Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1991 Feb 2;112(4):653–664. doi: 10.1083/jcb.112.4.653

Beta actin and its mRNA are localized at the plasma membrane and the regions of moving cytoplasm during the cellular response to injury

PMCID: PMC2288855  PMID: 1993736

Abstract

Previous work in our laboratory has shown that microvascular pericytes sort muscle and nonmuscle actin isoforms into discrete cytoplasmic domains (Herman, I. M., and P. A. D'Amore. 1985. J. Cell Biol. 101:43- 52; DeNofrio, D.T.C. Hoock, and I. M. Herman. J. Cell. Biol. 109:191- 202). Specifically, muscle (alpha-smooth) actin is present on the stress fibers while nonmuscle actins (beta and gamma) are located on stress fibers and in regions of moving cytoplasm (e.g., ruffles, lamellae). To determine the form and function of beta actin in microvascular pericytes and endothelial cells recovering from injury, we prepared isoform-specific antibodies and cDNA probes for immunolocalization, Western and Northern blotting, as well as in situ hybridization. Anti-beta actin IgG was prepared by adsorption and release of beta actin-specific IgG from electrophoretically purified pericyte beta actin bound to nitrocellulose paper. Anti-beta actin IgGs prepared by this affinity selection procedure showed exclusive binding to beta actin present in crude cell lysates containing all three actin isoforms. For controls, we localized beta actin as a bright rim of staining beneath the erythrocyte plasma membrane. Anti-beta actin IgG, absorbed with beta actin bound to nitrocellulose, failed to stain erythrocytes. Simultaneous localization of beta actin with the entire F- actin pool was performed on microvascular pericytes or endothelial cells and 3T3 fibroblasts recovering from injury using anti-beta actin IgG in combination with fluorescent phalloidin. Results of these experiments revealed that pericyte beta actin is localized beneath the plasma membrane in association with filopods, pseudopods, and fan lamellae. Additionally, we observed bright focal fluorescence within fan lamellae and in association with the ends of stress fibers that are preferentially associated with the ventral plasmalemma. Whereas fluorescent phalloidin staining along the stress fibers is continuous, anti-beta actin IgG localization is discontinuous. When injured endothelial and 3T3 cells were stained through wound closure, we localized beta actin only in motile cytoplasm at the wound edge. Staining disappeared as cells became quiescent upon monolayer restoration. Appearance of beta actin at the wound edge correlated with a two- to threefold increase in steady-state levels of beta actin mRNA, which rose within 15-60 min after injury and returned to noninjury levels during monolayer restoration. In situ hybridization revealed that transcripts encoding beta actin were localized at the wound edge in association with the repositioned protein. Results of these experiments indicate that beta actin and its encoded mRNA are polarized at the membrane-cytoskeletal interface within regions of moving cytoplasm.

Full Text

The Full Text of this article is available as a PDF (4.6 MB).

Selected References

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

  1. Adams R. J., Pollard T. D. Binding of myosin I to membrane lipids. Nature. 1989 Aug 17;340(6234):565–568. doi: 10.1038/340565a0. [DOI] [PubMed] [Google Scholar]
  2. Aebi U., Millonig R., Salvo H., Engel A. The three-dimensional structure of the actin filament revisited. Ann N Y Acad Sci. 1986;483:100–119. doi: 10.1111/j.1749-6632.1986.tb34502.x. [DOI] [PubMed] [Google Scholar]
  3. Antonelli-Orlidge A., Saunders K. B., Smith S. R., D'Amore P. A. An activated form of transforming growth factor beta is produced by cocultures of endothelial cells and pericytes. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4544–4548. doi: 10.1073/pnas.86.12.4544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Atkinson M. A., Morrow J. S., Marchesi V. T. The polymeric state of actin in the human erythrocyte cytoskeleton. J Cell Biochem. 1982;18(4):493–505. doi: 10.1002/jcb.1982.240180410. [DOI] [PubMed] [Google Scholar]
  5. Bandman E. Myosin isoenzyme transitions in muscle development, maturation, and disease. Int Rev Cytol. 1985;97:97–131. doi: 10.1016/s0074-7696(08)62349-9. [DOI] [PubMed] [Google Scholar]
  6. Barja F., Coughlin C., Belin D., Gabbiani G. Actin isoform synthesis and mRNA levels in quiescent and proliferating rat aortic smooth muscle cells in vivo and in vitro. Lab Invest. 1986 Aug;55(2):226–233. [PubMed] [Google Scholar]
  7. Bennett V. The membrane skeleton of human erythrocytes and its implications for more complex cells. Annu Rev Biochem. 1985;54:273–304. doi: 10.1146/annurev.bi.54.070185.001421. [DOI] [PubMed] [Google Scholar]
  8. Brenner S. L., Korn E. D. Spectrin/actin complex isolated from sheep erythrocytes accelerates actin polymerization by simple nucleation. Evidence for oligomeric actin in the erythrocyte cytoskeleton. J Biol Chem. 1980 Feb 25;255(4):1670–1676. [PubMed] [Google Scholar]
  9. Buchanan R. A., Wagner R. C. Associations between pericytes and capillary endothelium in the eel rete mirabile. Microvasc Res. 1990 Jan;39(1):60–76. doi: 10.1016/0026-2862(90)90059-z. [DOI] [PubMed] [Google Scholar]
  10. Buckingham M. E. Actin and myosin multigene families: their expression during the formation of skeletal muscle. Essays Biochem. 1985;20:77–109. [PubMed] [Google Scholar]
  11. Chamley-Campbell J., Campbell G. R., Ross R. The smooth muscle cell in culture. Physiol Rev. 1979 Jan;59(1):1–61. doi: 10.1152/physrev.1979.59.1.1. [DOI] [PubMed] [Google Scholar]
  12. Cohen C. M., Foley S. F. The role of band 4.1 in the association of actin with erythrocyte membranes. Biochim Biophys Acta. 1982 Jun 28;688(3):691–701. doi: 10.1016/0005-2736(82)90281-4. [DOI] [PubMed] [Google Scholar]
  13. Collins J. H., Borysenko C. W. The 110,000-dalton actin- and calmodulin-binding protein from intestinal brush border is a myosin-like ATPase. J Biol Chem. 1984 Nov 25;259(22):14128–14135. [PubMed] [Google Scholar]
  14. Craig S. W., Pardo J. V. Gamma actin, spectrin, and intermediate filament proteins colocalize with vinculin at costameres, myofibril-to-sarcolemma attachment sites. Cell Motil. 1983;3(5-6):449–462. doi: 10.1002/cm.970030513. [DOI] [PubMed] [Google Scholar]
  15. Das A., Frank R. N., Weber M. L., Kennedy A., Reidy C. A., Mancini M. A. ATP causes retinal pericytes to contract in vitro. Exp Eye Res. 1988 Mar;46(3):349–362. doi: 10.1016/s0014-4835(88)80025-3. [DOI] [PubMed] [Google Scholar]
  16. DeBiasio R. L., Wang L. L., Fisher G. W., Taylor D. L. The dynamic distribution of fluorescent analogues of actin and myosin in protrusions at the leading edge of migrating Swiss 3T3 fibroblasts. J Cell Biol. 1988 Dec;107(6 Pt 2):2631–2645. doi: 10.1083/jcb.107.6.2631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. DeNofrio D., Hoock T. C., Herman I. M. Functional sorting of actin isoforms in microvascular pericytes. J Cell Biol. 1989 Jul;109(1):191–202. doi: 10.1083/jcb.109.1.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fallon J. R., Nachmias V. T. Localization of cytoplasmic and skeletal myosins in developing muscle cells by double-label immunofluorescence. J Cell Biol. 1980 Oct;87(1):237–247. doi: 10.1083/jcb.87.1.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fowler V. M., Bennett V. Erythrocyte membrane tropomyosin. Purification and properties. J Biol Chem. 1984 May 10;259(9):5978–5989. [PubMed] [Google Scholar]
  20. Fowler V. M., Davis J. Q., Bennett V. Human erythrocyte myosin: identification and purification. J Cell Biol. 1985 Jan;100(1):47–55. doi: 10.1083/jcb.100.1.47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fowler V. M. Identification and purification of a novel Mr 43,000 tropomyosin-binding protein from human erythrocyte membranes. J Biol Chem. 1987 Sep 15;262(26):12792–12800. [PubMed] [Google Scholar]
  22. Fowler V. M. Tropomodulin: a cytoskeletal protein that binds to the end of erythrocyte tropomyosin and inhibits tropomyosin binding to actin. J Cell Biol. 1990 Aug;111(2):471–481. doi: 10.1083/jcb.111.2.471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Fyrberg E. A., Mahaffey J. W., Bond B. J., Davidson N. Transcripts of the six Drosophila actin genes accumulate in a stage- and tissue-specific manner. Cell. 1983 May;33(1):115–123. doi: 10.1016/0092-8674(83)90340-9. [DOI] [PubMed] [Google Scholar]
  24. Gabbiani G., Kocher O., Bloom W. S., Vandekerckhove J., Weber K. Actin expression in smooth muscle cells of rat aortic intimal thickening, human atheromatous plaque, and cultured rat aortic media. J Clin Invest. 1984 Jan;73(1):148–152. doi: 10.1172/JCI111185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Gabbiani G., Schmid E., Winter S., Chaponnier C., de Ckhastonay C., Vandekerckhove J., Weber K., Franke W. W. Vascular smooth muscle cells differ from other smooth muscle cells: predominance of vimentin filaments and a specific alpha-type actin. Proc Natl Acad Sci U S A. 1981 Jan;78(1):298–302. doi: 10.1073/pnas.78.1.298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Gadasi H., Korn E. D. Evidence for differential intracellular localization of the Acanthamoeba myosin isoenzymes. Nature. 1980 Jul 31;286(5772):452–456. doi: 10.1038/286452a0. [DOI] [PubMed] [Google Scholar]
  27. Hayden S. M., Wolenski J. S., Mooseker M. S. Binding of brush border myosin I to phospholipid vesicles. J Cell Biol. 1990 Aug;111(2):443–451. doi: 10.1083/jcb.111.2.443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Herman I. M., Castellot J. J., Jr Regulation of vascular smooth muscle cell growth by endothelial-synthesized extracellular matrices. Arteriosclerosis. 1987 Sep-Oct;7(5):463–469. doi: 10.1161/01.atv.7.5.463. [DOI] [PubMed] [Google Scholar]
  29. Herman I. M., D'Amore P. A. Microvascular pericytes contain muscle and nonmuscle actins. J Cell Biol. 1985 Jul;101(1):43–52. doi: 10.1083/jcb.101.1.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Herman I. M. Extracellular matrix-cytoskeletal interactions in vascular cells. Tissue Cell. 1987;19(1):1–19. doi: 10.1016/0040-8166(87)90052-8. [DOI] [PubMed] [Google Scholar]
  31. Herman I. M., Jacobson S. In situ analysis of microvascular pericytes in hypertensive rat brains. Tissue Cell. 1988;20(1):1–12. doi: 10.1016/0040-8166(88)90002-x. [DOI] [PubMed] [Google Scholar]
  32. Herman I. M., Newcomb P. M., Coughlin J. E., Jacobson S. Characterization of microvascular cell cultures from normotensive and hypertensive rat brains: pericyte-endothelial cell interactions in vitro. Tissue Cell. 1987;19(2):197–206. doi: 10.1016/0040-8166(87)90005-x. [DOI] [PubMed] [Google Scholar]
  33. Joyce N. C., Haire M. F., Palade G. E. Contractile proteins in pericytes. II. Immunocytochemical evidence for the presence of two isomyosins in graded concentrations. J Cell Biol. 1985 May;100(5):1387–1395. doi: 10.1083/jcb.100.5.1387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kelley C., D'Amore P., Hechtman H. B., Shepro D. Microvascular pericyte contractility in vitro: comparison with other cells of the vascular wall. J Cell Biol. 1987 Mar;104(3):483–490. doi: 10.1083/jcb.104.3.483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kordeli E., Davis J., Trapp B., Bennett V. An isoform of ankyrin is localized at nodes of Ranvier in myelinated axons of central and peripheral nerves. J Cell Biol. 1990 Apr;110(4):1341–1352. doi: 10.1083/jcb.110.4.1341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lawrence J. B., Singer R. H. Intracellular localization of messenger RNAs for cytoskeletal proteins. Cell. 1986 May 9;45(3):407–415. doi: 10.1016/0092-8674(86)90326-0. [DOI] [PubMed] [Google Scholar]
  37. Leavitt J., Bushar G., Kakunaga T., Hamada H., Hirakawa T., Goldman D., Merril C. Variations in expression of mutant beta actin accompanying incremental increases in human fibroblast tumorigenicity. Cell. 1982 Feb;28(2):259–268. doi: 10.1016/0092-8674(82)90344-0. [DOI] [PubMed] [Google Scholar]
  38. Leavitt J., Kakunaga T. Expression of a variant form of actin and additional polypeptide changes following chemical-induced in vitro neoplastic transformation of human fibroblasts. J Biol Chem. 1980 Feb 25;255(4):1650–1661. [PubMed] [Google Scholar]
  39. 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]
  40. Lin J. J., Hegmann T. E., Lin J. L. Differential localization of tropomyosin isoforms in cultured nonmuscle cells. J Cell Biol. 1988 Aug;107(2):563–572. doi: 10.1083/jcb.107.2.563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. McHugh K. M., Lessard J. L. The nucleotide sequence of a rat vascular smooth muscle alpha-actin cDNA. Nucleic Acids Res. 1988 May 11;16(9):4167–4167. doi: 10.1093/nar/16.9.4167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Murphy D. B., Wallis K. T. Isolation of microtubule protein from chicken erythrocytes and determination of the critical concentration for tubulin polymerization in vitro and in vivo. J Biol Chem. 1983 Jul 10;258(13):8357–8364. [PubMed] [Google Scholar]
  43. Noden D. M. Embryonic origins and assembly of blood vessels. Am Rev Respir Dis. 1989 Oct;140(4):1097–1103. doi: 10.1164/ajrccm/140.4.1097. [DOI] [PubMed] [Google Scholar]
  44. Olmsted J. B. Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples. J Biol Chem. 1981 Dec 10;256(23):11955–11957. [PubMed] [Google Scholar]
  45. Orlidge A., D'Amore P. A. Inhibition of capillary endothelial cell growth by pericytes and smooth muscle cells. J Cell Biol. 1987 Sep;105(3):1455–1462. doi: 10.1083/jcb.105.3.1455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Otey C. A., Kalnoski M. H., Lessard J. L., Bulinski J. C. Immunolocalization of the gamma isoform of nonmuscle actin in cultured cells. J Cell Biol. 1986 May;102(5):1726–1737. doi: 10.1083/jcb.102.5.1726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Owens G. K., Loeb A., Gordon D., Thompson M. M. Expression of smooth muscle-specific alpha-isoactin in cultured vascular smooth muscle cells: relationship between growth and cytodifferentiation. J Cell Biol. 1986 Feb;102(2):343–352. doi: 10.1083/jcb.102.2.343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Pardo J. V., Pittenger M. F., Craig S. W. Subcellular sorting of isoactins: selective association of gamma actin with skeletal muscle mitochondria. Cell. 1983 Apr;32(4):1093–1103. doi: 10.1016/0092-8674(83)90293-3. [DOI] [PubMed] [Google Scholar]
  49. Pinder J. C., Bray D., Gratzer W. B. Control of interaction of spectrin and actin by phosphorylation. Nature. 1977 Dec 22;270(5639):752–754. doi: 10.1038/270752a0. [DOI] [PubMed] [Google Scholar]
  50. Pinder J. C., Gratzer W. B. Structural and dynamic states of actin in the erythrocyte. J Cell Biol. 1983 Mar;96(3):768–775. doi: 10.1083/jcb.96.3.768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Pramanik S. K., Walsh R. W., Bag J. Association of messenger RNA with the cytoskeletal framework in rat L6 myogenic cells. Eur J Biochem. 1986 Oct 15;160(2):221–230. doi: 10.1111/j.1432-1033.1986.tb09960.x. [DOI] [PubMed] [Google Scholar]
  52. Pratt B. M., Harris A. S., Morrow J. S., Madri J. A. Mechanisms of cytoskeletal regulation. Modulation of aortic endothelial cell spectrin by the extracellular matrix. Am J Pathol. 1984 Dec;117(3):349–354. [PMC free article] [PubMed] [Google Scholar]
  53. Rovner A. S., Murphy R. A., Owens G. K. Expression of smooth muscle and nonmuscle myosin heavy chains in cultured vascular smooth muscle cells. J Biol Chem. 1986 Nov 5;261(31):14740–14745. [PubMed] [Google Scholar]
  54. Sato M., Schwarz W. H., Pollard T. D. Dependence of the mechanical properties of actin/alpha-actinin gels on deformation rate. 1987 Feb 26-Mar 4Nature. 325(6107):828–830. doi: 10.1038/325828a0. [DOI] [PubMed] [Google Scholar]
  55. Schwartz R. J., Rothblum K. N. Gene switching in myogenesis: differential expression of the chicken actin multigene family. Biochemistry. 1981 Jul 7;20(14):4122–4129. doi: 10.1021/bi00517a027. [DOI] [PubMed] [Google Scholar]
  56. Skalli O., Pelte M. F., Peclet M. C., Gabbiani G., Gugliotta P., Bussolati G., Ravazzola M., Orci L. Alpha-smooth muscle actin, a differentiation marker of smooth muscle cells, is present in microfilamentous bundles of pericytes. J Histochem Cytochem. 1989 Mar;37(3):315–321. doi: 10.1177/37.3.2918221. [DOI] [PubMed] [Google Scholar]
  57. Tsukita S., Tsukita S., Ishikawa H. Bidirectional polymerization of G-actin on the human erythrocyte membrane. J Cell Biol. 1984 Mar;98(3):1102–1110. doi: 10.1083/jcb.98.3.1102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wang Y. L. Mobility of filamentous actin in living cytoplasm. J Cell Biol. 1987 Dec;105(6 Pt 1):2811–2816. doi: 10.1083/jcb.105.6.2811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wang Y. L., Taylor D. L. Distribution of fluorescently labeled actin in living sea urchin eggs during early development. J Cell Biol. 1979 Jun;81(3):672–679. doi: 10.1083/jcb.81.3.672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Warembourg M., Tranchant O., Atger M., Milgrom E. Uteroglobin messenger ribonucleic acid: localization in rabbit uterus and lung by in situ hybridization. Endocrinology. 1986 Oct;119(4):1632–1640. doi: 10.1210/endo-119-4-1632. [DOI] [PubMed] [Google Scholar]
  61. Wong A. J., Kiehart D. P., Pollard T. D. Myosin from human erythrocytes. J Biol Chem. 1985 Jan 10;260(1):46–49. [PubMed] [Google Scholar]
  62. Yost J. C., Herman I. M. Substratum-induced stress fiber assembly in vascular endothelial cells during spreading in vitro. J Cell Sci. 1990 Mar;95(Pt 3):507–520. doi: 10.1242/jcs.95.3.507. [DOI] [PubMed] [Google Scholar]

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

RESOURCES