Analysing bioelectrical phenomena in the Drosophila ovary with genetic tools: tissue-specific expression of sensors for membrane potential and intracellular pH and RNAi-knockdown of mechanisms involved in ion exchange

摘要

Analysis of Vmem and pHi using the genetically-encoded fluorescent Vmem- and pHi-sensors ArcLight and pHluorin-Moesin, respectively. a Uniform expression of GFP in the FE using the soma-driver tj-Gal4 (control); WFM-images of typical tj-Gal4 > UAST-GFP follicles of vitellogenic stages S8-S12 (scale bars represent 50 μm). b, c Pseudocolour images of follicles expressing ArcLight (b) or pHluorin-Moesin (c) in the FE; median optical sections (SIM) of typical follicles of S8-S12 (scale bars represent 50 μm). d Analysis of fluorescence intensities in the FE (“mean grey value”; area marked in yellow); examples of four follicles of S10B (and one of S11) expressing pHluorin-Moesin (WFM-image; scale bar represents 200 μm). e Both the Vmem-sensor ArcLight and the pHi-sensor pHluorin-Moesin use the chromophore pHluorin which responds, in deprotonated state, to blue-light excitation (blue arrow) with the emission of green light (green arrow). The exact mechanism of ArcLight is not known, but is believed to involve voltage-dependent dimerisation leading to protonation of the chromophore (VSD, voltage-sensing domain; scheme inspired by [45]). In cells expressing the respective sensor, relative depolarisation or relative acidification is indicated by weaker fluorescence intensities, whereas relative hyperpolarisation or relative alkalisation is indicated by stronger fluorescence intensities