Spatially segregated distinct changes in PLCδ1PH-mRFP fluorescence upon strong membrane depolarization in fibers expressing Ci-VSP. (A) Confocal images of a portion of a muscle fiber expressing Ci-VSP and PLCδ1PH-mRFP. Images from left to right were taken before, during, and after a 5-s-long depolarizing pulse to 50 mV, respectively. The graph below each image shows the fluorescence intensity profile along the rectangular area highlighted in the image on the left. (B; left graph) Difference between the intensity at a given location of the fluorescence profile during the pulse (Fluo+50) and its corresponding intensity before the pulse (Fluo−80(1)) versus Fluo−80(1). (Right graph) Difference between the intensity at a given location of the fluorescence profile during the pulse (Fluo+50) and its corresponding intensity after the pulse (Fluo−80(2)) versus Fluo−80(2). (C) Result of the analysis similar to that shown in Fig. 9 C, except that pixels with high initial intensity and low initial intensity were separated; pixel selection was achieved by adjusting manually the threshold tool in ImageJ in the first image of the sequence. (D) Mean relative peak change in PLCδ1PH-mRFP fluorescence versus membrane potential calculated from three fibers that experienced the pulse protocol shown in C; closed circles, open squares, and open circles correspond to values obtained using all, high intensity, and low intensity initial pixels, respectively. Error bars represent ± SEM.
