Methodological improvements for fluorescence recordings in Xenopus laevis oocytes

Abstract

Xenopus laevis oocytes are used to study membrane proteins because of their ability to translate exogenous mRNA, but their high intrinsic fluorescence limits fluorescence recordings. Lee and Bezanilla present two methods to increase the amount of melanin and reduce background fluorescence in oocytes.

A novel voltage-clamp/dye uptake assay reveals saturable transport of molecules through CALHM1 and connexin channels

“…Detection of fluorescence in intact Xenopus oocytes is challenging because of their high and variable intrinsic autofluorescence and the high content of pigments and yolk granules that make difficult the detection of a fluorescent signal from the cytoplasm (Dumont, 1972;Lee and Bezanilla, 2019). To detect fluorescence emitted from the intracellular compartment, we rendered oocytes translucent.…”

Exploring structural dynamics of a membrane protein by combining bioorthogonal chemistry and cysteine mutagenesis

“…For both AF488-DBCO and AF488-C5-maleimide labeled oocytes (Figure 5A,B), the magnitude of maximal fluorescence response increases along with channel expression on the surface of oocytes (Figure 5C,D), although there is a considerable spread in both relationships. Variability in fluorescence responses is to be expected given the heterogeneity in the endogenous oocyte fluorescence around 480 nm excitation (Lee and Bezanilla, 2019). Nevertheless, comparison of the trends for AHA- and cysteine-mediated fluorescent labeling suggests that AHA-mediated labeling requires approximately two-fold higher protein expression when compared to cysteine-mediated labeling (Figure 5C,D).…”

A novel voltage clamp/dye uptake assay reveals saturable transport of molecules through CALHM1 and connexin channels

“…Detection of fluorescence in intact Xenopus oocytes is challenging because of their high variable intrinsic autofluorescence and the high content of pigments and yolk granules that make difficult the detection of a fluorescent signal from the cytoplasm (Dumont, 1972;Lee and Bezanilla, 2019). To detect fluorescence emitted from the intracellular compartment, we rendered oocytes translucent.…”

“…Oocytes have a variable intrinsic autofluorescence, which may limit the use of fluorescence to study heterologous expressed ion channels (Lee and Bezanilla, 2019). However, in the translucent oocytes, the fluorescence emitted by the interaction of DAPI, YO-PRO, or ethidium with exogenous salmon DNA in the intracellular compartment was easily distinguished from autofluorescence.…”