FRAP and FRET Methods to Study Nuclear Receptors in Living Cells

Royen, Martin E. van; Dinant, Christoffel; Farla, Pascal; Trapman, Jan; Houtsmuller, Adriaan B.

doi:10.1007/978-1-60327-575-0_5

Cited by 24 publications

(31 citation statements)

References 83 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The extent of immobilization is strongly correlated with chromatin-binding characteristics (27). For all receptors tested in our FRAP experiments, DHT induced nearly overlapping redistribution curves from which very similar immobile fractions can be calculated (Fig.…”

Section: Comparison Of the Intranuclear Mobility Of Ar Wt And Mutant mentioning

confidence: 60%

“…When the AR is activated by agonists it undergoes a conformational change that leads to close positioning of the NTD and LBD and thus interaction between these two domains (27,43). The enzalutamide-bound AR WT or F877L did not show any N/C interactions.…”

Section: N/c Interactions and Binding To Coregulator Peptidesmentioning

confidence: 93%

“…A Zeiss LSM510 META confocal laser scanning microscope equipped with a 40Â/1.3NA oilimmersion objective, an argon laser (30 mW) and an acoustooptic tunable filter was used. Fluorescence recovery after photobleaching (FRAP) analysis was performed according to the instructions by van Royen and colleagues (27). For each treatment group, 30 cells were measured by FRAP in two independent experiments.…”

Section: Fluorescence Recovery After Photobleachingmentioning

confidence: 99%

See 2 more Smart Citations

The Effect of F877L and T878A Mutations on Androgen Receptor Response to Enzalutamide

Prekovic

Royen

Voet

et al. 2016

Molecular Cancer Therapeutics

Self Cite

View full text Add to dashboard Cite

Treatment-induced mutations in the ligand-binding domain of the androgen receptor (AR) are known to change antagonists into agonists. Recently, the F877L mutation has been described to convert enzalutamide into an agonist. This mutation was seen to co-occur in the endogenous AR allele of LNCaP cells, next to the T878A mutation. Here, we studied the effects of enzalutamide on the F877L and T878A mutants, as well as the double-mutant AR (F877L/T878A). Molecular modeling revealed favorable structural changes in the double-mutant AR that lead to a decrease in steric clashes for enzalutamide. Ligand-binding assays confirmed that the F877L mutation leads to an increase in relative binding affinity for enzalutamide, but only the combination with the T878A mutation resulted in a strong agonistic activity. This correlated with changes in coregulator recruitment and chromatin interactions. Our data show that enzalutamide is only a very weak partial agonist of the AR F877L, and a strong partial agonist of the double-mutant AR.

show abstract

Section: Comparison Of the Intranuclear Mobility Of Ar Wt And Mutant mentioning

confidence: 60%

Section: N/c Interactions and Binding To Coregulator Peptidesmentioning

confidence: 93%

Section: Fluorescence Recovery After Photobleachingmentioning

confidence: 99%

See 1 more Smart Citation

The Effect of F877L and T878A Mutations on Androgen Receptor Response to Enzalutamide

Prekovic

Royen

Voet

et al. 2016

Molecular Cancer Therapeutics

Self Cite

View full text Add to dashboard Cite

show abstract

“…1F). In abFRET, the relative increase of the donor emission after acceptor photobleaching is a measure of the interaction between the tagged molecules under surveillance Karpova et al, 2003;Kenworthy, 2001;van Royen et al, 2009a). In the presence of the synthetic androgen, R1881, cells expressing double-tagged AR and cells expressing a combination of single-tagged YFP-AR and AR-CFP showed abFRET.…”

Section: Resultsmentioning

confidence: 99%

“…An argon laser was used for excitation of CFP and YFP at 458 and 514 nm, respectively. In all quantitative imaging experiments cells with a physiologically relevant expression level of tagged ARs were selected for analysis (van Royen et al, 2007;van Royen et al, 2009a). N/C interactions of double-tagged YFP-AR-CFP, or co-transfected YFP-AR and AR-CFP were assessed using YFP/CFP ratio imaging and acceptor photobleaching FRET (abFRET) (van Royen et al, 2009a and references therein).…”

Section: Confocal Imaging Yfp/cfp Ratio Imaging and Abfret Analysismentioning

confidence: 99%

Stepwise androgen receptor dimerization

Royen

Cappellen

Vos

et al. 2012

Journal of Cell Science

Self Cite

122

135

View full text Add to dashboard Cite

SummaryAndrogen-regulated gene expression is a highly coordinated dynamic process mediated by androgen receptor (AR) ligand binding and DNA binding, and by specific AR protein-protein interactions. The latter include DNA-binding domain (D-box) interactions in AR homodimers, and the interaction of the FQNLF motif in the AR N-terminal domain and the coactivator groove in the ligand-binding domain (N/C interaction). We have studied these interactions in AR homodimerization using quantitative imaging techniques. We found that the initial cytoplasmic intramolecular AR N/C interaction after ligand binding is followed by a D-box-dimerization-dependent transition to intermolecular N/C interaction in a proportion of nuclear ARs. The consecutive steps leading to homodimerization are initiated prior to DNA binding. Our data indicate the presence of nuclear pools of both AR homodimers and monomers. On the basis of AR-regulated reporter assays we propose specificity in regulation of gene expression by AR homodimers and monomers mediated by AR domain interactions. Moreover, our findings elucidate important steps in the spatiotemporal organization of AR intra-and intermolecular interactions.

show abstract

Recent advances in dynamic intravital multi‐photon microscopy

Niesner

Hauser

2011

Cytometry Pt A

View full text Add to dashboard Cite

Standard multiphoton laser scanning microscopy (MPLSM) has revolutionized our view of physiologic and pathologic processes in living organisms by enlightening different aspects of cellular choreography in immune responses, that is, cellular motility and co-localization. To understand cellular communication on a molecular level, novel transgenic reporter mice have been generated. In parallel, MPLSM systems have been developed, which make it possible for this technique to be more widely used to address crucial immunological questions. Here, we review the latest progress concerning transgenic mouse technology and multiphoton imaging capacities and discuss further developments which will enable us to visualize all around monitoring and quantification of cellular function at a molecular level directly in vivo. ' 2011 International Society for Advancement of CytometryKey terms multi-photon laser scanning microscopy; intravital imaging IN the past, cytometry of cellular and molecular mechanisms underlying immune reactions was mainly performed ex vivo. These analyses focused on characterization of the players in the immune system as single entities, without taking tissue context into account. On the other hand, microscopic analyses of intact tissues were restricted to fixed samples. Thus, the dynamic nature of the immune response, with cellular interactions and migration being two of its most prominent features, could not be analyzed until recently.The development of multiphoton intravital microscopy and its application to address immunological questions has changed this over the past decade. For instance, typical motility patterns of lymphocytes could be tracked for the first time in timelapse movies taken in living tissue. Lymphocytes in secondary lymphoid organs were shown to move with a median instantaneous three-dimensional (3D) velocity of between 5 and 10 lm/min, and are therefore able to cover distances of several hundred micrometers within a few hours (1,2). Compared with other imaging techniques (summarized in Table 1), multiphoton microscopy is the only technology able to capture the dynamic nature of these processes in deep tissue, as it allows fast acquisition of 3D image stacks (for example: 300 3 300 3 30 lm 3 with a resolution of 512 3 512 3 11 voxels can be recorded every 15 s). Until now, most of the work in the field has focused on the analysis of motility parameters (e.g., the velocity at which cells migrate, their displacement, directed versus random migration) and the duration and type of cellular interactions using fluorescent reporter mice to distinguish between different cell subsets. These studies have already contributed tremendously to our understanding on how the immune system works in vivo, and at the same time have sparked the desire to visualize subcellular events by intravital microscopy. As novel photoactivatable fluorescent proteins and genetically encoded Förster resonant energy transfer (FRET)-biosensors of sufficient brightness for in vivo imaging

show abstract

FRAP and FRET Methods to Study Nuclear Receptors in Living Cells

Cited by 24 publications

References 83 publications

The Effect of F877L and T878A Mutations on Androgen Receptor Response to Enzalutamide

The Effect of F877L and T878A Mutations on Androgen Receptor Response to Enzalutamide

Stepwise androgen receptor dimerization

Recent advances in dynamic intravital multi‐photon microscopy

Contact Info

Product

Resources

About