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. 1984 Sep 1;99(3):918–928. doi: 10.1083/jcb.99.3.918

Interaction of fluorescently-labeled contractile proteins with the cytoskeleton in cell models

PMCID: PMC2113417  PMID: 6540785

Abstract

To determine if a living cell is necessary for the incorporation of actin, alpha-actinin, and tropomyosin into the cytoskeleton, we have exposed cell models to fluorescently labeled contractile proteins. In this in vitro system, lissamine rhodamine-labeled actin bound to attachment plaques, ruffles, cleavage furrows and stress fibers, and the binding could not be blocked by prior exposure to unlabeled actin. Fluorescently labeled alpha-actinin also bound to ruffles, attachment plaques, cleavage furrows, and stress fibers. The periodicity of fluorescent alpha-actinin along stress fibers was wider in gerbil fibroma cells than it was in PtK2 cells. The fluorescent alpha-actinin binding in cell models could not be blocked by the prior addition of unlabeled alpha-actinin suggesting that alpha-actinin was binding to itself. While there was only slight binding of fluorescent tropomyosin to the cytoskeleton of interphase cells, there was stronger binding in furrow regions of models of dividing cells. The binding of fluorescently labeled tropomyosin could be blocked by prior exposure of the cell models to unlabeled tropomyosin. If unlabeled actin was permitted to polymerize in the stress fibers in cell models, fluorescently labeled tropomyosin stained the fibers. In contrast to the labeled contractile proteins, fluorescently labeled ovalbumin and BSA did not stain any elements of the cytoskeleton. Our results are discussed in terms of the structure and assembly of stress fibers and cleavage furrows.

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Selected References

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  1. Aubin J. E., Weber K., Osborn M. Analysis of actin and microfilament-associated proteins in the mitotic spindle and cleavage furrow of PtK2 cells by immunofluorescence microscopy. A critical note. Exp Cell Res. 1979 Nov;124(1):93–109. doi: 10.1016/0014-4827(79)90260-x. [DOI] [PubMed] [Google Scholar]
  2. Barak L. S., Nothnagel E. A., DeMarco E. F., Webb W. W. Differential staining of actin in metaphase spindles with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin and fluorescent DNase: is actin involved in chromosomal movement? Proc Natl Acad Sci U S A. 1981 May;78(5):3034–3038. doi: 10.1073/pnas.78.5.3034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barak L. S., Yocum R. R., Nothnagel E. A., Webb W. W. Fluorescence staining of the actin cytoskeleton in living cells with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin. Proc Natl Acad Sci U S A. 1980 Feb;77(2):980–984. doi: 10.1073/pnas.77.2.980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blikstad I., Eriksson S., Carlsson L. alpha-Actinin promotes polymerization of actin from profilactin. Eur J Biochem. 1980 Aug;109(2):317–323. doi: 10.1111/j.1432-1033.1980.tb04797.x. [DOI] [PubMed] [Google Scholar]
  5. Cande W. Z., Lazarides E., McIntosh J. R. A comparison of the distribution of actin and tubulin in the mammalian mitotic spindle as seen by indirect immunofluorescence. J Cell Biol. 1977 Mar;72(3):552–567. doi: 10.1083/jcb.72.3.552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Feramisco J. R., Burridge K. A rapid purification of alpha-actinin, filamin, and a 130,000-dalton protein from smooth muscle. J Biol Chem. 1980 Feb 10;255(3):1194–1199. [PubMed] [Google Scholar]
  7. Feramisco J. R. Microinjection of fluorescently labeled alpha-actinin into living fibroblasts. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3967–3971. doi: 10.1073/pnas.76.8.3967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Franke W. W., Grund C., Osborn M., Weber K. The intermediate-sized filaments in rat kangaroo PtK2 cells. I. Morphology in situ. Cytobiologie. 1978 Aug;17(2):365–391. [PubMed] [Google Scholar]
  9. Fujiwara K., Porter M. E., Pollard T. D. Alpha-actinin localization in the cleavage furrow during cytokinesis. J Cell Biol. 1978 Oct;79(1):268–275. doi: 10.1083/jcb.79.1.268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Füchtbauer A., Jockusch B. M., Maruta H., Kilimann M. W., Isenberg G. Disruption of microfilament organization after injection of F-actin capping proteins into living tissue culture cells. 1983 Jul 28-Aug 3Nature. 304(5924):361–364. doi: 10.1038/304361a0. [DOI] [PubMed] [Google Scholar]
  11. Geiger B. The association of rhodamine - labelled alpha-actinin with actin bundles in demembranated cells. Cell Biol Int Rep. 1981 Jun;5(6):627–634. doi: 10.1016/s0309-1651(81)80015-x. [DOI] [PubMed] [Google Scholar]
  12. Gordon S. R. The localization of actin in dividing corneal endothelial cells demonstrated with nitrobenzoxadiazole phallacidin. Cell Tissue Res. 1983;229(3):533–539. doi: 10.1007/BF00207696. [DOI] [PubMed] [Google Scholar]
  13. Gordon W. E., 3rd Immunofluorescent and ultrastructural studies of "sarcomeric" units in stress fibers of cultured non-muscle cells. Exp Cell Res. 1978 Dec;117(2):253–260. doi: 10.1016/0014-4827(78)90138-6. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Kreis T. E., Geiger B., Schlessinger J. Mobility of microinjected rhodamine actin within living chicken gizzard cells determined by fluorescence photobleaching recovery. Cell. 1982 Jul;29(3):835–845. doi: 10.1016/0092-8674(82)90445-7. [DOI] [PubMed] [Google Scholar]
  16. Kreis T. E., Winterhalter K. H., Birchmeier W. In vivo distribution and turnover of fluorescently labeled actin microinjected into human fibroblasts. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3814–3818. doi: 10.1073/pnas.76.8.3814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Lazarides E., Burridge K. Alpha-actinin: immunofluorescent localization of a muscle structural protein in nonmuscle cells. Cell. 1975 Nov;6(3):289–298. doi: 10.1016/0092-8674(75)90180-4. [DOI] [PubMed] [Google Scholar]
  19. Lazarides E. Tropomyosin antibody: the specific localization of tropomyosin in nonmuscle cells. J Cell Biol. 1975 Jun;65(3):549–561. doi: 10.1083/jcb.65.3.549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lazarides E., Weber K. Actin antibody: the specific visualization of actin filaments in non-muscle cells. Proc Natl Acad Sci U S A. 1974 Jun;71(6):2268–2272. doi: 10.1073/pnas.71.6.2268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Masaki T., Takaiti O. Some properties of chicken alpha-actinin. J Biochem. 1969 Nov;66(5):637–643. [PubMed] [Google Scholar]
  22. Maupin-Szamier P., Pollard T. D. Actin filament destruction by osmium tetroxide. J Cell Biol. 1978 Jun;77(3):837–852. doi: 10.1083/jcb.77.3.837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pochapin M. B., Sanger J. M., Sanger J. W. Microinjection of Lucifer yellow CH into sea urchin eggs and embryos. Cell Tissue Res. 1983;234(2):309–318. doi: 10.1007/BF00213770. [DOI] [PubMed] [Google Scholar]
  24. Pollard T. D., Weihing R. R. Actin and myosin and cell movement. CRC Crit Rev Biochem. 1974 Jan;2(1):1–65. doi: 10.3109/10409237409105443. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Sanger J. W. Changing patterns of actin localization during cell division. Proc Natl Acad Sci U S A. 1975 May;72(5):1913–1916. doi: 10.1073/pnas.72.5.1913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sanger J. W. Mitosis in beating cardiac myoblasts treated with cytochalasin-B. J Exp Zool. 1977 Sep;201(3):463–469. doi: 10.1002/jez.1402010313. [DOI] [PubMed] [Google Scholar]
  28. Sanger J. W., Mittal B., Sanger J. M. Analysis of myofibrillar structure and assembly using fluorescently labeled contractile proteins. J Cell Biol. 1984 Mar;98(3):825–833. doi: 10.1083/jcb.98.3.825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sanger J. W. Presence of actin during chromosomal movement. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2451–2455. doi: 10.1073/pnas.72.6.2451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sanger J. W., Sanger J. M., Jockusch B. M. Differences in the stress fibers between fibroblasts and epithelial cells. J Cell Biol. 1983 Apr;96(4):961–969. doi: 10.1083/jcb.96.4.961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sanger J. W., Sanger J. M., Jockusch B. M. Differential response of three types of actin filament bundles to depletion of cellular ATP levels. Eur J Cell Biol. 1983 Sep;31(2):197–204. [PubMed] [Google Scholar]
  32. Sanger J. W., Sanger J. M., Kreis T. E., Jockusch B. M. Reversible translocation of cytoplasmic actin into the nucleus caused by dimethyl sulfoxide. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5268–5272. doi: 10.1073/pnas.77.9.5268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sanger J. W., Sanger J. M. The cytoskeleton and cell division. Methods Achiev Exp Pathol. 1979;8:110–142. [PubMed] [Google Scholar]
  34. Smillie L. B. Preparation and identification of alpha- and beta-tropomyosins. Methods Enzymol. 1982;85(Pt B):234–241. doi: 10.1016/0076-6879(82)85023-4. [DOI] [PubMed] [Google Scholar]
  35. Soranno T., Bell E. Cytostructural dynamics of spreading and translocating cells. J Cell Biol. 1982 Oct;95(1):127–136. doi: 10.1083/jcb.95.1.127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Suzuki A., Goll D. E., Singh I., Allen R. E., Robson R. M., Stromer M. H. Some properties of purified skeletal muscle alpha-actinin. J Biol Chem. 1976 Nov 10;251(21):6860–6870. [PubMed] [Google Scholar]
  37. Wang K., Feramisco J. R., Ash J. F. Fluorescent localization of contractile proteins in tissue culture cells. Methods Enzymol. 1982;85(Pt B):514–562. doi: 10.1016/0076-6879(82)85050-7. [DOI] [PubMed] [Google Scholar]
  38. Wang Y. L., Heiple J. M., Taylor D. L. Fluorescent analog cytochemistry of contractile proteins. Methods Cell Biol. 1982;25(Pt B):1–11. [PubMed] [Google Scholar]
  39. 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]
  40. Wang Y. L., Taylor D. L. Preparation and characterization of a new molecular cytochemical probe: 5-iodoacetamidofluorescein-labeled actin. J Histochem Cytochem. 1980 Nov;28(11):1198–1206. doi: 10.1177/28.11.6107318. [DOI] [PubMed] [Google Scholar]
  41. Weber K., Groeschel-Stewart U. Antibody to myosin: the specific visualization of myosin-containing filaments in nonmuscle cells. Proc Natl Acad Sci U S A. 1974 Nov;71(11):4561–4564. doi: 10.1073/pnas.71.11.4561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wehland J., Weber K. Distribution of fluorescently labeled actin and tropomyosin after microinjection in living tissue culture cells as observed with TV image intensification. Exp Cell Res. 1980 Jun;127(2):397–408. doi: 10.1016/0014-4827(80)90444-9. [DOI] [PubMed] [Google Scholar]
  43. Woods E. F. Molecular weight and subunit structure of tropomyosin B. J Biol Chem. 1967 Jun 25;242(12):2859–2871. [PubMed] [Google Scholar]
  44. Zigmond S. H., Otto J. J., Bryan J. Organization of myosin in a submembranous sheath in well-spread human fibroblasts. Exp Cell Res. 1979 Mar 15;119(2):205–219. doi: 10.1016/0014-4827(79)90349-5. [DOI] [PubMed] [Google Scholar]

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