Movements of steroid receptors inside the cell nucleus unraveled
Advanced fluorescence microscopy techniques have revealed how steroid receptors move inside the nucleus. The results were published by a team from Leiden University and the Erasmus Medical Center in Rotterdam, led by IBL-researcher Marcel Schaaf.
Steroid hormones such as the male reproductive hormone testosterone and the stress hormone cortisol serve regulatory functions throughout the body through intracellular receptors. Upon activation, these steroid receptors act like transcription factors that can bind to target sites in the DNA and thereby activate gene transcription ultimately leading to gene expression.
Until recently it was unclear how steroid receptors and other transcription factors found their target sites. The new data reveal that transcription factors diffuse freely through the nucleus, and bind non-specifically to DNA. They are thereby ‘probing’ the entire DNA until they bind to a target site to which they stably bind for a longer period of time (approximately 2-5 seconds).
Three advanced fluorescence microscopy techniques were used to obtain these data: Fluorescence Recovery After Photobleaching (FRAP), Single-Molecule Microscopy (SMM) and Fluorescence Correlation Spectroscopy (FCS). Combining these three techniques enabled the acquisition of data at different time scales (from milliseconds to seconds), so both the fast diffusion of receptors through the nucleus and their binding to DNA could be investigated.
Dr. Marcel Schaaf has a long-standing collaboration with biophysicist Prof. Dr. Thomas Schmidt (LION), and in this project they joined forces with endocrinologists Prof. Dr. Ron de Kloet and Femke Groeneweg (LACDR/LUMC). The collaborators in Rotterdam were Dr. Martin van Royen and Prof. Dr. Adriaan Houtsmuller (Erasmus MC).
Figure 1. Single-molecule tracking in live cells with yellow fluorescent protein (YFP) tagged to the glucocorticoid receptor (GR) A) Fluorescence image of the nucleus of a COS cell with high expression level of YFP-GR. B) Fluorescence image of a nuclear area containing low concentration of fluorescent YFP-GR molecules. C) Fluorescence intensity peaks attributed to single molecules
Van Royen ME, van Cappellen WA, Geverts B, Schmidt T, Houtsmuller AB, Schaaf MJ. 2014. Androgen receptor complexes probe DNA for recognition sequences by short random interactions. Journal of Cell Science DOI: 10.1242/jcs.135228
Groeneweg FL, van Royen ME, Fenz S, Keizer VI, Geverts B, Prins J, de Kloet ER, Houtsmuller AB, Schmidt TS, Schaaf MJ. 2014. Quantitation of Glucocorticoid Receptor DNA-Binding Dynamics by Single-Molecule Microscopy and FRAP. PLoS One, DOI: 10.1371/journal.pone.0090532