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. 1988 Apr 1;106(4):1229–1240. doi: 10.1083/jcb.106.4.1229

Localization and mobility of gelsolin in cells

PMCID: PMC2115018  PMID: 2834402

Abstract

To investigate the physiologic role of gelsolin in cells, we have studied the location and mobility of gelsolin in a mouse fibroblast cell line (C3H). Gelsolin was localized by immunofluorescence of fixed and permeabilized cells and by fluorescent analog cytochemistry of living cells and cells that were fixed and/or permeabilized. Overall, the images show that in living cells gelsolin has a diffuse cytoplasmic distribution, but in fixed cells a minor fraction is associated with regions of the cell that are rich in actin filaments. The latter fraction is more prominent after permeabilization of the fixed cells because some diffuse gelsolin is not fixed and is therefore lost during permeabilization, confirmed by immunoblots. To determine quantitatively whether gelsolin is bound to actin filaments in living cells, we measured the mobility of microinjected fluorescent gelsolin by fluorescence photobleaching recovery. Gelsolin is fully mobile with a diffusion coefficient similar to that of control proteins. As a positive control, fluorescent phalloidin, which binds actin filaments, is totally immobile. These results are supported by immunoblots on cells permeabilized with detergent. All the endogenous gelsolin is extracted, and the half-time for the extraction is approximately 5 s, which is about the rate predicted for diffusion. Therefore, gelsolin is not tightly bound to actin filaments in cells. The most likely interpretation of the difference between living and fixed cells is that fixation traps a fraction of gelsolin that is associated with actin filaments in short-lived complexes.

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Selected References

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  1. Axelrod D., Koppel D. E., Schlessinger J., Elson E., Webb W. W. Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J. 1976 Sep;16(9):1055–1069. doi: 10.1016/S0006-3495(76)85755-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berg O. G. Effective diffusion rate through a polymer network: influence of nonspecific binding and intersegment transfer. Biopolymers. 1986 May;25(5):811–821. doi: 10.1002/bip.360250506. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Bryan J., Hwo S. Definition of an N-terminal actin-binding domain and a C-terminal Ca2+ regulatory domain in human brevin. J Cell Biol. 1986 Apr;102(4):1439–1446. doi: 10.1083/jcb.102.4.1439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carron C. P., Hwo S. Y., Dingus J., Benson D. M., Meza I., Bryan J. A re-evaluation of cytoplasmic gelsolin localization. J Cell Biol. 1986 Jan;102(1):237–245. doi: 10.1083/jcb.102.1.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chaponnier C., Janmey P. A., Yin H. L. The actin filament-severing domain of plasma gelsolin. J Cell Biol. 1986 Oct;103(4):1473–1481. doi: 10.1083/jcb.103.4.1473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cooper J. A., Bryan J., Schwab B., 3rd, Frieden C., Loftus D. J., Elson E. L. Microinjection of gelsolin into living cells. J Cell Biol. 1987 Mar;104(3):491–501. doi: 10.1083/jcb.104.3.491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cooper J. A. Effects of cytochalasin and phalloidin on actin. J Cell Biol. 1987 Oct;105(4):1473–1478. doi: 10.1083/jcb.105.4.1473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cooper J. A., Walker S. B., Pollard T. D. Pyrene actin: documentation of the validity of a sensitive assay for actin polymerization. J Muscle Res Cell Motil. 1983 Apr;4(2):253–262. doi: 10.1007/BF00712034. [DOI] [PubMed] [Google Scholar]
  10. Craig S. W., Lancashire C. L., Cooper J. A. Preparation of smooth muscle alpha-actinin. Methods Enzymol. 1982;85(Pt B):316–321. doi: 10.1016/0076-6879(82)85031-3. [DOI] [PubMed] [Google Scholar]
  11. Ey P. L., Ashman L. K. The use of alkaline phosphatase-conjugated anti-immunoglobulin with immunoblots for determining the specificity of monoclonal antibodies to protein mixtures. Methods Enzymol. 1986;121:497–509. doi: 10.1016/0076-6879(86)21050-2. [DOI] [PubMed] [Google Scholar]
  12. Fujiwara K., Pollard T. D. Fluorescent antibody localization of myosin in the cytoplasm, cleavage furrow, and mitotic spindle of human cells. J Cell Biol. 1976 Dec;71(3):848–875. doi: 10.1083/jcb.71.3.848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gibson W. Polyoma virus proteins: a description of the structural proteins of the virion based on polyacrylamide gel electrophoresis and peptide analysis. Virology. 1974 Dec;62(2):319–336. doi: 10.1016/0042-6822(74)90395-x. [DOI] [PubMed] [Google Scholar]
  14. Hunkapiller M. W., Lujan E., Ostrander F., Hood L. E. Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods Enzymol. 1983;91:227–236. doi: 10.1016/s0076-6879(83)91019-4. [DOI] [PubMed] [Google Scholar]
  15. Hwo S., Bryan J. Immuno-identification of Ca2+-induced conformational changes in human gelsolin and brevin. J Cell Biol. 1986 Jan;102(1):227–236. doi: 10.1083/jcb.102.1.227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jacobson K., Wojcieszyn J. The translational mobility of substances within the cytoplasmic matrix. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6747–6751. doi: 10.1073/pnas.81.21.6747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kwiatkowski D. J., Stossel T. P., Orkin S. H., Mole J. E., Colten H. R., Yin H. L. Plasma and cytoplasmic gelsolins are encoded by a single gene and contain a duplicated actin-binding domain. Nature. 1986 Oct 2;323(6087):455–458. doi: 10.1038/323455a0. [DOI] [PubMed] [Google Scholar]
  18. Nodes B. R., Shackelford J. E., Lebherz H. G. Synthesis and secretion of serum gelsolin by smooth muscle tissue. J Biol Chem. 1987 Apr 15;262(11):5422–5427. [PubMed] [Google Scholar]
  19. Pollard T. D., Cooper J. A. Actin and actin-binding proteins. A critical evaluation of mechanisms and functions. Annu Rev Biochem. 1986;55:987–1035. doi: 10.1146/annurev.bi.55.070186.005011. [DOI] [PubMed] [Google Scholar]
  20. Rouayrenc J. F., Fattoum A., Gabrion J., Audemard E., Kassab R. Muscle gelsolin: isolation from heart tissue and characterization as an integral myofibrillar protein. FEBS Lett. 1984 Feb 13;167(1):52–58. doi: 10.1016/0014-5793(84)80831-5. [DOI] [PubMed] [Google Scholar]
  21. Sanger J. W., Mittal B., Sanger J. M. Interaction of fluorescently-labeled contractile proteins with the cytoskeleton in cell models. J Cell Biol. 1984 Sep;99(3):918–928. doi: 10.1083/jcb.99.3.918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Scott R. E., Perkins R. G., Zschunke M. A., Hoerl B. J., Maercklein P. B. Plasma membrane vesiculation in 3T3 and SV3T3 cells. I. Morphological and biochemical characterization. J Cell Sci. 1979 Feb;35:229–243. doi: 10.1242/jcs.35.1.229. [DOI] [PubMed] [Google Scholar]
  23. Soua Z., Porte F., Harricane M. C., Feinberg J., Capony J. P. Bovine serum brevin. Purification by hydrophobic chromatography and properties. Eur J Biochem. 1985 Dec 2;153(2):275–287. doi: 10.1111/j.1432-1033.1985.tb09298.x. [DOI] [PubMed] [Google Scholar]
  24. Stossel T. P., Chaponnier C., Ezzell R. M., Hartwig J. H., Janmey P. A., Kwiatkowski D. J., Lind S. E., Smith D. B., Southwick F. S., Yin H. L. Nonmuscle actin-binding proteins. Annu Rev Cell Biol. 1985;1:353–402. doi: 10.1146/annurev.cb.01.110185.002033. [DOI] [PubMed] [Google Scholar]
  25. Tank D. W., Wu E. S., Webb W. W. Enhanced molecular diffusibility in muscle membrane blebs: release of lateral constraints. J Cell Biol. 1982 Jan;92(1):207–212. doi: 10.1083/jcb.92.1.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Taylor D. L., Wang Y. L. Fluorescently labelled molecules as probes of the structure and function of living cells. Nature. 1980 Apr 3;284(5755):405–410. doi: 10.1038/284405a0. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Wang E., Yin H. L., Krueger J. G., Caliguiri L. A., Tamm I. Unphosphorylated gelsolin is localized in regions of cell-substratum contact or attachment in Rous sarcoma virus-transformed rat cells. J Cell Biol. 1984 Feb;98(2):761–771. doi: 10.1083/jcb.98.2.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wu E. S., Tank D. W., Webb W. W. Unconstrained lateral diffusion of concanavalin A receptors on bulbous lymphocytes. Proc Natl Acad Sci U S A. 1982 Aug;79(16):4962–4966. doi: 10.1073/pnas.79.16.4962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Yin H. L., Albrecht J. H., Fattoum A. Identification of gelsolin, a Ca2+-dependent regulatory protein of actin gel-sol transformation, and its intracellular distribution in a variety of cells and tissues. J Cell Biol. 1981 Dec;91(3 Pt 1):901–906. doi: 10.1083/jcb.91.3.901. [DOI] [PMC free article] [PubMed] [Google Scholar]

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