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. 1993 Oct 1;123(1):173–181. doi: 10.1083/jcb.123.1.173

Localization and dynamics of nonfilamentous actin in cultured cells [published erratum appears in J Cell Biol 1993 Nov;123(3):following 767]

PMCID: PMC2119807  PMID: 8408196

Abstract

Although the distribution of filamentous actin is well characterized in many cell types, the distribution of nonfilamentous actin remains poorly understood. To determine the relative distribution of filamentous and nonfilamentous actin in cultured NRK cells, we have used a number of labeling agents that differ with respect to their specificities toward the filamentous or nonfilamentous form, including monoclonal and polyclonal anti-actin antibodies, vitamin D-binding protein (DBP), and fluorescent phalloidin. Numerous punctate structures were identified that bind poorly to phalloidin but stain positively with several anti-actin antibodies. These bead structures also stain with DBP, suggesting that they are enriched in nonfilamentous actin. Similar punctate structures were observed after the microinjection of fluorescently labeled actin into living cells, allowing us to examine their dynamics in living cells. The actin-containing punctate structures were observed predominantly in the region behind lamellipodia, particularly in spreading cells induced by wounding confluent monolayers. Time-lapse recording of cells injected with fluorescent actin indicated that they form continuously near the leading edge and move centripetally toward the nucleus. Our results suggest that at least part of the unpolymerized actin molecules are localized at discrete sites, possibly as complexes with monomer sequestering proteins. These structures may represent transient storage sites of G-actin within the cell which can be transformed rapidly into actin filaments upon stimulation by specific signals.

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Selected References

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  1. 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]
  2. Blikstad I., Markey F., Carlsson L., Persson T., Lindberg U. Selective assay of monomeric and filamentous actin in cell extracts, using inhibition of deoxyribonuclease I. Cell. 1978 Nov;15(3):935–943. doi: 10.1016/0092-8674(78)90277-5. [DOI] [PubMed] [Google Scholar]
  3. Bonder E. M., Fishkind D. J., Cotran N. M., Begg D. A. The cortical actin-membrane cytoskeleton of unfertilized sea urchin eggs: analysis of the spatial organization and relationship of filamentous actin, nonfilamentous actin, and egg spectrin. Dev Biol. 1989 Aug;134(2):327–341. doi: 10.1016/0012-1606(89)90105-x. [DOI] [PubMed] [Google Scholar]
  4. Bray D., Thomas C. Unpolymerized actin in fibroblasts and brain. J Mol Biol. 1976 Aug 25;105(4):527–544. doi: 10.1016/0022-2836(76)90233-3. [DOI] [PubMed] [Google Scholar]
  5. Burgess D. R., Schroeder T. E. Polarized bundles of actin filaments within microvilli of fertilized sea urchin eggs. J Cell Biol. 1977 Sep;74(3):1032–1037. doi: 10.1083/jcb.74.3.1032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cao L. G., Wang Y. L. Mechanism of the formation of contractile ring in dividing cultured animal cells. I. Recruitment of preexisting actin filaments into the cleavage furrow. J Cell Biol. 1990 Apr;110(4):1089–1095. doi: 10.1083/jcb.110.4.1089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cooper J. A. The role of actin polymerization in cell motility. Annu Rev Physiol. 1991;53:585–605. doi: 10.1146/annurev.ph.53.030191.003101. [DOI] [PubMed] [Google Scholar]
  8. Coué M., Constans J., Olomucki A. Effects of serum vitamin-D-binding protein on actin in the presence of plasma gelsolin. Eur J Biochem. 1986 Oct 15;160(2):273–277. doi: 10.1111/j.1432-1033.1986.tb09967.x. [DOI] [PubMed] [Google Scholar]
  9. D'Andrea L., Fishkind D. J., Begg D. A., Bonder E. M. Isolation and localization of a spectrin-like protein from echinoderm sperm. Cell Motil Cytoskeleton. 1991;19(1):49–61. doi: 10.1002/cm.970190107. [DOI] [PubMed] [Google Scholar]
  10. DuBose D. A., Haugland R. Comparisons of endothelial cell G- and F-actin distribution in situ and in vitro. Biotech Histochem. 1993 Jan;68(1):8–16. doi: 10.3109/10520299309105570. [DOI] [PubMed] [Google Scholar]
  11. Fishkind D. J., Cao L. G., Wang Y. L. Microinjection of the catalytic fragment of myosin light chain kinase into dividing cells: effects on mitosis and cytokinesis. J Cell Biol. 1991 Sep;114(5):967–975. doi: 10.1083/jcb.114.5.967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Giuliano K. A., Taylor D. L. Formation, transport, contraction, and disassembly of stress fibers in fibroblasts. Cell Motil Cytoskeleton. 1990;16(1):14–21. doi: 10.1002/cm.970160104. [DOI] [PubMed] [Google Scholar]
  13. Goldschmidt-Clermont P. J., Galbraith R. M., Emerson D. L., Marsot F., Nel A. E., Arnaud P. Distinct sites on the G-actin molecule bind group-specific component and deoxyribonuclease I. Biochem J. 1985 Jun 1;228(2):471–477. doi: 10.1042/bj2280471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hartwig J. H., Kwiatkowski D. J. Actin-binding proteins. Curr Opin Cell Biol. 1991 Feb;3(1):87–97. doi: 10.1016/0955-0674(91)90170-4. [DOI] [PubMed] [Google Scholar]
  15. Heath J. P., Holifield B. F. Cell locomotion: new research tests old ideas on membrane and cytoskeletal flow. Cell Motil Cytoskeleton. 1991;18(4):245–257. doi: 10.1002/cm.970180402. [DOI] [PubMed] [Google Scholar]
  16. Herman I. M., Pollard T. D. Actin localization in fixed dividing cells stained with fluorescent heavy meromyosin. Exp Cell Res. 1978 Jun;114(1):15–25. doi: 10.1016/0014-4827(78)90030-7. [DOI] [PubMed] [Google Scholar]
  17. Holifield B. F., Jacobson K. Mapping trajectories of Pgp-1 membrane protein patches on surfaces of motile fibroblasts reveals a distinct boundary separating capping on the lamella and forward transport on the retracting tail. J Cell Sci. 1991 Feb;98(Pt 2):191–203. doi: 10.1242/jcs.98.2.191. [DOI] [PubMed] [Google Scholar]
  18. Hoock T. C., Newcomb P. M., Herman I. M. Beta actin and its mRNA are localized at the plasma membrane and the regions of moving cytoplasm during the cellular response to injury. J Cell Biol. 1991 Feb;112(4):653–664. doi: 10.1083/jcb.112.4.653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Knowles G. C., McCulloch C. A. Simultaneous localization and quantification of relative G and F actin content: optimization of fluorescence labeling methods. J Histochem Cytochem. 1992 Oct;40(10):1605–1612. doi: 10.1177/40.10.1527379. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Lee W. M., Galbraith R. M. The extracellular actin-scavenger system and actin toxicity. N Engl J Med. 1992 May 14;326(20):1335–1341. doi: 10.1056/NEJM199205143262006. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Lin J. J. Monoclonal antibodies against myofibrillar components of rat skeletal muscle decorate the intermediate filaments of cultured cells. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2335–2339. doi: 10.1073/pnas.78.4.2335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McKenna N. M., Wang Y. L., Konkel M. E. Formation and movement of myosin-containing structures in living fibroblasts. J Cell Biol. 1989 Sep;109(3):1163–1172. doi: 10.1083/jcb.109.3.1163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mckenna N. M., Wang Y. L. Culturing cells on the microscope stage. Methods Cell Biol. 1989;29:195–205. doi: 10.1016/s0091-679x(08)60195-8. [DOI] [PubMed] [Google Scholar]
  26. Mitchison T., Kirschner M. Cytoskeletal dynamics and nerve growth. Neuron. 1988 Nov;1(9):761–772. doi: 10.1016/0896-6273(88)90124-9. [DOI] [PubMed] [Google Scholar]
  27. Nachmias V. T. Small actin-binding proteins: the beta-thymosin family. Curr Opin Cell Biol. 1993 Feb;5(1):56–62. doi: 10.1016/s0955-0674(05)80008-0. [DOI] [PubMed] [Google Scholar]
  28. Pinder J. C., Gratzer W. B. Investigation of the actin-deoxyribonuclease I interaction using a pyrene-conjugated actin derivative. Biochemistry. 1982 Sep 28;21(20):4886–4890. doi: 10.1021/bi00263a009. [DOI] [PubMed] [Google Scholar]
  29. Podolski J. L., Steck T. L. Association of deoxyribonuclease I with the pointed ends of actin filaments in human red blood cell membrane skeletons. J Biol Chem. 1988 Jan 15;263(2):638–645. [PubMed] [Google Scholar]
  30. Safer D. The interaction of actin with thymosin beta 4. J Muscle Res Cell Motil. 1992 Jun;13(3):269–271. doi: 10.1007/BF01766454. [DOI] [PubMed] [Google Scholar]
  31. Satterwhite L. L., Pollard T. D. Cytokinesis. Curr Opin Cell Biol. 1992 Feb;4(1):43–52. doi: 10.1016/0955-0674(92)90057-j. [DOI] [PubMed] [Google Scholar]
  32. Singer R. H. The cytoskeleton and mRNA localization. Curr Opin Cell Biol. 1992 Feb;4(1):15–19. doi: 10.1016/0955-0674(92)90053-f. [DOI] [PubMed] [Google Scholar]
  33. Spudich A., Wrenn J. T., Wessells N. K. Unfertilized sea urchin eggs contain a discrete cortical shell of actin that is subdivided into two organizational states. Cell Motil Cytoskeleton. 1988;9(1):85–96. doi: 10.1002/cm.970090109. [DOI] [PubMed] [Google Scholar]
  34. Theriot J. A., Mitchison T. J. Actin microfilament dynamics in locomoting cells. Nature. 1991 Jul 11;352(6331):126–131. doi: 10.1038/352126a0. [DOI] [PubMed] [Google Scholar]
  35. Tilney L. G. The polymerization of actin. II. How nonfilamentous actin becomes nonrandomly distributed in sperm: evidence for the association of this actin with membranes. J Cell Biol. 1976 Apr;69(1):51–72. doi: 10.1083/jcb.69.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tilney L. G. The polymerization of actin. III. Aggregates of nonfilamentous actin and its associated proteins: a storage form of actin. J Cell Biol. 1976 Apr;69(1):73–89. doi: 10.1083/jcb.69.1.73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Van Baelen H., Bouillon R., De Moor P. Vitamin D-binding protein (Gc-globulin) binds actin. J Biol Chem. 1980 Mar 25;255(6):2270–2272. [PubMed] [Google Scholar]
  38. Wang E., Goldberg A. R. Binding of deoxyribonuclease I to actin: a new way to visualize microfilament bundles in nonmuscle cells. J Histochem Cytochem. 1978 Sep;26(9):745–749. doi: 10.1177/26.9.361884. [DOI] [PubMed] [Google Scholar]
  39. Wang Y. L. Exchange of actin subunits at the leading edge of living fibroblasts: possible role of treadmilling. J Cell Biol. 1985 Aug;101(2):597–602. doi: 10.1083/jcb.101.2.597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wang Y. L. Reorganization of actin filament bundles in living fibroblasts. J Cell Biol. 1984 Oct;99(4 Pt 1):1478–1485. doi: 10.1083/jcb.99.4.1478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wieland T. Modification of actins by phallotoxins. Naturwissenschaften. 1977 Jun;64(6):303–309. doi: 10.1007/BF00446784. [DOI] [PubMed] [Google Scholar]
  42. Wulf E., Deboben A., Bautz F. A., Faulstich H., Wieland T. Fluorescent phallotoxin, a tool for the visualization of cellular actin. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4498–4502. doi: 10.1073/pnas.76.9.4498. [DOI] [PMC free article] [PubMed] [Google Scholar]

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