Application of Staining Intensity Correlation Analysis to Visualize Protein Colocalizationat a Cellular Level
Authors: Permphan Dharmasaroja
Mutations of the telomeric copy of the survival motor neuron 1 (SMN1) gene cause spinal muscular atrophy. A deletion of the Eef1a2 gene leads to lower motor neuron degeneration in wasted mice. Indirect evidences have been shown that the eEF1A protein family may interact with SMN, and our previous study showed that abnormalities of neuromuscular junctions in wasted mice were similar to those of Smn mutant mice. To determine potential colocalization between SMN and tissue-specific translation elongation factor 1A2 (eEF1A2), an immunochemical analysis of HeLa cells transfected with the plasmid pcDNA3.1(+)C-hEEF1A2- myc and a new quantitative test of colocalization by intensity correlation analysis (ICA) was used to explore the association of SMN and eEF1A2. Here the results showed that eEF1A2 redistributed from the cytoplasm to the nucleus in response to serum and epidermal growth factor. In the cytoplasm, compelling evidence showed that staining for myc-tagged eEF1A2 varied in synchrony with that for SMN, consistent with the formation of a SMN-eEF1A2 complex in the cytoplasm of HeLa cells. These findings suggest that eEF1A2 may colocalize with SMN in the cytoplasm and may be a component of the SMN complex. However, the limitation of the ICA method is an inability to resolve colocalization in components of small organelles such as the nucleus.
Digital Object Identifier (DOI): doi.org/10.5281/zenodo.1077987Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1208
 S. Lefebvre, L. Burglen, S. Reboullet, O. Clermont, P. Burlet, L. Viollet, B. Benichou, C. Cruaud, P. Millasseau, M. Zeviani et al, "Identification and characterization of a spinal muscular atrophy-determining gene." Cell, vol. 80, pp. 155-165, Jan. 1995.
 D.D. Coovert, T.T. Le, P.E. McAndrew, J. Strasswimmer, T.O. Crawford, J.R. Mendell, S.E. Coulson, E.J. Androphy, T.W. Prior, and A.H. Burghes, "The survival motor neuron protein in spinal muscular atrophy." Hum. Mol. Genet., vol. 6, pp. 1205-1214, Aug. 1997.
 S. Lefebvre, P. Burlet, Q. Liu, S. Bertrandy, O. Clermont, A. Munnich, G. Dreyfuss, and J. Melki, "Correlation between severity and SMN protein level in spinal muscular atrophy." Nat. Genet., vol. 16, pp. 265- 269, July 1997.
 L. Gangwani, M. Mikrut, S. Theroux, M. Sharma, and R.J. Davis, "Spinal muscular atrophy disrupts the interaction of ZPR1 with the SMN protein." Nat. Cell Biol., vol. 3, pp. 376-383, Apr. 2001.
 S.M. Knudsen, J. Frydenberg, B.F. Clark, and H. Leffers, "Tissuedependent variation in the expression of elongation factor-1 alpha isoforms: isolation and characterisation of a cDNA encoding a novel variant of human elongation-factor 1 alpha." Eur. J. Biochem., vol. 215, pp. 549-554, Aug. 1993.
 S. Kahns, A. Lund, P. Kristensen, C.R. Knudsen, B.F. Clark, J. Cavallius, and W.C. Merrick, "The elongation factor 1 A-2 isoform from rabbit: cloning of the cDNA and characterization of the protein." Nucleic Acids Res., vol. 26, pp. 1884-1890, Apr. 1998.
 S. Lee, A. LeBlanc, A. Duttaroy, and E. Wang, "Terminal differentiation-dependent alteration in the expression of translation elongation factor-1 alpha and its sister gene, S1, in neurons." Exp. Cell Res., vol. 219, pp. 589-597, Aug. 1995.
 S. Lee, L.A. Wolfraim, and E. Wang, "Differential expression of S1 and elongation factor-1 alpha during rat development." J. Biol. Chem., vol. 268, pp. 24453-24459, Nov. 1993.
 S. Lee, A.M. Francoeur, S. Liu, and E. Wang, "Tissue-specific expression in mammalian brain, heart, and muscle of S1, a member of the elongation factor-1 alpha gene family." J. Biol. Chem., vol. 267, pp. 24064-24068, Nov. 1992.
 D.M. Chambers, J. Peters, and C.M. Abbott, "The lethal mutation of the mouse wasted (wst) is a deletion that abolishes expression of a tissuespecific isoform of translation elongation factor 1alpha, encoded by the Eef1a2 gene." Proc. Natl. Acad. Sci. U S A, vol. 95, pp. 4463-4468, Apr. 1998.
 H.L. Lutsep and M. Rodriguez, "Ultrastructural, morphometric, and immunocytochemical study of anterior horn cells in mice with "wasted" mutation." J. Neuropath. Exp. Neurol., vol. 48, pp. 519-533, Sep. 1998.
 H.J. Newbery, T.H. Gillingwater, P. Dharmasaroja, J. Peters, S.B. Wharton, D. Thomson, et al, "Progressive loss of motor neuron function in wasted mice: effects of a spontaneous null mutation in the gene for the eEF1 A2 translation factor." J. Neuropath. Exp. Neurol., vol. 64, pp. 295-303, Apr. 2005.
 B.T. Edmonds, J. Wyckoff, Y.G. Yeung, Y. Wang, E.R. Stanley, J. Jones, J. Segall, J. Condeelis, "Elongation factor-1 alpha is an overexpressed actin binding protein in metastatic rat mammary adenocarcinoma." J. Cell Sci., vol. 109, pp. 2705-2714, Nov. 1996.
 O. Minella, O. Mulner-Lorillon, V. De Smedt, S. Hourdez, P. Cormier, and R. Bellé, "Major intracellular localization of elongation factor-1." Cell Mol. Biol., vol. 42, pp. 805-810, Sep. 1996.
 L. Gangwani, M. Mikrut, Z. Galcheva-Gargova, and R.J. Davis, "Interaction of ZPR1 with translation elongation factor-1alpha in proliferating cells." J. Cell Biol., vol. 143, pp. 1471-1484, Dec. 1998.
 Z. Galcheva-Gargova, K.N. Konstantinov, I-H. Wu, F.G. Klier, T. Barrett, and R.J. Davis, "Binding of Zinc Finger Protein ZPR1 to the Epidermal Growth Factor Receptor." Science, vol. 272, pp. 1797-1802, June 1996.
 B. Charroux, L. Pellizzoni, R.A. Perkinson, J. Yong, A. Shevchenko, M. Mann, and G. Dreyfuss, "Gemin4: A Novel Component of the SMN Complex That Is Found in both Gems and Nucleoli." J. Cell Biol., vol. 148, pp. 1177-1186, Mar. 2000.
 T. Carvalho, F. Almeida, A. Calapez, M. Lafarga, M.T. Berciano, and M. Carmo-Fonseca, "The spinal muscular atrophy disease gene product, SMN: A link between snRNP biogenesis and the Cajal (coiled) body." .J Cell Biol., vol. 147, pp. 715-728, Nov. 1999.
 Q. Liu, and G. Dreyfuss, "A novel nuclear structure containing the survival of motor neurons protein." EMBO J., vol. 15, pp. 3555-3565, July 1996.
 A.G. Matera, and M.R. Frey, "Coiled bodies and gems: Janus or gemini?" Am. J. Hum. Genet., vol. 63, pp. 317-321, Aug. 1998.
 Q. Li, A. Lau, T.J. Morris, L. Guo, C.B. Fordyce, and E.F. Stanley, "A syntaxin 1, Galpha(o), and N-type calcium channel complex at a presynaptic nerve terminal: analysis by quantitative immunocolocalization." J. Neurosci., vol. 24, pp. 4070-4081, Apr. 2004.
 J. Sanders, M. Brandsma, G.M. Janssen, J. Dijk, and W. Möller, "Immunofluorescence studies of human fibroblasts demonstrate the presence of the complex of elongation factor-1 beta gamma delta in the endoplasmic reticulum." J. Cell Sci., vol. 109, pp. 1113-1117, May 1996.
 M.T. Bohnsack, K. Regener, B. Schwappach, R. Saffrich, E. Paraskeva, E. Hartmann, and D. Görlich, "Exp5 exports eEF1A via tRNA from nuclei and synergizes with other transport pathways to confine translation to the cytoplasm." EMBO J., vol. 21, pp. 6205-6215, Nov. 2000.
 A. Calado, N. Treichel, E.C. Muller, A. Otto, and U. Kutay, "Exportin- 5-mediated nuclear export of eukaryotic elongation factor 1A and tRNA." EMBO J., vol. 21, pp. 6216-6224, Nov. 2002.
 F.J. Iborra, D.A. Jackson, and P.R. Cook, "Coupled transcription and translation within nuclei of mammalian cells." Science, vol. 293, pp. 1139-1142, Aug. 2001.
 P.J. Young, T.T. Le, N. thi Man, A.H. Burghes, and G.E. Morris, "The relationship between SMN, the spinal muscular atrophy protein, and nuclear coiled bodies in differentiated tissues and cultured cells." Exp. Cell Res., vol. 256, pp. 365-374, May 2001.
 A.G. Matera, and M.D. Hebert, "The survival motor neurons protein uses its ZPR for nuclear localization." Nat. Cell Biol., Vol. 3, pp. E93- E95, Apr. 2001.
 C. Cifuentes-Diaz, S. Nicole, M.E. Velasco, C. Borra-Cebrian, C. Panozzo, T. Frugier, et al, "Neurofilament accumulation at the motor endplate and lack of axonal sprouting in a spinal muscular atrophy mouse model." Hum. Mol. Genet., vol. 11, pp. 1439-1447, June 2002.