Nucleocytoplasmic shuttling revealed by FRAP and FLIP technologies.

Protein mobility within cells is of key importance for many cellular functions. Although immunostaining can reveal protein locations in the steady-state, this might not represent the full picture and provides no information about protein movements. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are two techniques that enable the dynamics of intracellular protein mobility to be studied. These technologies have been successfully used to analyze the nucleocytoplasmic shuttling of STAT1, an intracellular signal transducer and activator of transcription, and can applied to the study of other proteins. Furthermore, FRAP and FLIP approaches have the added advantage of not affecting cell viability and might find application in the imaging of intracellular events in certain tissues and live animals.

[1]  Kevin M. McBride,et al.  Regulated nuclear import of the STAT1 transcription factor by direct binding of importin‐α , 2002, The EMBO journal.

[2]  J. Lippincott-Schwartz,et al.  Diffusional Mobility of Golgi Proteins in Membranes of Living Cells , 1996, Science.

[3]  J. Lippincott-Schwartz,et al.  Studying protein dynamics in living cells , 2001, Nature Reviews Molecular Cell Biology.

[4]  P. Heinrich,et al.  Real Time Analysis of STAT3 Nucleocytoplasmic Shuttling* , 2004, Journal of Biological Chemistry.

[5]  Bernhard Schmierer,et al.  Analysis of Smad nucleocytoplasmic shuttling in living cells , 2004, Journal of Cell Science.

[6]  D. Clapham,et al.  Real-time imaging of nuclear permeation by EGFP in single intact cells. , 2003, Biophysical journal.

[7]  Kevin M. McBride,et al.  The Ins and Outs of STAT1 Nuclear Transport , 2003, Science's STKE.

[8]  I. Kerr,et al.  STAT1 from the cell membrane to the DNA , 2001, The EMBO journal.

[9]  T. Hope,et al.  An N‐terminal nuclear export signal is required for the nucleocytoplasmic shuttling of IκBα , 1999 .

[10]  A. Houtsmuller,et al.  Action of DNA repair endonuclease ERCC1/XPF in living cells. , 1999, Science.

[11]  A. Miyawaki,et al.  An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[12]  J. Darnell,et al.  The rapid inactivation of nuclear tyrosine phosphorylated Stat1 depends upon a protein tyrosine phosphatase. , 1996, The EMBO journal.

[13]  T. Misteli,et al.  High mobility of proteins in the mammalian cell nucleus , 2000, Nature.

[14]  R. Truant,et al.  Live-cell nucleocytoplasmic protein shuttle assay utilizing laser confocal microscopy and FRAP. , 2002, BioTechniques.

[15]  I. Lödige,et al.  Constitutive and IFN‐γ‐induced nuclear import of STAT1 proceed through independent pathways , 2002 .

[16]  Andreas Marg,et al.  DNA binding controls inactivation and nuclear accumulation of the transcription factor Stat1. , 2003, Genes & development.

[17]  W. Webb,et al.  Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. , 1976, Biophysical journal.

[18]  A. Verkman,et al.  Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion. , 1997, Biophysical journal.

[19]  Matthias Weiss,et al.  Challenges and Artifacts in Quantitative Photobleaching Experiments , 2004, Traffic.

[20]  George H. Patterson,et al.  A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells , 2002, Science.

[21]  G. Patterson,et al.  Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. , 1997, Biophysical journal.

[22]  H. Hauser,et al.  Dynamic redistribution of STAT1 protein in IFN signaling visualized by GFP fusion proteins. , 1999, European journal of biochemistry.

[23]  W. Webb,et al.  Constrained diffusion or immobile fraction on cell surfaces: a new interpretation. , 1996, Biophysical journal.

[24]  Gustavo Carrero,et al.  Using FRAP and mathematical modeling to determine the in vivo kinetics of nuclear proteins. , 2003, Methods.

[25]  J. Darnell,et al.  Signalling: STATs: transcriptional control and biological impact , 2002, Nature Reviews Molecular Cell Biology.

[26]  Jan Ellenberg,et al.  Dynamics and retention of misfolded proteins in native ER membranes , 2000, Nature Cell Biology.

[27]  T. Reich,et al.  Nucleocytoplasmic Trafficking of Steroid-free Glucocorticoid Receptor* , 1999, The Journal of Biological Chemistry.