Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Mar 1;108(3):997–1008. doi: 10.1083/jcb.108.3.997

Concurrent collapse of keratin filaments, aggregation of organelles, and inhibition of protein synthesis during the heat shock response in mammary epithelial cells

PMCID: PMC2115390  PMID: 2466040

Abstract

The sequence of heat shock-induced perturbations in protein synthesis and cytoskeletal organization was investigated in primary cultures of mouse mammary epithelial cells (MMEC). Exposure of the cells to 45 degrees C for 15 min caused a marked inhibition of protein synthesis through 2 h after heart. Resumption of protein synthesis began by 4 h, was complete by 8 h, and was accompanied by induction of four major heat shock proteins (HSPs) of 68, 70, 89, and 110 kD. Fluorescent cytochemistry studies indicated that heat shock elicited a reversible change in the organization of keratin filaments (KFs) and actin filaments but had a negligible effect on microtubules. Changes in the organization of KFs progressed gradually with maximal retraction and collapse into the perinuclear zone occurring at 1-2 h after heat followed by restoration to the fully extended state at 8 h. In contrast, actin filaments disappeared immediately after heat treatment and then rapidly returned within 30-60 min to their original appearance. The translocation of many organelles first into and then away from the juxtanuclear area along with the disruption and reformation of polyribosomes were concurrent with the sequential changes in distribution of KFs. The recovery of the arrangement of KFs coincided with but was independent of the resumption of protein synthesis and induction of HSPs. Thermotolerance could be induced in protein synthesis and KFs, but not in actin filaments, by a conditioning heat treatment. Neither protein synthesis nor induction of HSPs was necessary for the acquisition of thermotolerance in the KFs. The results are compatible with the possibility that protein synthesis may depend on the integrity of the KF network in MMEC. Heat shock thus can efficiently disarrange the KF system in a large population of epithelial cells, thereby facilitating studies on the functions of this cytoskeletal component.

Full Text

The Full Text of this article is available as a PDF (4.9 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Asch B. B., Burstein N. A., Vidrich A., Sun T. T. Identification of mouse mammary epithelial cells by immunofluorescence with rabbit and guinea pig antikeratin antisera. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5643–5647. doi: 10.1073/pnas.78.9.5643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Asch B. B., Medina D. Concanavalin A-induced agglutinability of normal, preneoplastic, and neoplastic mouse mammary cells. J Natl Cancer Inst. 1978 Dec;61(6):1423–1430. [PubMed] [Google Scholar]
  3. Asch H. L., Asch B. B. Heterogeneity of keratin expression in mouse mammary hyperplastic alveolar nodules and adenocarcinomas. Cancer Res. 1985 Jun;45(6):2760–2768. [PubMed] [Google Scholar]
  4. Ashburner M., Bonner J. J. The induction of gene activity in drosophilia by heat shock. Cell. 1979 Jun;17(2):241–254. doi: 10.1016/0092-8674(79)90150-8. [DOI] [PubMed] [Google Scholar]
  5. Ball E. H., Singer S. J. Mitochondria are associated with microtubules and not with intermediate filaments in cultured fibroblasts. Proc Natl Acad Sci U S A. 1982 Jan;79(1):123–126. doi: 10.1073/pnas.79.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cervera M., Dreyfuss G., Penman S. Messenger RNA is translated when associated with the cytoskeletal framework in normal and VSV-infected HeLa cells. Cell. 1981 Jan;23(1):113–120. doi: 10.1016/0092-8674(81)90276-2. [DOI] [PubMed] [Google Scholar]
  7. Chen L. B., Summerhayes I. C., Johnson L. V., Walsh M. L., Bernal S. D., Lampidis T. J. Probing mitochondria in living cells with rhodamine 123. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 1):141–155. doi: 10.1101/sqb.1982.046.01.018. [DOI] [PubMed] [Google Scholar]
  8. Collier N. C., Schlesinger M. J. The dynamic state of heat shock proteins in chicken embryo fibroblasts. J Cell Biol. 1986 Oct;103(4):1495–1507. doi: 10.1083/jcb.103.4.1495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Collot M., Louvard D., Singer S. J. Lysosomes are associated with microtubules and not with intermediate filaments in cultured fibroblasts. Proc Natl Acad Sci U S A. 1984 Feb;81(3):788–792. doi: 10.1073/pnas.81.3.788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Coss R. A., Dewey W. C., Bamburg J. R. Effects of hyperthermia on dividing Chinese hamster ovary cells and on microtubules in vitro. Cancer Res. 1982 Mar;42(3):1059–1071. [PubMed] [Google Scholar]
  11. Drummond I. A., McClure S. A., Poenie M., Tsien R. Y., Steinhardt R. A. Large changes in intracellular pH and calcium observed during heat shock are not responsible for the induction of heat shock proteins in Drosophila melanogaster. Mol Cell Biol. 1986 May;6(5):1767–1775. doi: 10.1128/mcb.6.5.1767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Eckert B. S. Alteration of intermediate filament distribution in PtK1 cells by acrylamide. Eur J Cell Biol. 1985 May;37:169–174. [PubMed] [Google Scholar]
  13. Eckert B. S. Alteration of the distribution of intermediate filaments in PtK1 cells by acrylamide. II: Effect on the organization of cytoplasmic organelles. Cell Motil Cytoskeleton. 1986;6(1):15–24. doi: 10.1002/cm.970060104. [DOI] [PubMed] [Google Scholar]
  14. Eckert B. S., Yeagle P. L. Acrylamide treatment of PtK1 cells causes dephosphorylation of keratin polypeptides. Cell Motil Cytoskeleton. 1988;11(1):24–30. doi: 10.1002/cm.970110104. [DOI] [PubMed] [Google Scholar]
  15. Falkner F. G., Saumweber H., Biessmann H. Two Drosophila melanogaster proteins related to intermediate filament proteins of vertebrate cells. J Cell Biol. 1981 Oct;91(1):175–183. doi: 10.1083/jcb.91.1.175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fey E. G., Wan K. M., Penman S. Epithelial cytoskeletal framework and nuclear matrix-intermediate filament scaffold: three-dimensional organization and protein composition. J Cell Biol. 1984 Jun;98(6):1973–1984. doi: 10.1083/jcb.98.6.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Franke W. W., Hergt M., Grund C. Rearrangement of the vimentin cytoskeleton during adipose conversion: formation of an intermediate filament cage around lipid globules. Cell. 1987 Apr 10;49(1):131–141. doi: 10.1016/0092-8674(87)90763-x. [DOI] [PubMed] [Google Scholar]
  18. Franke W. W., Schmid E., Osborn M., Weber K. Different intermediate-sized filaments distinguished by immunofluorescence microscopy. Proc Natl Acad Sci U S A. 1978 Oct;75(10):5034–5038. doi: 10.1073/pnas.75.10.5034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Georgatos S. D., Blobel G. Two distinct attachment sites for vimentin along the plasma membrane and the nuclear envelope in avian erythrocytes: a basis for a vectorial assembly of intermediate filaments. J Cell Biol. 1987 Jul;105(1):105–115. doi: 10.1083/jcb.105.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gerner E. W., Schneider M. J. Induced thermal resistance in HeLa cells. Nature. 1975 Aug 7;256(5517):500–502. doi: 10.1038/256500a0. [DOI] [PubMed] [Google Scholar]
  21. Glass J. R., DeWitt R. G., Cress A. E. Rapid loss of stress fibers in Chinese hamster ovary cells after hyperthermia. Cancer Res. 1985 Jan;45(1):258–262. [PubMed] [Google Scholar]
  22. Goldman R., Goldman A., Green K., Jones J., Lieska N., Yang H. Y. Intermediate filaments: possible functions as cytoskeletal connecting links between the nucleus and the cell surface. Ann N Y Acad Sci. 1985;455:1–17. doi: 10.1111/j.1749-6632.1985.tb50400.x. [DOI] [PubMed] [Google Scholar]
  23. Green K. J., Goldman R. D. Evidence for an interaction between the cell surface and intermediate filaments in cultured fibroblasts. Cell Motil Cytoskeleton. 1986;6(4):389–405. doi: 10.1002/cm.970060405. [DOI] [PubMed] [Google Scholar]
  24. Grossi de Sa M. F., Martins de Sa C., Harper F., Olink-Coux M., Huesca M., Scherrer K. The association of prosomes with some of the intermediate filament networks of the animal cell. J Cell Biol. 1988 Oct;107(4):1517–1530. doi: 10.1083/jcb.107.4.1517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hall B. G. Yeast thermotolerance does not require protein synthesis. J Bacteriol. 1983 Dec;156(3):1363–1365. doi: 10.1128/jb.156.3.1363-1365.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Henle K. J., Leeper D. B. Interaction of hyperthermia and radiation in CHO cells: recovery kinetics. Radiat Res. 1976 Jun;66(3):505–518. [PubMed] [Google Scholar]
  27. Howe J. G., Hershey J. W. Translational initiation factor and ribosome association with the cytoskeletal framework fraction from HeLa cells. Cell. 1984 May;37(1):85–93. doi: 10.1016/0092-8674(84)90303-9. [DOI] [PubMed] [Google Scholar]
  28. Johnson L. V., Walsh M. L., Chen L. B. Localization of mitochondria in living cells with rhodamine 123. Proc Natl Acad Sci U S A. 1980 Feb;77(2):990–994. doi: 10.1073/pnas.77.2.990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Klymkowsky M. W., Miller R. H., Lane E. B. Morphology, behavior, and interaction of cultured epithelial cells after the antibody-induced disruption of keratin filament organization. J Cell Biol. 1983 Feb;96(2):494–509. doi: 10.1083/jcb.96.2.494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Knapp L. W., O'Guin W. M., Sawyer R. H. Drug-induced alterations of cytokeratin organization in cultured epithelial cells. Science. 1983 Feb 4;219(4584):501–503. doi: 10.1126/science.6186022. [DOI] [PubMed] [Google Scholar]
  31. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  32. Landry J., Bernier D., Chrétien P., Nicole L. M., Tanguay R. M., Marceau N. Synthesis and degradation of heat shock proteins during development and decay of thermotolerance. Cancer Res. 1982 Jun;42(6):2457–2461. [PubMed] [Google Scholar]
  33. Lazarides E. Intermediate filaments as mechanical integrators of cellular space. Nature. 1980 Jan 17;283(5744):249–256. doi: 10.1038/283249a0. [DOI] [PubMed] [Google Scholar]
  34. Leicht B. G., Biessmann H., Palter K. B., Bonner J. J. Small heat shock proteins of Drosophila associate with the cytoskeleton. Proc Natl Acad Sci U S A. 1986 Jan;83(1):90–94. doi: 10.1073/pnas.83.1.90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lenk R., Ransom L., Kaufmann Y., Penman S. A cytoskeletal structure with associated polyribosomes obtained from HeLa cells. Cell. 1977 Jan;10(1):67–78. doi: 10.1016/0092-8674(77)90141-6. [DOI] [PubMed] [Google Scholar]
  36. Lepock J. R. Involvement of membranes in cellular responses to hyperthermia. Radiat Res. 1982 Dec;92(3):433–438. [PubMed] [Google Scholar]
  37. Lessard J. L. Two monoclonal antibodies to actin: one muscle selective and one generally reactive. Cell Motil Cytoskeleton. 1988;10(3):349–362. doi: 10.1002/cm.970100302. [DOI] [PubMed] [Google Scholar]
  38. Li G. C., Werb Z. Correlation between synthesis of heat shock proteins and development of thermotolerance in Chinese hamster fibroblasts. Proc Natl Acad Sci U S A. 1982 May;79(10):3218–3222. doi: 10.1073/pnas.79.10.3218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Lindquist S. The heat-shock response. Annu Rev Biochem. 1986;55:1151–1191. doi: 10.1146/annurev.bi.55.070186.005443. [DOI] [PubMed] [Google Scholar]
  40. Mangeat P., Burridge K. Actin-membrane interaction in fibroblasts: what proteins are involved in this association? J Cell Biol. 1984 Jul;99(1 Pt 2):95s–103s. doi: 10.1083/jcb.99.1.95s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Matteoni R., Kreis T. E. Translocation and clustering of endosomes and lysosomes depends on microtubules. J Cell Biol. 1987 Sep;105(3):1253–1265. doi: 10.1083/jcb.105.3.1253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. McKenzie S. L., Henikoff S., Meselson M. Localization of RNA from heat-induced polysomes at puff sites in Drosophila melanogaster. Proc Natl Acad Sci U S A. 1975 Mar;72(3):1117–1121. doi: 10.1073/pnas.72.3.1117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Mirande M., Le Corre D., Louvard D., Reggio H., Pailliez J. P., Waller J. P. Association of an aminoacyl-tRNA synthetase complex and of phenylalanyl-tRNA synthetase with the cytoskeletal framework fraction from mammalian cells. Exp Cell Res. 1985 Jan;156(1):91–102. doi: 10.1016/0014-4827(85)90264-2. [DOI] [PubMed] [Google Scholar]
  44. Mooseker M. S., Coleman T. R., Conzelman K. A. Calcium and the regulation of cytoskeletal assembly, structure and contractility. Ciba Found Symp. 1986;122:232–249. doi: 10.1002/9780470513347.ch14. [DOI] [PubMed] [Google Scholar]
  45. Mose-Larsen P., Bravo R., Fey S. J., Small J. V., Celis J. E. Putative association of mitochondria with a subpopulation of intermediate-sized filaments in cultured human skin fibroblasts. Cell. 1982 Dec;31(3 Pt 2):681–692. doi: 10.1016/0092-8674(82)90323-3. [DOI] [PubMed] [Google Scholar]
  46. Nishida E., Koyasu S., Sakai H., Yahara I. Calmodulin-regulated binding of the 90-kDa heat shock protein to actin filaments. J Biol Chem. 1986 Dec 5;261(34):16033–16036. [PubMed] [Google Scholar]
  47. Ohtsuka K., Tanabe K., Nakamura H., Sato C. Possible cytoskeletal association of 69,000- and 68,000-dalton heat shock proteins and structural relations among heat shock proteins in murine mastocytoma cells. Radiat Res. 1986 Oct;108(1):34–42. [PubMed] [Google Scholar]
  48. Ornelles D. A., Fey E. G., Penman S. Cytochalasin releases mRNA from the cytoskeletal framework and inhibits protein synthesis. Mol Cell Biol. 1986 May;6(5):1650–1662. doi: 10.1128/mcb.6.5.1650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Poglazov B. F. Actin and coordination of metabolic processes. Biochem Int. 1983 Jun;6(6):757–765. [PubMed] [Google Scholar]
  50. Pollard T. D., Cooper J. A. Actin and actin-binding proteins. A critical evaluation of mechanisms and functions. Annu Rev Biochem. 1986;55:987–1035. doi: 10.1146/annurev.bi.55.070186.005011. [DOI] [PubMed] [Google Scholar]
  51. Reiter T., Penman S. "Prompt" heat shock proteins: translationally regulated synthesis of new proteins associated with the nuclear matrix-intermediate filaments as an early response to heat shock. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4737–4741. doi: 10.1073/pnas.80.15.4737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Schamhart D. H., van Walraven H. S., Wiegant F. A., Linnemans W. A., van Rijn J., van den Berg J., van Wijk R. Thermotolerance in cultured hepatoma cells: cell viability, cell morphology, protein synthesis, and heat-shock proteins. Radiat Res. 1984 Apr;98(1):82–95. [PubMed] [Google Scholar]
  53. Sciandra J. J., Subjeck J. R. Heat shock proteins and protection of proliferation and translation in mammalian cells. Cancer Res. 1984 Nov;44(11):5188–5194. [PubMed] [Google Scholar]
  54. Sharpe A. H., Chen L. B., Murphy J. R., Fields B. N. Specific disruption of vimentin filament organization in monkey kidney CV-1 cells by diphtheria toxin, exotoxin A, and cycloheximide. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7267–7271. doi: 10.1073/pnas.77.12.7267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Subjeck J. R., Sciandra J. J., Johnson R. J. Heat shock proteins and thermotolerance; a comparison of induction kinetics. Br J Radiol. 1982 Aug;55(656):579–584. doi: 10.1259/0007-1285-55-656-579. [DOI] [PubMed] [Google Scholar]
  56. Subjeck J. R., Shyy T. T. Stress protein systems of mammalian cells. Am J Physiol. 1986 Jan;250(1 Pt 1):C1–17. doi: 10.1152/ajpcell.1986.250.1.C1. [DOI] [PubMed] [Google Scholar]
  57. Sun T. T., Green H. Immunofluorescent staining of keratin fibers in cultured cells. Cell. 1978 Jul;14(3):469–476. doi: 10.1016/0092-8674(78)90233-7. [DOI] [PubMed] [Google Scholar]
  58. Tanguay R. M. Genetic regulation during heat shock and function of heat-shock proteins: a review. Can J Biochem Cell Biol. 1983 Jun;61(6):387–394. doi: 10.1139/o83-053. [DOI] [PubMed] [Google Scholar]
  59. Taylor D. L., Wang Y. L. Fluorescently labelled molecules as probes of the structure and function of living cells. Nature. 1980 Apr 3;284(5755):405–410. doi: 10.1038/284405a0. [DOI] [PubMed] [Google Scholar]
  60. Thomas G. P., Welch W. J., Mathews M. B., Feramisco J. R. Molecular and cellular effects of heat-shock and related treatments of mammalian tissue-culture cells. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 2):985–996. doi: 10.1101/sqb.1982.046.01.092. [DOI] [PubMed] [Google Scholar]
  61. Wang E., Goldman R. D. Functions of cytoplasmic fibers in intracellular movements in BHK-21 cells. J Cell Biol. 1978 Dec;79(3):708–726. doi: 10.1083/jcb.79.3.708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Welch W. J., Feramisco J. R., Blose S. H. The mammalian stress response and the cytoskeleton: alterations in intermediate filaments. Ann N Y Acad Sci. 1985;455:57–67. doi: 10.1111/j.1749-6632.1985.tb50403.x. [DOI] [PubMed] [Google Scholar]
  63. Welch W. J., Feramisco J. R. Disruption of the three cytoskeletal networks in mammalian cells does not affect transcription, translation, or protein translocation changes induced by heat shock. Mol Cell Biol. 1985 Jul;5(7):1571–1581. doi: 10.1128/mcb.5.7.1571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Welch W. J., Mizzen L. A. Characterization of the thermotolerant cell. II. Effects on the intracellular distribution of heat-shock protein 70, intermediate filaments, and small nuclear ribonucleoprotein complexes. J Cell Biol. 1988 Apr;106(4):1117–1130. doi: 10.1083/jcb.106.4.1117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Welch W. J., Suhan J. P. Morphological study of the mammalian stress response: characterization of changes in cytoplasmic organelles, cytoskeleton, and nucleoli, and appearance of intranuclear actin filaments in rat fibroblasts after heat-shock treatment. J Cell Biol. 1985 Oct;101(4):1198–1211. doi: 10.1083/jcb.101.4.1198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Widelitz R. B., Magun B. E., Gerner E. W. Effects of cycloheximide on thermotolerance expression, heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts. Mol Cell Biol. 1986 Apr;6(4):1088–1094. doi: 10.1128/mcb.6.4.1088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Wiegant F. A., van Bergen en Henegouwen P. M., van Dongen G., Linnemans W. A. Stress-induced thermotolerance of the cytoskeleton of mouse neuroblastoma N2A cells and rat Reuber H35 hepatoma cells. Cancer Res. 1987 Mar 15;47(6):1674–1680. [PubMed] [Google Scholar]
  68. van Bergen en Henegouwen P. M., Jordi W. J., van Dongen G., Ramaekers F. C., Amesz H., Linnemans W. A. Studies on a possible relationship between alterations in the cytoskeleton and induction of heat shock protein synthesis in mammalian cells. Int J Hyperthermia. 1985 Jan-Mar;1(1):69–83. doi: 10.3109/02656738509029275. [DOI] [PubMed] [Google Scholar]
  69. van Bergen en Henegouwen P. M., Linnemans A. M. Heat shock gene expression and cytoskeletal alterations in mouse neuroblastoma cells. Exp Cell Res. 1987 Aug;171(2):367–375. doi: 10.1016/0014-4827(87)90169-8. [DOI] [PubMed] [Google Scholar]
  70. van Venrooij W. J., Sillekens P. T., van Eekelen C. A., Reinders R. J. On the association of mRNA with the cytoskeleton in uninfected and adenovirus-infected human KB cells. Exp Cell Res. 1981 Sep;135(1):79–91. doi: 10.1016/0014-4827(81)90301-3. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES