Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1981 May 1;89(2):362–367. doi: 10.1083/jcb.89.2.362

Detection of actin assembly by fluorescence energy transfer

PMCID: PMC2111691  PMID: 6894758

Abstract

Fluorescence energy transfer was used to measure the assembly and disassembly of actin filaments. Actin was labeled at cysteine 373 with an energy donor (5-iodoacetamidofluorescein) or an energy acceptor (tetramethylrhodamine iodoacetamide or eosin iodoacetamide). Donor- labeled actin and acceptor-labeled actin were coassembled. The dependence of the transfer efficiency on the mole fraction of acceptor- labeled actin showed that the radial coordinate of the label at cysteine 373 is approximately 35 A, which means that this site is located near the outer surface of the filament. The distance between a donor and the closest acceptor in such a filament is 58 A. The increase in fluorescence after the mixing of actin filaments containing both donor and acceptor with unlabeled filaments showed that there is a slow continuous exchange of actin units. The rate of exchange was markedly accelerated when the filaments were sonicated. The rapid loss of energy transfer caused by mechanical shear probably resulted from an increase in the number of filament ends, which in turn accelerated the exchange of monomeric actin units. Energy transfer promises to be a valuable tool in characterizing the assembly and dynamics of actin and other cytoskeletal and contractile proteins in vitro and in intact cells.

Full Text

The Full Text of this article is available as a PDF (543.6 KB).

Selected References

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

  1. ASAKURA S. F-actin adenosine triphosphatase activated under sonic vibration. Biochim Biophys Acta. 1961 Sep 2;52:65–75. doi: 10.1016/0006-3002(61)90904-0. [DOI] [PubMed] [Google Scholar]
  2. Brenner S. L., Korn E. D. Substoichiometric concentrations of cytochalasin D inhibit actin polymerization. Additional evidence for an F-actin treadmill. J Biol Chem. 1979 Oct 25;254(20):9982–9985. [PubMed] [Google Scholar]
  3. Brown S. S., Spudich J. A. Cytochalasin inhibits the rate of elongation of actin filament fragments. J Cell Biol. 1979 Dec;83(3):657–662. doi: 10.1083/jcb.83.3.657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cheung H. C., Cooke R., Smith L. The G-actin is greater than F-actin transformation as studied by the fluorescence of bound dansyl cystine. Arch Biochem Biophys. 1971 Jan;142(1):333–339. doi: 10.1016/0003-9861(71)90291-8. [DOI] [PubMed] [Google Scholar]
  5. Cooke R. The role of the bound nucleotide in the polymerization of actin. Biochemistry. 1975 Jul 15;14(14):3250–3256. doi: 10.1021/bi00685a035. [DOI] [PubMed] [Google Scholar]
  6. Fairclough R. H., Cantor C. R. The use of singlet-singlet energy transfer to study macromolecular assemblies. Methods Enzymol. 1978;48:347–379. doi: 10.1016/s0076-6879(78)48019-x. [DOI] [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. Fernandez S. M., Berlin R. D. Cell surface distribution of lectin receptors determined by resonance energy transfer. Nature. 1976 Dec 2;264(5585):411–415. doi: 10.1038/264411a0. [DOI] [PubMed] [Google Scholar]
  9. Flanagan M. D., Lin S. Cytochalasins block actin filament elongation by binding to high affinity sites associated with F-actin. J Biol Chem. 1980 Feb 10;255(3):835–838. [PubMed] [Google Scholar]
  10. Hartwig J. H., Stossel T. P. Cytochalasin B and the structure of actin gels. J Mol Biol. 1979 Nov 5;134(3):539–553. doi: 10.1016/0022-2836(79)90366-8. [DOI] [PubMed] [Google Scholar]
  11. Ikkai T., Wahl P., Auchet J. C. Anisotropy decay of labelled actin. Evidence of the flexibility of the peptide chain in F-actin molecules. Eur J Biochem. 1979 Jan 15;93(2):397–408. doi: 10.1111/j.1432-1033.1979.tb12836.x. [DOI] [PubMed] [Google Scholar]
  12. Kawasaki Y., Mihashi K., Tanaka H., Ohnuma H. Fluorescence study of N-(3-pyrene)maleimide conjugated to rabbit skeletal F-actin and plasmodium actin polymers. Biochim Biophys Acta. 1976 Sep 28;446(1):166–178. doi: 10.1016/0005-2795(76)90108-2. [DOI] [PubMed] [Google Scholar]
  13. Knight P., Offer G. p-NN'-phenylenebismaleimide, a specific cross-linking agent for F-actin. Biochem J. 1978 Dec 1;175(3):1023–1032. doi: 10.1042/bj1751023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. LATT S. A., CHEUNG H. T., BLOUT E. R. ENERGY TRANSFER. A SYSTEM WITH RELATIVELY FIXED DONOR-ACCEPTOR SEPARATION. J Am Chem Soc. 1965 Mar 5;87:995–1003. doi: 10.1021/ja01083a011. [DOI] [PubMed] [Google Scholar]
  16. Lin T. I. Fluorimetric studies of actin labeled with dansyl aziridine. Arch Biochem Biophys. 1978 Jan 30;185(2):285–299. doi: 10.1016/0003-9861(78)90170-4. [DOI] [PubMed] [Google Scholar]
  17. MacLean-Fletcher S., Pollard T. D. Mechanism of action of cytochalasin B on actin. Cell. 1980 Jun;20(2):329–341. doi: 10.1016/0092-8674(80)90619-4. [DOI] [PubMed] [Google Scholar]
  18. Miki M., Mihashi K. Fluorescence energy transfer between epsilon-ATP at the nucleotide binding site and N-(4-dimethylamino-3,5-dinitrophenyl)-maleimide at Cys-373 of G-actin. Biochim Biophys Acta. 1978 Mar 28;533(1):163–172. doi: 10.1016/0005-2795(78)90560-3. [DOI] [PubMed] [Google Scholar]
  19. Nakaoka Y., Kasai M. Behaviour of sonicated actin polymers: adenosine triphosphate splitting and polymerization. J Mol Biol. 1969 Sep 14;44(2):319–332. doi: 10.1016/0022-2836(69)90178-8. [DOI] [PubMed] [Google Scholar]
  20. Porter M., Weber A. Non-cooperative response of actin-cystein 373 in cooperatively behaving regulated actin filaments. FEBS Lett. 1979 Sep 15;105(2):259–262. doi: 10.1016/0014-5793(79)80624-9. [DOI] [PubMed] [Google Scholar]
  21. STRYER L. Intramolecular resonance transfer of energy in proteins. Biochim Biophys Acta. 1959 Sep;35:242–244. doi: 10.1016/0006-3002(59)90355-5. [DOI] [PubMed] [Google Scholar]
  22. Simpson P. A., Spudich J. A. ATP-driven steady-state exchange of monomeric and filamentous actin from Dictyostelium discoideum. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4610–4613. doi: 10.1073/pnas.77.8.4610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Stone D. B., Prevost S. C., Botts J. Studies on spin-labeled actin. Biochemistry. 1970 Sep 29;9(20):3937–3947. doi: 10.1021/bi00822a012. [DOI] [PubMed] [Google Scholar]
  24. Stryer L., Haugland R. P. Energy transfer: a spectroscopic ruler. Proc Natl Acad Sci U S A. 1967 Aug;58(2):719–726. doi: 10.1073/pnas.58.2.719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Takashi R. Fluorescence energy transfer between subfragment-1 and actin points in the rigor complex of actosubfragment-1. Biochemistry. 1979 Nov 13;18(23):5164–5169. doi: 10.1021/bi00590a021. [DOI] [PubMed] [Google Scholar]
  26. Tao T., Cho J. Fluorescence lifetime quenching studies on the accessibilities of actin sulfhydryl sites. Biochemistry. 1979 Jun 26;18(13):2759–2765. doi: 10.1021/bi00580a011. [DOI] [PubMed] [Google Scholar]
  27. Taylor D. L., Condeelis J. S. Cytoplasmic structure and contractility in amoeboid cells. Int Rev Cytol. 1979;56:57–144. doi: 10.1016/s0074-7696(08)61821-5. [DOI] [PubMed] [Google Scholar]
  28. Taylor D. L., Wang Y. L., Heiple J. M. Contractile basis of ameboid movement. VII. The distribution of fluorescently labeled actin in living amebas. J Cell Biol. 1980 Aug;86(2):590–598. doi: 10.1083/jcb.86.2.590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Taylor D. L., Wang Y. L. Molecular cytochemistry: incorporation of fluorescently labeled actin into living cells. Proc Natl Acad Sci U S A. 1978 Feb;75(2):857–861. doi: 10.1073/pnas.75.2.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Uyemura D. G., Brown S. S., Spudich J. A. Biochemical and structural characterization of actin from Dictyostelium discoideum. J Biol Chem. 1978 Dec 25;253(24):9088–9096. [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Wegner A. Head to tail polymerization of actin. J Mol Biol. 1976 Nov;108(1):139–150. doi: 10.1016/s0022-2836(76)80100-3. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Yguerabide J. Nanosecond fluorescence spectroscopy of macromolecules. Methods Enzymol. 1972;26:498–578. doi: 10.1016/s0076-6879(72)26026-8. [DOI] [PubMed] [Google Scholar]
  36. Yin H. L., Stossel T. P. Control of cytoplasmic actin gel-sol transformation by gelsolin, a calcium-dependent regulatory protein. Nature. 1979 Oct 18;281(5732):583–586. doi: 10.1038/281583a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES