Figure 1
Dynorphins differently accumulate in the plasma membrane of PC12 cells. Confocal fluorescence imaging and FCS show that dynorphin peptides differently interact with live PC12 cells, which do not express opioid receptors.67 (A) Fluorescently labeled Big Dyn and Dyn A, but not Dyn B, associate with the plasma membrane of live PC12, as evident from the increased intensity of Big Dyn and Dyn A fluorescence in the membrane compared with the medium. Confocal images of live PC12 cells were taken after 30 min incubation with fluorescently labeled dynorphins added to the culture medium at 100 nM concentration. After incubation, the concentrations of TAMRA-Dyn B, TAMRA-Dyn A and TAMRA-Big Dyn in the bulk medium measured by FCS were 100, 70 and 30 nM, respectively. Scale bars, 20 μm. (B) Bright fluorescent domains of TAMRA-Big Dyn and TAMRA-Dyn A, but not TAMRA-Dyn B, observed in the plasma membrane of live PC12 cells after dynorphin peptides washout. Scale bars, 10 μm. (C, a) Two- (2D) and (b) three-dimensional (3D) distribution of TRITC-labeled Dyn A on the surface of live PC12 cells. Confocal images were taken after 15 min incubation of the cells with 250 nM fluorescently labeled Dyn A. (c) The time course of fluorescence intensity rise at the plasma membrane during 15 min of incubation is shown below (bottom). Scale bar, 10 μm. (D) FCS measurements performed after 30 min incubation of PC12 cells with 300 or 150 nM TAMRA-Big Dyn or TAMRA-Dyn A, respectively. (a) Fluorescence intensity fluctuations recorded in the bulk medium (red) and the plasma membrane of live PC12 cells incubated with TAMRA-Dyn A (green). (b) Autocorrelation curves reflecting TAMRA-Dyn A dynamics and concentration in the bulk medium (red dots) and the plasma membrane of live PC12 cells (green dots) showing that TAMRA-Dyn A accumulates in the plasma membrane, as evident from the increased average number of molecules in the plasma membrane, Npm=(105±15), as compared with the medium, Nmed=(20±2). (c) Autocorrelation curves recorded in the medium (red) and the plasma membrane (green) normalized to the same amplitude (Gn(τ)=1 at τ=10 μs), showing a marked shift of the autocorrelation curve towards longer characteristic times because of TAMRA-Dyn A interactions with, and its reduced mobility in, the plasma membrane. Inset: PCHs show that photon counting distribution recorded in the plasma membrane (green circles) deviates from the Poisson distribution (black line, mean=5.17) more than the photon counting distribution observed in the cell culture medium (red circles). (d) Autocorrelation curves reflecting TAMRA-Big Dyn dynamics and concentration in the bulk medium (red dots) and the plasma membrane of live PC12 cells (green dots). The average number of TAMRA-Big Dyn molecules at the plasma membrane, Npm=(119±19), is several times higher than the average number of molecules in the bulk cell culturing medium, Nmed=(45±6). (e) Autocorrelation curves normalized to the same amplitude (Gn(τ)=1 at τ=10 μs) show that TAMRA-Big Dyn lateral mobility is significantly reduced, as evident from the appearance of a second component with a significantly longer diffusion time. Inset: PCHs show that photon counting distribution recorded in the plasma membrane (green dots, mean=3.93) deviates more from the Poisson distribution (black line, mean=3.68) than the photon counting histogram recorded in the cell culture medium (red dots), which could be fitted by Poisson distribution (black line, mean=3.16)
