Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Jul 1;109(1):191–202. doi: 10.1083/jcb.109.1.191

Functional sorting of actin isoforms in microvascular pericytes

PMCID: PMC2115462  PMID: 2745546

Abstract

We characterized the form and distribution of muscle and nonmuscle actin within retinal pericytes. Antibodies with demonstrable specificities for the actin isoforms were used in localization and immunoprecipitation experiments to identify those cellular domains that were enriched or deficient in one or several actin isoforms. Living pericyte behavior was monitored with phase-contract video microscopy before fixation to identify those cellular areas that might preferentially be stained with either of the fluorescent antiactins or phallotoxins. Antibody and phallotoxin staining of pericytes revealed that nonmuscle actin is present within membrane ruffles, pseudopods, and stress fibers. In contrast, muscle actin could be convincingly localized in stress fibers, but not within specific motile areas of pericyte cytoplasm. To confirm and quantitatively extend the results obtained by fluorescence microscopy, nonionic and ionic detergents were used to selectively extract the motile or immobilized (stress fiber- containing) regions of biosynthetically labeled pericyte cytoplasm. Immunoprecipitated actins that were present within these discrete cellular domains were subjected to isoelectric focusing in urea- polyacrylamide gels before fluorographic analysis. Scanning laser densitometry of the focused actins could not reveal any detectable alpha-actin within those beta- and gamma-actin-enriched motile regions extracted with nonionic detergents. Moreover, when pericyte stress fibers are completely dissolved by ionic detergent lysis, three actin isoforms can be quantified to be present in a ratio of 1:2.75:3 (alpha:beta:gamma). These biochemical findings on biosynthetically labeled and immunoprecipitated pericyte actins confirm the fluorescent localization studies. While the regulatory events governing this actin sorting are unknown, it seems possible that such events may play important roles in controlling cell shape, adhesion, or the promotion of localized cell spreading.

Full Text

The Full Text of this article is available as a PDF (5.1 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. Propulsion of organelles isolated from Acanthamoeba along actin filaments by myosin-I. Nature. 1986 Aug 21;322(6081):754–756. doi: 10.1038/322754a0. [DOI] [PubMed] [Google Scholar]
  2. Amato P. A., Taylor D. L. Probing the mechanism of incorporation of fluorescently labeled actin into stress fibers. J Cell Biol. 1986 Mar;102(3):1074–1084. doi: 10.1083/jcb.102.3.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Broadwell R. D., Salcman M. Expanding the definition of the blood-brain barrier to protein. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7820–7824. doi: 10.1073/pnas.78.12.7820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bulinski J. C., Kumar S., Titani K., Hauschka S. D. Peptide antibody specific for the amino terminus of skeletal muscle alpha-actin. Proc Natl Acad Sci U S A. 1983 Mar;80(6):1506–1510. doi: 10.1073/pnas.80.6.1506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Buzney S. M., Frank R. N., Varma S. D., Tanishima T., Gabbay K. H. Aldose reductase in retinal mural cells. Invest Ophthalmol Vis Sci. 1977 May;16(5):392–396. [PubMed] [Google Scholar]
  6. Byers H. R., White G. E., Fujiwara K. Organization and function of stress fibers in cells in vitro and in situ. A review. Cell Muscle Motil. 1984;5:83–137. doi: 10.1007/978-1-4684-4592-3_2. [DOI] [PubMed] [Google Scholar]
  7. COGAN D. G., TOUSSAINT D., KUWABARA T. Retinal vascular patterns. IV. Diabetic retinopathy. Arch Ophthalmol. 1961 Sep;66:366–378. doi: 10.1001/archopht.1961.00960010368014. [DOI] [PubMed] [Google Scholar]
  8. Cebra J. J., Goldstein G. Chromatographic purification of tetramethylrhodamine-immune globulin conjugates and their use in the cellular localization of rabbit gamma-globulin polypeptide chains. J Immunol. 1965 Aug;95(2):230–245. [PubMed] [Google Scholar]
  9. Clowes A. W., Clowes M. M., Kocher O., Ropraz P., Chaponnier C., Gabbiani G. Arterial smooth muscle cells in vivo: relationship between actin isoform expression and mitogenesis and their modulation by heparin. J Cell Biol. 1988 Nov;107(5):1939–1945. doi: 10.1083/jcb.107.5.1939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Crocker D. J., Murad T. M., Geer J. C. Role of the pericyte in wound healing. An ultrastructural study. Exp Mol Pathol. 1970 Aug;13(1):51–65. doi: 10.1016/0014-4800(70)90084-5. [DOI] [PubMed] [Google Scholar]
  11. Garrels J. I., Gibson W. Identification and characterization of multiple forms of actin. Cell. 1976 Dec;9(4 Pt 2):793–805. doi: 10.1016/0092-8674(76)90142-2. [DOI] [PubMed] [Google Scholar]
  12. Gitlin J. D., D'Amore P. A. Culture of retinal capillary cells using selective growth media. Microvasc Res. 1983 Jul;26(1):74–80. doi: 10.1016/0026-2862(83)90056-0. [DOI] [PubMed] [Google Scholar]
  13. Herman I. M., Crisona N. J., Pollard T. D. Relation between cell activity and the distribution of cytoplasmic actin and myosin. J Cell Biol. 1981 Jul;90(1):84–91. doi: 10.1083/jcb.90.1.84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Herman I. M., D'Amore P. A. Capillary endothelial cell migration: loss of stress fibres in response to retina-derived growth factor. J Muscle Res Cell Motil. 1984 Dec;5(6):697–709. doi: 10.1007/BF00713928. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Herman I. M., Pollard T. D. Comparison of purified anti-actin and fluorescent-heavy meromyosin staining patterns in dividing cells. J Cell Biol. 1979 Mar;80(3):509–520. doi: 10.1083/jcb.80.3.509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Herman I. M., Pollard T. D., Wong A. J. Contractile proteins in endothelial cells. Ann N Y Acad Sci. 1982;401:50–60. doi: 10.1111/j.1749-6632.1982.tb25706.x. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. KUWABARA T., COGAN D. G. Retinal vascular patterns. VI. Mural cells of the retinal capillaries. Arch Ophthalmol. 1963 Apr;69:492–502. doi: 10.1001/archopht.1963.00960040498013. [DOI] [PubMed] [Google Scholar]
  20. Kuroda M. Change of actin isomers during differentiation of smooth muscle. Biochim Biophys Acta. 1985 Dec 13;843(3):208–213. doi: 10.1016/0304-4165(85)90141-2. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Lark M. W., Laterra J., Culp L. A. Close and focal contact adhesions of fibroblasts to a fibronectin-containing matrix. Fed Proc. 1985 Feb;44(2):394–403. [PubMed] [Google Scholar]
  23. Lewis L., Verna J. M., Levinstone D., Sher S., Marek L., Bell E. The relationship of fibroblast translocations to cell morphology and stress fibre density. J Cell Sci. 1982 Feb;53:21–36. doi: 10.1242/jcs.53.1.21. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. McKenna N., Meigs J. B., Wang Y. L. Identical distribution of fluorescently labeled brain and muscle actins in living cardiac fibroblasts and myocytes. J Cell Biol. 1985 Jan;100(1):292–296. doi: 10.1083/jcb.100.1.292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  28. Otey C. A., Kalnoski M. H., Bulinski J. C. Identification and quantification of actin isoforms in vertebrate cells and tissues. J Cell Biochem. 1987 Jun;34(2):113–124. doi: 10.1002/jcb.240340205. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. 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]
  32. Sanger J. M., Sanger J. W. Banding and polarity of actin filaments in interphase and cleaving cells. J Cell Biol. 1980 Aug;86(2):568–575. doi: 10.1083/jcb.86.2.568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Spudich J. A., Watt S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem. 1971 Aug 10;246(15):4866–4871. [PubMed] [Google Scholar]
  34. Tilton R. G., Kilo C., Williamson J. R., Murch D. W. Differences in pericyte contractile function in rat cardiac and skeletal muscle microvasculatures. Microvasc Res. 1979 Nov;18(3):336–352. doi: 10.1016/0026-2862(79)90042-6. [DOI] [PubMed] [Google Scholar]
  35. Tilton R. G., Kilo C., Williamson J. R. Pericyte-endothelial relationships in cardiac and skeletal muscle capillaries. Microvasc Res. 1979 Nov;18(3):325–335. doi: 10.1016/0026-2862(79)90041-4. [DOI] [PubMed] [Google Scholar]
  36. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wieland T., Faulstich H. Amatoxins, phallotoxins, phallolysin, and antamanide: the biologically active components of poisonous Amanita mushrooms. CRC Crit Rev Biochem. 1978 Dec;5(3):185–260. doi: 10.3109/10409237809149870. [DOI] [PubMed] [Google Scholar]
  38. Young W. C., Herman I. M. Extracellular matrix modulation of endothelial cell shape and motility following injury in vitro. J Cell Sci. 1985 Feb;73:19–32. doi: 10.1242/jcs.73.1.19. [DOI] [PubMed] [Google Scholar]

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

RESOURCES