Skip to main content
SpringerLink
Log in

Comparative analysis of subcellular fractionation methods for revealing α-actinin 1 and α-actinin 4 in A431 cells

  • Published:
Cell and Tissue Biology Aims and scope Submit manuscript

Abstract

α-Actinin 1 and α-actinin 4 are actin-binding proteins with shared structural functions that are responsible for the regulation of several processes in the cell. Based on previous data on the different distribution of these proteins in the nucleus and cytoplasm, we have studied in detail the presence of α-actinin 1 and α-actinin 4 in subcellular fractions in the A431 cells spread on fibronectin. The detection of α-actinins in some particular fractions has been shown to depend on the method of lysis, as well as whether the preliminary low-temperature freezing of cells was used. The application of various fractionation methods has allowed us to conclude that α-actinin 4 is present in all cytoplasmic and nuclear subfractions, whereas, in addition to in the cytoplasm, α-actinin 1 can also be revealed in the nuclear envelope fraction.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Are, A.F., Pinaev, G.P., Burova, E., and Lindberg, U., Attachment of A-431 Cells on Immobilized Antibodies to the EGF Receptor Promotes Cell Spreading and Reorganization of the Microfilament System, Cell Motil. Cytoskelet., 2001, vol. 48, pp. 24–36.

    Article  CAS  Google Scholar 

  • Babakov, V.N., Bobkov, D.E., Petukhova, O.A., Turoverova, L.V., Kropacheva, I.V., Podol’skaya, E.P., and Pinaev, G.P., Alpha-Actinin-4 and p65/RelA Subunit of NF-kappaB Transcription Factor Are Co-Localized and Migrate Together into the Nucleus in EGF-Stimulated A431 cell, Tsitologiia, 2004, vol. 46, no. 12, pp. 1064–1072.

    PubMed  CAS  Google Scholar 

  • Babakov, V.N., Kropacheva, I.V., Petukhova, O.A., Turoverova, L.V., and Pinaev, G.P., Intracellular Distribution of Tyrosine-Phosphorylated Actin-Binding Proteins in A431 Cells Spread on Different Ligands, Tsitologiia, 2004, vol. 46, no. 12, pp. 1055–1063.

    PubMed  CAS  Google Scholar 

  • Babakov, V.N., Petukhova, O.A., Turoverova, L.V., Kropacheva, I.V., Tentler, D.G., Bolshakova, A.V., Podolskaya, E.P., Magnusson, K.E., and Pinaev, G.P., RelA / NF-KappaB Transcription Factor Associates with Alpha-Actinin-4, Exp Cell Res., 2008, vol. 314, pp. 1030–1038.

    Article  PubMed  CAS  Google Scholar 

  • Batchelor, C.L., Woodward, A.M., and Crouch, D.H., Nuclear ERM (Ezrin, Radixin, Moesin) Proteins: Regulation by Cell Density and Nuclear Import, Exp. Cell Res., 2004, vol. 296, pp. 208–222.

    Article  PubMed  CAS  Google Scholar 

  • Benmerah, A., Scott, M., Poupon, V., and Marullo, S., Nuclear Functions for Plasma Membrane-Associated Proteins, Traffic, 2003, vol. 4, pp. 503–511.

    PubMed  CAS  Google Scholar 

  • Bershadsky, A.D. and Vasiliev, J.M., Cytoskeleton, New York: Plenum, 1988, p. 298.

    Google Scholar 

  • Blessing, C.A., Ugrinova, G.T., and Goodson, H.V., Actin and ARPs: Action in the Nucleus, Trends Cell Biol., 2004, vol. 14, pp. 435–442.

    Article  PubMed  CAS  Google Scholar 

  • Blobel, G. and Potter, V.R., Nuclei from Rat Liver: Isolation Method that Combines Purity with High Yield, Science, 1966, vol. 154, pp. 1662–1665.

    Article  PubMed  CAS  Google Scholar 

  • Bolshakova, A., Petukhova, O., Turoverova, L., Tentler, D., Babakov, V., Magnusson, K.-E., and Pinaev, G., Extra-Cellular Matrix Proteins Induce Re-Distribution of α-Actinin 1 and α-Actinin-4 in A431 Cells, Cell Biol. Int., 2007, vol. 31, pp. 360–365.

    Article  PubMed  CAS  Google Scholar 

  • Bredford, M.M., A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Proteins Utilizing the Principle of Protein-Dye Binding, Anal. Biochem., 1976, vol. 72, pp. 248–254.

    Article  Google Scholar 

  • Chakraborty, S., Reineke, E.L., Lam, M., Li, X., Liu, Y., Gao, C., Khurana, S., and Kao, H.Y., Alpha-Aactinin 4 Potentiates Myocyte Enhancer Factor-2 Transcription Activity by Antagonizing Histone Deacetylase 7, J. Biol. Chem., 2006, vol. 281, pp. 35 070–35 080.

    CAS  Google Scholar 

  • Chiang, E.T., Lim, M.J., Patton, W.F., and Shepro, D., NF-kappaB Translocation in Human Microvessel Endothelial Cells Using a Four-Compartment Subcellular Protein Redistribution Assay, J. Biochem. Biophys. Methods, 2000, vol. 46, pp. 53–68.

    Article  PubMed  CAS  Google Scholar 

  • Communal, C., Sumandea, M., De Tombe, P., Narula, J., Solaro, R.J., and Hajjar, H., Functional Consequences of Caspase Activation in Cardiac Myocytes, Proc. Natl. Acad. Sci. USA, 2002, vol. 99, pp. 6252–6256.

    Article  PubMed  CAS  Google Scholar 

  • Dwyer, N. and Blobel, G., A Modified Procedure for the Isolation of Pore Complex-Lamina Fraction from Rat Liver Nuclei, The J. Cell Biol., 1976, vol. 70, pp. 581–591.

    Article  CAS  Google Scholar 

  • Fazal, F., Minhajuddin, M., Bijli, K.M., McGrath, J.L., and Rahman, A., Evidence for Actin Cytoskeleton-Dependent and -Independent Pathways for RelA p65 Nuclear Translocation in Endothelial Cells, J. Biol. Chem., 2007, vol. 282, pp. 3940–3950.

    Article  PubMed  CAS  Google Scholar 

  • Fey, E.G., Wan, K.M., and Penman, S., Epithelial Cytoskeletal Framework and Nuclear Matrix-Intermediate Filament Scaffold: Three-Dimensional Organization and protein composition, J. Cell Biol., 1984, vol. 98, pp. 1973–1984.

    Article  PubMed  CAS  Google Scholar 

  • Fricker, M., Hollinshead, M., White, N., and Vaux, D., Interphase Nuclei of Many Mammalian Cell Typer Contain Deep, Dynamic, Tubular Membrane-Bound Invaginations of the Nuclear Envelope, J. Cel Biol., 1997, vol. 136, pp. 531–544.

    Article  CAS  Google Scholar 

  • Gluck, U. and Ben-Ze’ev A. Modulation of Alpha-Actinin Levels Affects Cell Motility and Confers Tumorigenicity on 3T3 Cells, J. Nell Sci., 1994, vol. 107, pp. 1773–1782.

    CAS  Google Scholar 

  • Goffart, S., Franko, A., Clemen, C.S., and Wiesner, R.J., Alpha-Actinin and BAT 1 Interaction with Cytochrome c Promoter upon Skeletal Muscle Differentiation, Current Genetics, 2006, vol. 49, pp. 125–135.

    Article  PubMed  CAS  Google Scholar 

  • Gubanova, N.V. and Kiseleva, E.V., Structural Organization and Function of Nuclear Envelope, Tsitologiia, 2007, vol. 49, no. 4, pp. 257–269.

    PubMed  CAS  Google Scholar 

  • Harrington, E.O., Smeglin, A., Newton, J., Ballard, G., and Rounds, S., Protein Tyrosine Phosphatase-Dependent Proteolysis of Focal Adhesion Complexes in Endothelial Cell Apoptosis, Am. J. Physiol. Lung Cell Mol. Physiol., 2001, vol. 280, pp. 342–353.

    Google Scholar 

  • Honda, K., Yamada, T., Endo, R., Ino, Y., Gotoh, M., Tsuda, Y., Yamada, Y., Chiba, H., and Hirohashi, S., Actinin-4, a Novel Actin-Bundling Protein Associated with Cell Motility and Cancer Invasion, J. Cell Biol., 1998, vol. 140, pp. 1383–1393.

    Article  PubMed  CAS  Google Scholar 

  • Immure, M., Endo, T., Kuroda, M., Tanaka, T., and Masaki, T., Substructure and Higher Structure of Chicken Smooth Muscle α-Actinin Molecule, J. Biol. Chem., 1988, vol. 263, pp. 7800–7805.

    Google Scholar 

  • Laemmli, U.K., Cleavage of Structural Proteins During the Assembly of the Head of the Bacteriophage T4, Nature, 1970, vol. 227, pp. 680–685.

    Article  PubMed  CAS  Google Scholar 

  • Liu, B.-L. and McGrath, J., Response of Cytoskeleton of Murine Osteoblast Cultures to Two-Step Freezing, Acta Biochim. Biophys., 2005, vol. 37, pp. 814–818.

    CAS  Google Scholar 

  • Luikart, S., Masri, M., Wahl, D., Hinkel, T., Beck, J.M., Gyetko, M.R., Gupta, P., and Oegema, T., Urokinase is Required for the Formation of Mactinin, an Alpha-Actinin Fragment that Promotes Monocyte/Macrophage Maturation, Biochim. Biophys Acta, 2002, vol. 1591, pp. 99–107.

    Article  PubMed  CAS  Google Scholar 

  • Marzlav, U. and Khuan, R., RNA Transcription in Isolated Nuclei: Transcription and Translation, Moscow: Mir, 1987, pp. 111–159 [in Russian].

    Google Scholar 

  • McMahon, L.W., Sangerman, J., Goodman, S.R., Kumaresan, K., and Lambert, M.W., Human Alpha Spectrin II and the FANCA, FANCC, and FANCG Proteins Bind to DNA Containing Psoralen Interstrand Cross-Links, Biochemistry, 2001, vol. 40, pp. 7025–7034.

    Article  PubMed  CAS  Google Scholar 

  • McMahon, L.W., Walsh, C.E., and Lambert, M.W., Human Alpha Spectrin II and the Fanconi Anemia Proteins FANCA and FANCC Interact to Form a Nuclear Complex, Biol. Chem., 1999, vol. 274, pp. 32 904–32 908.

    CAS  Google Scholar 

  • Meredith, J., Jr., Mu, Z., Saido, T., and Du, X., Cleavage of the Cytoplasmic Domain of the Integrin Beta3 Subunit During Endothelial Cell Apoptosis, J. Biol. Chem., 1998, vol. 273, pp. 19 525–19 531.

    CAS  Google Scholar 

  • Otey, C.A. and Carpen, O., α-Actinin Revisited: A Fresh Look at an Old Player. Cell Motility, Cytoskeleton, 2004, vol. 58, pp. 104–111.

    Article  CAS  Google Scholar 

  • Papakonstanti, E.A., Vardaki, E.A., and Stournaras, C., Actin Cytoskeleton: A Signaling Sensor in Cell Volume Regulation, Cell Physiol. Biochem., 2000, vol. 10, pp. 257–264.

    Article  PubMed  CAS  Google Scholar 

  • Pavalko, F.M., Chen, N.X., Turner, C.H., Burr, D.B., Atkinson, S., Hsieh, Y.F., Qiu, J., and Duncan, R.L., Fluid Shear-Induced Mechanical Signaling in MC3T3-E1 Osteoblasts Requires Cytoskeleton-Integrin Interactions, Am. J. Physiol., 1998, vol. 275, pp. 1591–1601.

    Google Scholar 

  • Pederson, T. and Aebi, U., Actin in the Nucleus: What Form and what for?, J. Struct. Biol., 2002, vol. 140, pp. 3–9.

    Article  PubMed  CAS  Google Scholar 

  • Petukhova, O.A., Turoverova, L.V., Emel’yanov, A.N., Kropacheva, I.V., and Pinaev, G. PSRE, NF-kappaB, and AP-1 DNA-Binding Activities Induced by A431 Cell Adhesion Correlate with Actin Cytoskeleton Reorganization, Tsitologiia, 2005, vol. 47, no. 2, pp. 175–183.

    PubMed  CAS  Google Scholar 

  • Petukhova, O.A., Turoverova, L.V., Emel’yanov, A.N., Kropacheva, I.V., and Pinaev, G.P., Morphology of Epidermoid Carcinoma A431 Cells Spread on Immobilized Ligands, Tsitologiia, 2004, vol. 46, no. 1, pp. 5–15.

    PubMed  CAS  Google Scholar 

  • Poch, M.T., Al-Kassim, L., Smolinski, S.M., and Hines, R.N., Two Distinct Classes of CCAAT Box Elements that Bind Muclear Factor-Y/α-actinin-4: Potential Role in Human CYP1A1 Regulation, Toxicol. Applied Pharmacol., 2004, vol. 199, pp. 239–250.

    Article  CAS  Google Scholar 

  • Ramsby, M.L., Makowski, G.S., and Khairallah, E.A., Differential Detergent Fractionation of Isolated Hepatocytes: Biochemical, Immunochemical, and Two-Dimensional Gel Electrophoresis Characterization of Cytoskeletal and Noncytoskeletal Compartments, Electrophoresis, 1994, vol. 15, pp. 265–277.

    Article  PubMed  CAS  Google Scholar 

  • Schmidt, A. and Hall, M.N., Signaling to Actin Cytoskeleton, Annu. Rev. Cell Develop. Biol., 1998, vol. 14, pp. 305–338.

    Article  CAS  Google Scholar 

  • Schütze, S., Nottrott, S., Pfizenmaier, K., and Kronke, M., Tumor Necrosis Factor Signal Transduction, Cell-type-Specific Activation and Translocation of Protein Kinase C, J. Immunol., 1990, vol. 144, pp. 2604–2608.

    PubMed  Google Scholar 

  • Selliah, N., Brooks, W.H., and Roszman, T.L., Proteolytic Cleavage of Alpha-Actinin by Calpain in T Cells Stimulated with Anti-CD3 Monoclonal Antibody, J. Immunol., 1996, vol. 156, pp. 3215–3221.

    PubMed  CAS  Google Scholar 

  • Shalimov, S.A., Keisevich, L.V., Litvinenko, A.A., Volchenskova, I.I., Nikishin, L.F., Potebnya, G.P., Semernikov, V.A., and Kavetskogo, R.E., Cryosurgical Methods of Treatment of Inoperable Tumors of the Abdominal Cavity. (Beginning), Med. gazeta “Zdorov’e Ukrainy” (http://www.health-ua.com/articles/269.html), 2003.

  • Sridharan, D., Brown, M., Lambert, W.C., McMahon, L.W., and Lambert, M.W., Nonerythroid AlphaII Spectrin is Required for Recruitment of FANCA and XPF to Nuclear Foci Induced by DNA Interstrand Cross-Links, J. Cell Sci., 2003, vol. 116, pp. 823–835.

    Article  PubMed  CAS  Google Scholar 

  • Towbin, H., Staehelin, T., and Gordon, J., Electrophoretic Transfer of Proteins from Polyacrylamide Gels to Nitrocellulose Sheets: Procedure and some Applications, Proc. Nat. Acad. Sci. USA, 1979, vol. 76, pp. 4350–4354.

    Article  PubMed  CAS  Google Scholar 

  • Vincent, C., Pruliere, G., Pajot-Augy, E., Campion, E., Garnier, V., and Renard, J.P., Effects of Cryoprotectants on Actin Filaments During the Cryopreservation of One-Cell Rabbit Embryos, Cryobiology, 1990, vol. 27, pp. 9–23.

    Article  PubMed  CAS  Google Scholar 

  • Young, K.G. and Kothary, R., Specrtin Repeat Proteins in the Nucleus, BioEssays, 2005, vol. 27, pp. 144–152.

    Article  PubMed  CAS  Google Scholar 

  • Zhang, Q., Skepper, J.N., Yang, F., Davies, J.D., Hegyi, L., Roberts, R.G., Weissberg, P.L., Ellis, J.A., and Shanahan, C.M., Nesprins: a Novel Family of Spectrin-Repeat-Containing Proteins That Localize to the Nuclear Membrane in Multiple Tissues, J. Cell Sci., 2001, vol. 114, pp. 4485–4498.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia

    A. V. Bol’shakova, O. A. Petukhova & G. P. Pinaev

  2. Linkoping University, Linkoping, Sweden

    K. -E. Magnusson

Authors
  1. A. V. Bol’shakova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. O. A. Petukhova
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. G. P. Pinaev
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. K. -E. Magnusson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to A. V. Bol’shakova.

Additional information

Original Russian Text © A.V. Bol’shakova, O.A. Petukhova, G.P. Pinaev, K.-E. Magnusson, 2009, published in Tsitologiya, Vol. 51, No. 2, 2009, pp. 122–129.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bol’shakova, A.V., Petukhova, O.A., Pinaev, G.P. et al. Comparative analysis of subcellular fractionation methods for revealing α-actinin 1 and α-actinin 4 in A431 cells. Cell Tiss. Biol. 3, 188–197 (2009). https://doi.org/10.1134/S1990519X09020114

Download citation

  • Received:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S1990519X09020114

Key words

Search

Navigation