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EGF-induced dynamics of NF-κB and F-actin in A431 cells spread on fibronectin

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Abstract

To evaluate the role of actin cytoskeleton in the regulation of NF-κB transcription factor, we analyzed its involvement in the intracellular transport and nuclear translocation of the NF-κB RelA/p65 subunit in A431 epithelial cells stimulated with fibronectin and EGF. Live cell imaging and confocal microscopy showed that EGF activated the movement of RelA/p65 in the cytoplasm. Upon cell adhesion to fibronectin, RelA/p65 concentrated onto stress fibers, and EGF stimulated its subsequent allocation to membrane ruffles, newly organized stress fibers, and discrete cytoplasmic actin-rich patches. These patches also contained α-actinin-1 and α-actinin-4, vinculin, paxillin, α-tubulin, and PI3-kinase. Cytochalasin D treatment resulted in RelA/p65 redistribution to actin-containing aggregates, with the number of cells with RelA/p65-containing clusters in the cytoplasm increasing under the effect of EGF. Furthermore, EGF proved to induce RelA/p65 accumulation in the nucleus after cell pretreatment with actin-stabilizing and actin-destabilizing agents, which was accompanied by changes in its DNA-binding activity after either EGF stimulation or cytochalasin D treatment. Thus, EGF treatment of A431 cells results in simultaneous nuclear RelA/p65 translocation and cytoplasmic redistribution, with part of RelA/p65 pool forming a very tight association with actin-rich structures. Apparently, nuclear transport is independent on drug stabilization or destabilization of the actin.

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Acknowledgments

We are grateful to Dr. Theodorus Gadella (Section of Molecular Cytology, van Leeuwenhock Centre for Advanced Microscopy, Swammerdam Institute of Life Sciences, University of Amsterdam, Amsterdam, The Netherlands) for kindly supplying us with the ptagRFP-LifeAct vector. This research was supported by Grants from the Swedish Institute [Visby program 879/2009], Swedish Research Council [2010-3045], postdoctoral fellowship at the Faculty of Health Science, Linköping University (to A.B.), European Science Foundation (TraPPs Euromembrane project), Molecular and Cellular Biology Program of Russian Academy of Sciences, and Russian Foundation for Basic Research [13-04-00497].

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Authors and Affiliations

  1. Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Ave., St. Petersburg, 194064, Russia

    Anastasia Bolshakova, George Pinaev & Olga Petukhova

  2. Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Linköping University, 581 85, Linköping, Sweden

    Anastasia Bolshakova & Karl-Eric Magnusson

  3. Department of Medical Physics, St. Petersburg State Polytechnical University, 29 Polytechnicheskaya St., St. Petersburg, 195251, Russia

    Anastasia Bolshakova

Authors
  1. Anastasia Bolshakova
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  2. Karl-Eric Magnusson
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  3. George Pinaev
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  4. Olga Petukhova
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Corresponding author

Correspondence to Olga Petukhova.

Electronic supplementary material

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Supplementary Video: A431 cell transfected with GFP-RelA/p65 (green) and tagRFP-LifeAct (red) vectors. Images were taken of cells spread on fibronectin for 1 h and then treated with EGF (100 ng/ml). Time between frames was 30 sec. During attachment to fibronectin, the cells acquired a polygonal shape and a fine microfibrillar banding pattern throughout the cell body. Subsequent EGF stimulation resulted in F-actin reorganization with the formation of distinct lamellae within 5–15 min and their subsequent shortening by 30 min after adding EGF. The results of live cell imaging (epifluorescence and optical section images) are presented. In epifluorescent images (on the left), the fluorescence intensity of RelA/p65 in the lamella is increased after EGF stimulation, indicating p65 translocation to this area (MPEG 1108 kb)

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Bolshakova, A., Magnusson, KE., Pinaev, G. et al. EGF-induced dynamics of NF-κB and F-actin in A431 cells spread on fibronectin. Histochem Cell Biol 144, 223–235 (2015). https://doi.org/10.1007/s00418-015-1331-5

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