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FRAP reveals that mobility of oestrogen receptor-α is ligand- and proteasome-dependent

Nature Cell Biology volume 3, pages 15–23 (2001)Cite this article

Abstract

Here we report the use of fluorescence recovery after photobleaching (FRAP) to examine the intranuclear dynamics of fluorescent oestrogen receptor-α (ER). After bleaching, unliganded ER exhibits high mobility (recovery t1/2 < 1 s). Agonist (oestradiol; E2) or partial antagonist (4-hydroxytamoxifen) slows ER recovery (t1/2 ∼5–6 s), whereas the pure antagonist (ICI 182,780) and, surprisingly, proteasome inhibitors each immobilize ER to the nuclear matrix. Dual FRAP experiments show that fluorescent ER and SRC-1 exhibit similar dynamics only in the presence of E2. In contrast to reports that several nuclear proteins show uniform dynamics, ER exhibits differential mobility depending upon several factors that are linked to its transcription function.

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Figure 1: Characterization of CFP–ER and YFP–SRC-1.
Figure 2: FRAP analysis of ER following ligand treatment.
Figure 3: Dual FRAP analysis of ER and SRC-1.
Figure 4: Western blots of ER turnover and nuclear-matrix association.
Figure 5: Effects of proteasome inhibition, actinomycin D and ATP depletion on ER dynamics.
Figure 6: Helix 12 is required for ER immobilization.

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Acknowledgements

We thank J. Nickerson, A. Belmont, Z. D. Sharp and D. DeFranco for critical reviews of the manuscript, E. Schwoebel and M. S. Moore for assistance with ATP-depletion experiments, and A. Cooney and Z. Nawaz for discussions. This work was supported by NIH grant RO1 DK55622 and a National American Heart Association Scientist Development Award (9630033N) to M.A.M., an NIH postdoctoral fellowship (1F32DK09787) to D.L.S., NIH grant RO1 DK53002 to C.L.S., and funding from the Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas.

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  1. Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, 77030, Texas, USA

    David L. Stenoien, Kavita Patel, Maureen G. Mancini, Martin Dutertre, Carolyn L. Smith, Bert W. O'Malley & Michael A. Mancini

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Correspondence to Michael A. Mancini.

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Stenoien, D., Patel, K., Mancini, M. et al. FRAP reveals that mobility of oestrogen receptor-α is ligand- and proteasome-dependent. Nat Cell Biol 3, 15–23 (2001). https://doi.org/10.1038/35050515

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