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Analysis of Smad nucleocytoplasmic shuttling in living cells

(2004) JOURNAL OF CELL SCIENCE. 117(18). p.4113-4125
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Abstract
Transforming growth factor beta (TGF-beta) signalling leads to phosphorylation and activation of receptor-regulated Smad2 and Smad3, which form complexes with Smad4 and accumulate in the nucleus. The Smads, however, do not seem to reside statically in the cytoplasm in the absence of signalling or in the nucleus upon TGF-beta stimulation, but have been suggested to shuttle continuously between these cellular compartments in both the absence and presence of TGF-beta. Here we investigate this nucleocytoplasmic shuttling in detail in living cells using fusions of Smad2 and Smad4 with enhanced GFP. We first establish that the GFPSmad fusions behave like wild-type Smads in a variety of cellular assays. We go on to demonstrate directly, using photobleaching experiments, that Smad2 and Smad4 shuttle between the cytoplasm and nucleus in both TGF-beta-induced cells and in uninduced cells. In uninduced cells, GFPSmad2 is less mobile in the cytoplasm than is GFPSmad4, suggesting that it may be tethered there. In addition, we show that both GFPSmad2 and GFPSmad4 undergo a substantial decrease in mobility in the nucleus upon TGF-beta stimulation, suggesting that active complexes of Smads are tethered in the nucleus, whereas unactivated Smads are more freely diffusible. We propose that regulated cytoplasmic and nuclear retention may play a role in determining the distribution of Smads between the cytoplasm and the nucleus in both uninduced cells and upon TGF-beta induction.
Keywords
Smad, TGF-beta, nucleocytoplasmic shuttling, GFP, nuclear import, nuclear export, photobleaching, GROWTH-FACTOR-BETA, NUCLEAR-LOCALIZATION SIGNAL, GLUCOCORTICOID-RECEPTOR, TRANSCRIPTION FACTORS, INTERACTION MOTIF, PROTEIN DYNAMICS, EARLY ENDOSOMES, FYVE DOMAIN, TUMOR-CELLS, I RECEPTOR

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Citation

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MLA
Nicolás, Francisco J, Karolien De Bosscher, Bernhard Schmierer, et al. “Analysis of Smad Nucleocytoplasmic Shuttling in Living Cells.” JOURNAL OF CELL SCIENCE 117.18 (2004): 4113–4125. Print.
APA
Nicolás, F. J., De Bosscher, K., Schmierer, B., & Hill, C. S. (2004). Analysis of Smad nucleocytoplasmic shuttling in living cells. JOURNAL OF CELL SCIENCE, 117(18), 4113–4125.
Chicago author-date
Nicolás, Francisco J, Karolien De Bosscher, Bernhard Schmierer, and Caroline S Hill. 2004. “Analysis of Smad Nucleocytoplasmic Shuttling in Living Cells.” Journal of Cell Science 117 (18): 4113–4125.
Chicago author-date (all authors)
Nicolás, Francisco J, Karolien De Bosscher, Bernhard Schmierer, and Caroline S Hill. 2004. “Analysis of Smad Nucleocytoplasmic Shuttling in Living Cells.” Journal of Cell Science 117 (18): 4113–4125.
Vancouver
1.
Nicolás FJ, De Bosscher K, Schmierer B, Hill CS. Analysis of Smad nucleocytoplasmic shuttling in living cells. JOURNAL OF CELL SCIENCE. 2004;117(18):4113–25.
IEEE
[1]
F. J. Nicolás, K. De Bosscher, B. Schmierer, and C. S. Hill, “Analysis of Smad nucleocytoplasmic shuttling in living cells,” JOURNAL OF CELL SCIENCE, vol. 117, no. 18, pp. 4113–4125, 2004.
@article{2955635,
  abstract     = {Transforming growth factor beta (TGF-beta) signalling leads to phosphorylation and activation of receptor-regulated Smad2 and Smad3, which form complexes with Smad4 and accumulate in the nucleus. The Smads, however, do not seem to reside statically in the cytoplasm in the absence of signalling or in the nucleus upon TGF-beta stimulation, but have been suggested to shuttle continuously between these cellular compartments in both the absence and presence of TGF-beta. Here we investigate this nucleocytoplasmic shuttling in detail in living cells using fusions of Smad2 and Smad4 with enhanced GFP. We first establish that the GFPSmad fusions behave like wild-type Smads in a variety of cellular assays. We go on to demonstrate directly, using photobleaching experiments, that Smad2 and Smad4 shuttle between the cytoplasm and nucleus in both TGF-beta-induced cells and in uninduced cells. In uninduced cells, GFPSmad2 is less mobile in the cytoplasm than is GFPSmad4, suggesting that it may be tethered there. In addition, we show that both GFPSmad2 and GFPSmad4 undergo a substantial decrease in mobility in the nucleus upon TGF-beta stimulation, suggesting that active complexes of Smads are tethered in the nucleus, whereas unactivated Smads are more freely diffusible. We propose that regulated cytoplasmic and nuclear retention may play a role in determining the distribution of Smads between the cytoplasm and the nucleus in both uninduced cells and upon TGF-beta induction.},
  author       = {Nicolás, Francisco J and De Bosscher, Karolien and Schmierer, Bernhard and Hill, Caroline S},
  issn         = {0021-9533},
  journal      = {JOURNAL OF CELL SCIENCE},
  keywords     = {Smad,TGF-beta,nucleocytoplasmic shuttling,GFP,nuclear import,nuclear export,photobleaching,GROWTH-FACTOR-BETA,NUCLEAR-LOCALIZATION SIGNAL,GLUCOCORTICOID-RECEPTOR,TRANSCRIPTION FACTORS,INTERACTION MOTIF,PROTEIN DYNAMICS,EARLY ENDOSOMES,FYVE DOMAIN,TUMOR-CELLS,I RECEPTOR},
  language     = {eng},
  number       = {18},
  pages        = {4113--4125},
  title        = {Analysis of Smad nucleocytoplasmic shuttling in living cells},
  url          = {http://dx.doi.org/10.1242/jcs.01289},
  volume       = {117},
  year         = {2004},
}

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