A FRET-based cGMP biosensor in Drosophila

Abstract

CUTie2 is a FRET-based cGMP biosensor tested so far only in cells. To expand its use to multicellular organisms we generated two transgenic Drosophila melanogaster strains that express the biosensor in a tissue-dependent manner. CUTie2 expression and subcellular localization was verified by confocal microscopy. The performance of CUTie2 was analyzed on dissected larval brains by hyperspectral microscopy and flow cytometry. Both approaches confirmed its responsivity, and the latter showed a rapid and reversible change in the fluorescence of the FRET acceptor upon cGMP treatment. This validated reporter system may prove valuable for studying cGMP signaling at organismal level.

Figure 1.

A. Schematic representation of the construct used to introduce the CUTie2 biosensor under UAS control in flies; B. Single confocal plane of the left hemisphere of a larval brain of the insc-GAL4 x UAS-CUTie2 F1 marked with anti-Dpn (top) with an AlexaFluor™568-conjugated anti-guinea pig secondary antibody or anti-Pros (bottom) with Cy5-conjugated anti-mouse secondary antibody to stain neuroblasts and ganglion mother cells (green), respectively and the YFP module of CUTie2; C. Schematic depicting the allosteric change of CUTie2 upon exposure to cGMP. Fluorescent modules get close enough for FRET to occur under CFP excitation with 458nm light. D. Hyperspectral FRET images of CUTie2 in the larval brain neuroepithelium. Dissected brains from GAL4 ^c855a x UAS-CUTie2 F1 were incubated for 10 min with 5 µM ODQ to block endogenous soluble guanylyl cyclases and imaged. Then, 1 mM 8Br-cGMP was added and imaged after 1 min. The color-coded images were generated by spectral phasor analysis. E. Response of CUTie2 to cGMP. Flow cytometry analysis of disaggregated brain cells from insc-GAL4 x UAS-CUTie2 F1 brains upon addition of 100 μM 8Br-cGMP. The scatter plot shows the data-driven gate (area 2) used for selection of CFP+/YFP+ cells. The monoparametric density plots depict their fluorescence intensity on the FRET channel before and after 1 min and 5 min from the addition of 8Br-cGMP. The black vertical line indicates the median fluorescence intensity of these cells without addition of 8Br-cGMP. Scale bar: 20µm.