF 402 KB)

References

  1. Albright JD, DeVries VG, Largis EE, Miner TG, Reich MF, Schaffer SA, Shepherd RG, Upeslacis J (1983) Potential antiatherosclerotic agents. 2. (Aralalkylamino)- and (alkylamino) benzoic acid analogues of cetaben. J Med Chem 26:1378–1393PubMedGoogle Scholar
  2. Allan VJ, Schroer TA (1999) Membrane motors. Curr Opin Cell Biol 11:476–482CrossRefPubMedGoogle Scholar
  3. Angermüller S, Fahimi HD (1981) Selective cytochemical localization of peroxidase, cytochrome oxidase and catalase in rat liver with 3,3′-diaminobenzidine. Histochemistry 71:33–44PubMedGoogle Scholar
  4. Beier K, Völkl A, Hashimoto T, Fahimi HD (1988) Selective induction of peroxisomal enzymes by the hypolipidemic drug bezafibrate. Detection of modulations by automatic image analysis in conjunction with immunoelectron microscopy and immunoblotting. Eur J Cell Biol 46:383–393PubMedGoogle Scholar
  5. Brown MS, Goldstein JL (1999) A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood. Proc Natl Acad Sci U S A 96:11041–11048CrossRefPubMedGoogle Scholar
  6. Burkhardt JK, Echeverri CJ, Nilsson T, Vallee RB (1997) Overexpression of the dynamitin (p50) subunit of the dynactin complex disrupts dynein-dependent maintenance of membrane organelle distribution. J Cell Biol 139:469–484CrossRefPubMedGoogle Scholar
  7. Chandoga J, Rojekova I, Hampl L, Hocman G (1994a) Cetaben and fibrates both influence the activities of peroxisomal enzymes in different ways. Biochem Pharm 47:515–519CrossRefPubMedGoogle Scholar
  8. Chandoga J, Hampl L, Turecky L, Rojekova I, Uhlikova E, Hocman G (1994b) Cetaben is an exceptional type of peroxisome proliferator. Int J Biochem 26:679–696CrossRefPubMedGoogle Scholar
  9. Cole NB, Lippincott-Schwartz J (1995) Organization of organelles and membrane traffic by microtubules. Curr Opin Cell Biol 7:55–64CrossRefPubMedGoogle Scholar
  10. de Figueiredo P, Brown WJ (1999) Clofibrate inhibits membrane trafficking to the Golgi complex and induces its retrograde movement to the endoplasmic reticulum. Cell Biol Toxicol 15:311–323CrossRefPubMedGoogle Scholar
  11. de Pace DM, Esfahani M (1987) The effects of cholesterol depletion on cellular morphology. Anat Rec 219:135–143PubMedGoogle Scholar
  12. Duden R, Griffiths G, Frank R, Argos P, Kreis TE (1991) Beta-COP, a 110 kd protein associated with non-clathrin-coated vesicles and the Golgi complex, shows homology to beta-adaptin. Cell 64:649–665CrossRefPubMedGoogle Scholar
  13. Engfelt WH, Shackelford JE, Aboushadi N, Jessani N, Masuda K, Paton VG, Keller GA, Krisans SK (1997) Characterization of UT2 cells. The induction of peroxisomal 3-hydroxy-3-methylglutaryl coenzyme A reductase. J Biol Chem 272:24579–24587CrossRefPubMedGoogle Scholar
  14. Eskelinen E-L, Tanaka Y, Saftig P (2003) At the acidic edge: emerging functions for lysosomal membrane proteins. Trends Cell Biol 13:137–145CrossRefPubMedGoogle Scholar
  15. Farquhar MG, Palade GE (1998) The Golgi apparatus: 100 years of progress and controversy. Trends Cell Biol 8:2–10CrossRefPubMedGoogle Scholar
  16. Gaynor EC, Graham TR, Emr SD (1998) COPI in ER/Golgi and intra-Golgi transport: do yeast COPI mutants point the way? Biochim Biophys Acta 1404:33–51CrossRefPubMedGoogle Scholar
  17. Gorvel JP, Chavrier P, Zerial M, Gruenberg J (1991) Rab5 controls early endosome fusion in vitro. Cell 64:915–925CrossRefPubMedGoogle Scholar
  18. Grimmer S, Iversen TG, van Deurs B, Sandvig K (2000) Endosome to Golgi transport of ricin is regulated by cholesterol. Mol Biol Cell 11:4205–4216PubMedGoogle Scholar
  19. Hansen GH, Niels-Christiansen L-L, Thorsen E, Immerdal L, Danielsen EM (2000) Cholesterol depletion of enterocytes. J Biol Chem 275:5136–5142CrossRefPubMedGoogle Scholar
  20. Hirschberg K, Lippincott-Schwartz J (1999) Secretory pathway kinetics and in vivo analysis of protein traffic from the Golgi complex to the cell surface. FASEB J 13(suppl 2):S251–S256Google Scholar
  21. Ho WC, Allan VJ, Van Meer G, Berger EG, Kreis TE (1989) Reclustering of scattered Golgi elements occurs along microtubules. Eur J Cell Biol 48:250–263PubMedGoogle Scholar
  22. Ihrke G, Neufeld EB, Meads T, Shanks MR, Cassio D, Laurent M, Schroer TA, Pagano RE, Hubbard AL (1993) WIF-B cells: an in vitro model for studies of hepatocyte polarity. J Cell Biol 123:1761–1775CrossRefPubMedGoogle Scholar
  23. Ihrke G, Martin GV, Shanks MR, Schrader M, Schroer TA, Hubbard AL (1998) Apical plasma membrane proteins and endolyn-78 travel through a subapical compartment in polarized WIF-B hepatocytes. J Cell Biol 141:115–133CrossRefPubMedGoogle Scholar
  24. Keller P, Simons K (1998) Cholesterol is required for surface transport of influenza virus hemagglutinin. J Cell Biol 140:1357–1367CrossRefPubMedGoogle Scholar
  25. Klausner RD, Donaldson JG, Lippincott-Schwartz J (1992) Brefeldin A: insights into the control of membrane traffic and organelle structure. J Cell Biol 116:1071–1080CrossRefPubMedGoogle Scholar
  26. Kovacs W, Walter I, Stangl H (2001) Cetaben-induced changes on the morphology and peroxisomal enzymes in MH1C1 rat hepatoma cells and HepG2 human hepatoblastoma cells. Histochem Cell Biol 115:509–519PubMedGoogle Scholar
  27. Kreis TE (1990) Role of microtubules in the organisation of the Golgi apparatus. Cell Motil Cytoskeleton 15:67–70PubMedGoogle Scholar
  28. Krisans SK, Ericsson J, Edwards PA, Keller GA (1994) Farnesyl-diphosphate synthase is localized in peroxisomes. J Biol Chem 269:14165–14169PubMedGoogle Scholar
  29. Laemmli UK (1970) Cleavage of the structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedGoogle Scholar
  30. Linstedt AD, Hauri HD (1993) Giantin, a novel conserved Golgi membrane protein containing a cytoplasmic domain of at least 350 kDa. Mol Biol Cell 4:679–693PubMedGoogle Scholar
  31. Locke M, Huie P (1983) The mystery of the unstained Golgi complex cisternae. J Histochem Cytochem 31:1019–1032PubMedGoogle Scholar
  32. Mayor S, Sabharanjak S, Maxfield FR (1998) Cholesterol-dependent retention of GPI-anchored proteins in endosomes. EMBO J 17:4626–4638CrossRefPubMedGoogle Scholar
  33. Miwako I, Yamamoto A, Kitamura T, Nagayama K, Ohashill M (2001) Cholesterol requirement for cation-independent mannose 6-phosphate receptor exit from multivesicular late endosomes to the Golgi. J Cell Sci 114:1765–1776PubMedGoogle Scholar
  34. Mu FT, Callaghan JM, Steele-Mortimer O, Stenmark H, Parton RG, Campbell PL, McCluskey J, Yeo JP, Tock EP, Toh BH (1995) EEA1, an early endosome-associated protein. EEA1 is a conserved alpha-helical peripheral membrane protein flanked by cysteine “fingers” and contains a calmodulin-binding IQ motif. J Biol Chem 270:13503–13511CrossRefPubMedGoogle Scholar
  35. Paton VG, Shackelford JE, Krisans SK (1997) Cloning and subcellular localization of hamster and rat isopentenyl diphosphate dimethylallyl diphosphate isomerase. J Biol Chem 272:18945–18950CrossRefPubMedGoogle Scholar
  36. Pecot MY, Malhotra V (2004) Golgi membranes remain segregated from the endoplasmic reticulum during mitosis in mammalian cells. Cell 116:99–107CrossRefPubMedGoogle Scholar
  37. Pelham HR (1996) The dynamic organisation of the secretory pathway. Cell Struct Funct 21:413–419PubMedGoogle Scholar
  38. Ponnambalam S, Girotti M, Yaspo ML, Owen CE, Perry AC, Suganuma T, Nilsson T, Fried M, Banting G, Warren G (1996) Primate homologues of rat TGN38: primary structure, expression and functional implications. J Cell Sci 109:675–685PubMedGoogle Scholar
  39. Prescott AR, Lucocq JM, James J, Lister JM, Ponnambalam S (1997) Distinct compartmentalization of TGN46 and beta 1,4-galactosyltransferase in HeLa cells. Eur J Cell Biol 72:238–246PubMedGoogle Scholar
  40. Presley JF, Cole NB, Schroer TA, Hirschberg K, Zaal KJ, Lippincott-Schwartz J (1997) ER-to-Golgi transport visualized in living cells. Nature 389:81–85CrossRefPubMedGoogle Scholar
  41. Rapp S, Saffrich R, Anton M, Jäkle U, Ansorge W, Gorgas K, Just WW (1996) Microtubule-based peroxisome movement. J Cell Sci 109:837–849PubMedGoogle Scholar
  42. Rogalski AA, Singer SJ (1984) Associations of elements of the Golgi apparatus with microtubules. J Cell Biol 99:1092–1100CrossRefPubMedGoogle Scholar
  43. Sandoval IV, Bonifacino JS, Klausner RD, Henkart M, Wehland J (1984) Role of microtubules in the organization and localization of the Golgi apparatus. J Cell Biol 99:113s–118sCrossRefPubMedGoogle Scholar
  44. Schmidt K, Schrader M, Kern HF, Kleene R (2001) Regulated apical secretion of zymogens in rat pancreas. Involvement of the glycosylphosphatidylinositol-anchored glycoprotein GP-2, the lectin ZG16p, and cholesterol-glycosphingolipid-enriched microdomains. J Biol Chem 276:14315–14323PubMedGoogle Scholar
  45. Schrader M, Baumgart E, Völkl A, Fahimi HD (1994) Heterogeneity of peroxisomes in human hepatoblastoma cell line HepG2. Evidence of distinct subpopulations. Eur J Cell Biol 64:281–294PubMedGoogle Scholar
  46. Schrader M, Burkhardt JK, Baumgart E, Lüers G, Spring H, Völkl A, Fahimi HD (1996) Interaction of microtubules with peroxisomes. Tubular and spherical peroxisomes in HepG2 cells and their alterations induced by microtubule-active drugs. Eur J Cell Biol 69:24–35PubMedGoogle Scholar
  47. Schrader M, King SJ, Stroh TA, Schroer TA (2000) Real time imaging reveals a peroxisomal reticulum in living cells. J Cell Sci 113:3663–3671PubMedGoogle Scholar
  48. Shanks MR, Cassio D, Lecoq O, Hubbard AL (1994) An improved polarized rat hepatoma hybrid cell line. Generation and comparison with its hepatoma relatives and hepatocytes in vivo. J Cell Sci 107:813–825PubMedGoogle Scholar
  49. Shechter I, Klinger E, Rucker ML, Engstrom RG, Spirito JA, Islam MA, Boettcher BR, Weinstein DB (1992) Solubilization, purification, and characterization of a truncated form of rat hepatic squalene synthetase. J Biol Chem 267:8628–8635PubMedGoogle Scholar
  50. Stamellos KD, Shackelford JE, Shechter I, Jiang G, Conrad D, Keller GA, Krisans SK (1993) Subcellular localization of squalene synthase in rat hepatic cells. J Biol Chem 268:12825–12836PubMedGoogle Scholar
  51. Stangl H, Kovacs W, Böck P, Kremser K (1995) Differential induction of peroxisomal enzymes by hypolipidemics in human (HepG2) and rat (MH1C1) hepatoma cell lines. Eur J Clin Chem Clin Biochem 33:775–783PubMedGoogle Scholar
  52. Tanaka RD, Edwards PA, Lan SF, Fogelman AM (1983) Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in avian myoblasts. Mode of action of 25-hydroxycholesterol. J Biol Chem 258:13331–13339PubMedGoogle Scholar
  53. Thyberg J, Moskalewski S (1985) Microtubules and the organization of the Golgi complex. Exp Cell Res 159:1–16PubMedGoogle Scholar
  54. Turner JR, Tartakoff AM (1989) The response of the Golgi complex to microtubule alterations: the roles of metabolic energy and membrane traffic in Golgi complex organization. J Cell Biol 109:2081–2088CrossRefPubMedGoogle Scholar
  55. Vaisberg EA, Grissom PM, McIntosh JR (1996) Mammalian cells express three distinct dynein heavy chains that are localized to different cytoplasmic organelles. J Cell Biol 133:831–842CrossRefPubMedGoogle Scholar
  56. Ward TH, Polishchuk RS, Caplan S, Hirschberg K, Lippincott-Schwartz J (2001) Maintenance of Golgi structure and function depends on the integrity of ER export. J Cell Biol 155:557–570CrossRefPubMedGoogle Scholar
  57. Warren G, Levine T, Misteli T (1995) Mitotic disassembly of the mammalian Golgi apparatus. Trends Cell Biol 5:413–416CrossRefPubMedGoogle Scholar
  58. Wells WA (2001) Let’s make Golgi. J Cell Biol 155:498–499CrossRefPubMedGoogle Scholar
  59. Wiemer EA, Wenzel T, Deerinck TJ, Ellisman MH, Subramani S (1997) Visualization of the peroxisomal compartment in living mammalian cells: dynamic behavior and association with microtubules. J Cell Biol 136:71–80CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Werner J. Kovacs
    • 1
    • 4
  • Michael Schrader
    • 2
  • Ingrid Walter
    • 3
  • Herbert Stangl
    • 1
    Email author
  1. 1.Institute of Medical ChemistryUniversity of ViennaViennaAustria
  2. 2.Department of Cytobiology and CytopathologyUniversity of MarburgMarburgGermany
  3. 3.Institute of Histology and EmbryologyVeterinarian University of ViennaViennaAustria
  4. 4.Department of BiologySan Diego State UniversitySan DiegoUSA

Personalised recommendations