Identification of permissive insertion sites for generating functional fluorescent mineralocorticoid receptors.

The mineralocorticoid receptor (MR), a member of the nuclear receptor superfamily of transcription factors, is activated by aldosterone and mediates its natriferic action in tight epithelia. MR is also expressed in nonepithelial tissues. Importantly, it mediates the deleterious effects of inappropriately high aldosterone levels in the heart, in which it induces the development of cardiac fibrosis. Antagonism of MR in humans is useful in the treatment of severe cardiac failure and some forms of hypertension. Despite the important pathophysiological and pharmacological role of this receptor, many important questions about its cellular biology and functional roles remain unanswered. A major challenge in the study of MR is the unavailability of fully functional fluorescent derivatives of the receptor. In this study we have created a library of MR mutants with insertions of the yellow fluorescent protein in various internal locations in the receptor using a random-insertion transposon-based technique. Screening of this library using a transactivation assay allowed us to identify several fluorescent constructs that retain functionality. Detailed characterization of one of these construct showed that it induces aldosterone-target genes such as the epithelial Na(+) channel subunits and the serum and glucocorticoid-induced kinase 1 at physiological concentrations of aldosterone to an equal extent than the wild-type receptor. Furthermore, aldosterone affinity, hormone-induced nuclear translocation, DNA binding and regulation of nongenomic pathways are all indistinguishable from the wild-type receptor. This new set of fluorescent MR derivatives provides a useful tool for studying the cell biology of the receptor.

Optimizing the fluorescent yield in two-photon laser scanning microscopy with dispersion compensation.

A challenge for nonlinear imaging in living tissue is to maximize the total fluorescent yield from each fluorophore. We investigated the emission rates of three fluorophores-rhodamine B, a red fluorescent protein, and CdSe quantum dots-while manipulating the phase of the laser excitation pulse at the focus. In all cases a transform-limited pulse maximized the total yield to insure the highest signal-to-noise ratio. Further, we find evidence of fluorescence antibleaching in quantum dot samples.