Figure 9.
Figure 9. Visualization of vesicles and ribbons by TIRFM in rod terminals. The figure shows the synaptic terminals of rods in which vesicles were loaded with 3-kD Alexa Fluor 488 and ribbons were labeled by introducing a Ribeye-binding peptide conjugated to tetramethylrhodamine through the patch pipette. (A) Terminal of an isolated rod in control conditions (0.1% DMSO). (B) Rod terminal in the presence of dynasore (80 µM). The green fluorescence images at the left of A and B show vesicles loaded with 3-kD Alexa Fluor 488 after averaging the entire trial (150 frames, 40 ms apiece). Differences in vesicle brightness in these average images were largely caused by differences in the amount of time that a vesicle spent near the membrane during the acquisition period rather than intrinsic differences in vesicle brightness. The magenta images in the middle show the location of ribbons labeled by tetramethylrhodamine-conjugated Ribeye-binding peptide. The 561-nm laser was tilted to deepen penetration of the evanescent field into the cell and illuminate tetramethylrhodamine-labeled ribbons more effectively. As illustrated in the merged image at the right of A, we measured the distance between the center of a vesicle to the edge of the nearest ribbon. Bar, 5 µm. (C) Fates of newly approaching vesicles in control conditions. The relative frequency histogram of vesicle events as a function of distance from the nearest ribbon was binned at 350-nm intervals and adjusted for radial density (see Materials and methods). The likelihood of observing a newly approaching vesicle was consistently greater in the bin closest to the ribbon. However, the probability that fusion of a newly approaching vesicle (n = 48 total events in 9 rods) would occur in the bin closest to the ribbon rather than farther away was significantly greater than the probability that a newly approaching vesicle would dock at the membrane close to the ribbon (n = 17; P = 0.023, z test) or depart from ribbon-proximal membrane without fusion (n = 157; P < 0.0001, z test). (D) Fates of vesicles that began a trial as membrane-associated or docked vesicles. Fusion of previously docked vesicles was significantly more likely to occur in the bin closest to the ribbon (n = 30 total events; P = 0.00014) than retreat from the membrane (n = 35). The likelihood that a vesicle would remain docked throughout the trial was also significantly more likely in the bin closest to the ribbon (n = 28; P < 0.0001) than retreat.

Visualization of vesicles and ribbons by TIRFM in rod terminals. The figure shows the synaptic terminals of rods in which vesicles were loaded with 3-kD Alexa Fluor 488 and ribbons were labeled by introducing a Ribeye-binding peptide conjugated to tetramethylrhodamine through the patch pipette. (A) Terminal of an isolated rod in control conditions (0.1% DMSO). (B) Rod terminal in the presence of dynasore (80 µM). The green fluorescence images at the left of A and B show vesicles loaded with 3-kD Alexa Fluor 488 after averaging the entire trial (150 frames, 40 ms apiece). Differences in vesicle brightness in these average images were largely caused by differences in the amount of time that a vesicle spent near the membrane during the acquisition period rather than intrinsic differences in vesicle brightness. The magenta images in the middle show the location of ribbons labeled by tetramethylrhodamine-conjugated Ribeye-binding peptide. The 561-nm laser was tilted to deepen penetration of the evanescent field into the cell and illuminate tetramethylrhodamine-labeled ribbons more effectively. As illustrated in the merged image at the right of A, we measured the distance between the center of a vesicle to the edge of the nearest ribbon. Bar, 5 µm. (C) Fates of newly approaching vesicles in control conditions. The relative frequency histogram of vesicle events as a function of distance from the nearest ribbon was binned at 350-nm intervals and adjusted for radial density (see Materials and methods). The likelihood of observing a newly approaching vesicle was consistently greater in the bin closest to the ribbon. However, the probability that fusion of a newly approaching vesicle (n = 48 total events in 9 rods) would occur in the bin closest to the ribbon rather than farther away was significantly greater than the probability that a newly approaching vesicle would dock at the membrane close to the ribbon (n = 17; P = 0.023, z test) or depart from ribbon-proximal membrane without fusion (n = 157; P < 0.0001, z test). (D) Fates of vesicles that began a trial as membrane-associated or docked vesicles. Fusion of previously docked vesicles was significantly more likely to occur in the bin closest to the ribbon (n = 30 total events; P = 0.00014) than retreat from the membrane (n = 35). The likelihood that a vesicle would remain docked throughout the trial was also significantly more likely in the bin closest to the ribbon (n = 28; P < 0.0001) than retreat.

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