Chromosome Research

, 16:427 | Cite as

Dynamics of the CapG actin-binding protein in the cell nucleus studied by FRAP and FCS



FRAP (fluorescence recovery after photobleaching) and FCS (fluorescence correlation spectroscopy) are spectroscopic methods for monitoring the dynamic distribution of proteins inside the nucleus of living cells. As an example we report our studies on the intracellular mobility of the actin-binding protein CapG in live breast cancer cells. This Gelsolin-related protein is a putative oncogene. It appears to be overexpressed especially in metastasizing breast cancer. Furthermore, the CapG protein is known to be involved in the motility control of non-muscle benign cells. Its increased expression triggers an increase in cell motility of benign cells. Thus it can be expected that in cancer cells overexpressing the CapG protein, motility, invasiveness and metastasis might be particularly promoted. Since the nuclear CapG fraction seems to be pivotal to the increase in cell motility, we focused our studies on the CapG mobility in cell nuclei of live breast cancer cells. Using FCS and FRAP we showed that the eGFP-tagged CapG is monomeric and characterized its diffusional properties on the microsecond to minute timescale. This information about the mobility and compartmentalization of CapG might help to provide insight into its function within the cell nucleus and give clues about its altered cellular function in malignant dedifferentiation.

Key words

actin-binding protein breast cancer intranuclear mobility live cell analysis 


  1. Axelrod D, Koppel DE, Schlessinger J, Elson EL, Webb WW (1976) Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J 16: 1055–1069.PubMedGoogle Scholar
  2. Dahl E, Sadr-Nabavi A, Klopocki E et al. (2005) Systematic identification and molecular characterization of genes differentially expressed in breast and ovarian cancer. J Pathol 205: 21–28.PubMedCrossRefGoogle Scholar
  3. De Corte V, Van Impe K, Bruyneel E et al. (2004) Increased importin-beta-dependent nuclear import of the actin modulating protein CapG promotes cell invasion. J Cell Sci 117: 5283–5292.PubMedCrossRefGoogle Scholar
  4. Elson EL (1986) Fluorescence photobleaching and correlation spectroscopy for translational diffusion in biological systems. Biochem Soc Trans 14: 839–841.PubMedGoogle Scholar
  5. Elson EL, Qian H (1989) Interpretation of fluorescence correlation spectroscopy and photobleaching recovery in terms of molecular interactions. Methods Cell Biol 30: 307–332.PubMedGoogle Scholar
  6. Gettemans J, Van Impe K, Delanote V, Hubert T, Vandekerckhove J, De Corte V (2005) Nuclear actin-binding proteins as modulators of gene transcription. Traffic 6: 847–857.PubMedCrossRefGoogle Scholar
  7. Goerisch SM, Wachsmuth M, Toth KF, Lichter P, Rippe K (2005) Histone acetylation increases chromatin accessibility. J Cell Sci 118: 5825–5834.CrossRefGoogle Scholar
  8. Lippincott-Schwartz J, Snapp E, Kennworthy A (2001) Studying protein dynamics in living cells. Nat Rev Mol Cell Biol 2(6): 444–456.PubMedCrossRefGoogle Scholar
  9. Lu PJ, Hsu AL, Wang DS, Yan HY, Yin HL, Chen CS (1998) Phosphoinositide 3-kinase in rat liver nuclei. Biochemistry 37: 5738–5745.PubMedCrossRefGoogle Scholar
  10. Onoda K, Yu FX, Yin HL (1993) gCap39 is a nuclear and cytoplasmatic protein. Cell Motil Cytoskel 26(3): 227–238.CrossRefGoogle Scholar
  11. Parikh SS, Litherland SA, Clare-Salzler MJ, Li W, Gulig PA, Southwick FS (2003) CapG(−/−) mice have specific host defense defects that render them more susceptible than CapG (+/+) mice to Listeria monocytogenes infection but not to Salmonella enterica serovar Typhimurium infection. Infect Immun 71(11): 6582–6590.PubMedCrossRefGoogle Scholar
  12. Pellieux C, Desgeorges A, Pigeon CH et al. (2003) CapG, a gelsolin family protein modulating protective effects of unidirectional shear stress. J Biol Chem 278: 29136–29144.PubMedCrossRefGoogle Scholar
  13. Petersen NO, Elson EL (1986) Measurements of diffusion and chemical kinetics by fluorescence photobleaching recovery and fluorescence correlation spectroscopy. Methods Enzymol 130: 454–484.PubMedGoogle Scholar
  14. Prendergast GC, Ziff EB (1991) Mbh1: a novel gelsolin/severin-related protein which binds actin in vitro and exhibits nuclear localization. EMBO J 10(4): 757–766.PubMedGoogle Scholar
  15. Qian H, Elson EL, Frieden C (1992) Studies on the structure of actin gels using time correlation spectroscopy of fluorescent beads. Biophys J 63: 1000–1010.PubMedGoogle Scholar
  16. Rabut G, Ellenberg J (2005) Photobleaching techniques to study mobility and molecular dynamics of proteins in live cells: FRAP, iFRAP, and FLIP. In: Goldman R, Spector DL, eds. Live Cell Imaging. A Laboratory Manual. New York: Cold Spring Harbor Press, pp. 101–126.Google Scholar
  17. Renz M, Betz B, Niederacher D, Bender HG, Langowski J (2007) Invasive breast cancer cells exhibit increased mbility of actin-binding protein CapG. Int J Cancer 122(7): 1476–1482.CrossRefGoogle Scholar
  18. Silacci P, Mazzolai L, Gauci C, Stergiopulos N, Yin HL, Hayoz D (2004) Gelsolin superfamily proteins: key regulators of cellular functions. Cell Mol Life Sci 61: 2614–2623.PubMedCrossRefGoogle Scholar
  19. Southwick S, DiNubile MJ (1986) Rabbit alveolar macrophages contain a Ca2+-sensitive, 41,000-Dalton protein which reversibly blocks the “barbed” ends of actin filaments but does not sever them. J Biol Chem 261(30): 14191–14195.PubMedGoogle Scholar
  20. Sun HQ, Kwiatkowska K, Wooten DC, Yin HL (1995) Effects of CapG overexpression on agonist-induced motility and second messenger generation. J Cell Biol 129(1): 147–156.PubMedCrossRefGoogle Scholar
  21. Thompson CC, Ashcroft FJ, Patel S et al. (2007) Pancreatic cancer cells overexpress gelsolin family capping proteins which contribute to their cell motility. Gut 56(1): 95–106.PubMedCrossRefGoogle Scholar
  22. Van Impe K, De Corte V, Eichinger L et al. (2003) The nucleo-cytoplasmic actin-binding protein CapG lacks a nuclear export sequence present in structurally related proteins. J Biol Chem 278(20): 17945–17952.PubMedCrossRefGoogle Scholar
  23. Vukojevic V, Pramanik A, Yakovleva T, Rigler R, Terenius L, Bakalkin G (2005) Study of molecular events in cells by fluorescence correlation spectroscopy. Cell Mol Life Sci 62(5): 535–550.PubMedCrossRefGoogle Scholar
  24. Wachsmuth M, Waldeck W, Langowski J (2000) Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatiall-resolved fluorescence correlation spectroscopy. J Mol Biol 298(4): 677–689.PubMedCrossRefGoogle Scholar
  25. Wachsmuth M, Weidemann T, Müller G et al. (2003) Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching. Biophys J 84(5): 3353–3363.PubMedCrossRefGoogle Scholar
  26. Weidemann T, Wachsmuth M, Knoch TA, Müller G, Waldeck W, Langowski J (2003) Counting nucleosomes in living cells with a combination of fluorescence correlation spectroscopy and confocal imaging. J Mol Biol 334(2): 229–240.PubMedCrossRefGoogle Scholar
  27. Witke W, Li W, Kwiatkwoski DJ, Southwick FS (2001) Comparisons of CapG and gelsolin-null macrophages: demonstration of a unique role for CapG in receptor-mediated ruffling, phagocytosis, and vesicle rocketing. J Cell Biol 154(4): 775–784.PubMedCrossRefGoogle Scholar
  28. Zhang Y, Vorobiev SM, Gibson BG et al. (2006) A CapG gain-of-function mutant reveals critical structural and functional determinants for actin filament severing. EMBO J 25: 4458–4467.PubMedCrossRefGoogle Scholar

Copyright information

© Springer 2008

Authors and Affiliations

  1. 1.German Cancer Research Center, Division Biophysics of MacromoleculesIm Neuenheimer Feld 580HeidelbergGermany
  2. 2.Department of Obstetrics and GynecologyHeinrich-Heine-University DüsseldorfDüsseldorfGermany

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