Article Figures & Data
Figures
- Figure 1.
Specific peptide labeling of structures at the surface of retinal bipolar cell terminals imaged by TIRFM. A, Sequences of fluorescent peptides used in this study. Bold indicates CtBP binding domain binding. B, Images of four retinal bipolar cell terminals loaded with 27.7 μm fluorescein-labeled peptide and imaged using TIRFM. C, Epifluorescence image of a bipolar cell terminal loaded with the peptide.
- Figure 2.
Ribbon displacement in the synaptic terminal. A, Ribbons were imaged using TRFM microscopy and tracked using a software tracking algorithm (MetaMorph; Universal Imaging) and their displacement from the location where they were first visible plotted as a function of time. In general, displacement increased with time. B, To estimate the speed of ribbon movement, the plot displacement as a function of time was best least-squares fit to a straight line through the origin, and the slopes of those lines are plotted as a histogram. Although ribbons were relatively immobile on the time scale of our experiments, ribbons were twice observed to make comparatively rapid movements in bipolar cells. C, An example of a bipolar cell with a ribbon undergoing a rapid movement. Images are consecutive frames taken in 30 sec intervals. D, Same as in C, except at higher magnification.
- Figure 3.
Measurements of peptide affinity for spots. Bipolar cells were loaded with 415 μm fluorescein-labeled peptide and imaged using TIRFM. The fluorescence intensity of spots were measured (Fspot) and normalized to the maximum fluorescence for that spot (Fspot, max) over the time course of cell loading. The fluorescence of the cytoplasm outside the fluorescent spots was taken as an index of cytosolic peptide concentration. A, Fspot/Fspot, max is plotted as a function of peptide concentration ([peptide]) within the cell. Inset shows one example of a plot of fluorescence as a function of time for a spot (open circles) and in the cytoplasm (filled squares). The right axis in the inset shows the conversion of cytoplasmic fluorescence to peptide concentration, assuming that the asymptotic concentration is the same as the pipette concentration. Note that fluorescence of the spot saturates sooner than the cytoplasmic fluorescence. B, Plot of Fspot, max/Fspot versus [peptide]-1. First-order binding predicts a linear relationship with a slope equal to the Kd (see Materials and Methods). Slope of linear least-squares fit indicates a Kd of 27 μm.
- Figure 4.
Selective peptide-labeling of structures in the synaptic terminal of ribbon-containing neurons. Each picture shows planar projections of confocal optical sections through the entire depth of the cell. A, Bipolar cell. B, Cone photoreceptor. C, Horizontal cell. D, Cultured hippocampal neuron. The cell body of the labeled hippocampal neuron was above the field of view, and a labeled process followed the principal process of a nearby unlabeled cell, the position of which is indicated by the outline.
- Figure 5.
Spatial correspondence of labeled structures in confocal optical sections with ribbons in electron micrographs of the same synaptic terminal. Arrows indicate fluorescent spots and corresponding ribbons. The insets show close-up views of the ribbon clusters indicated by boxes. Clusters were rare and were only observed within the interior of the terminal of the one cell shown here. Asterisks indicate fluorescent spots that corresponded to ribbons in nearby semithin sections.
- Figure 6.
Ribbons colocalize with calcium entry sites. Bipolar cells were loaded with the fluorescent CtBP-binding peptide (28 μm), Fluo-5f (100 μm), and 10 mm EGTA and imaged using TIRFM. TIRFM images the region of the cell that is adherent to the glass, and in this example the cell adhered in two locations. A, Fluorescence collected and averaged for 250 msec just before a depolarization to 0 mV. B, Fluorescence collected and averaged during a 250 msec depolarization to 0 mV. C, Fluorescence collected for 250 msec immediately after return of the membrane potential to its resting potential of -60 mV. D, Image of B subtracted by the average of the images in A and C. E, Top shows fluorescence in regions marked in A. Note the rapid change in fluorescence at locations near spots seen at rest but not at other locations within the terminal.
Additional Files
Supplemental data
Files in this Data Supplement:
- supplemental material - Movie 1.� Ribbon movement in a retinal bipolar cell terminal.� Movie of two ribbons in a single bipolar cell terminal imaged using TIRF microscopy.� One ribbon moves disappears during the time course of the movie, presumably representing movement away from the membrane.� Images are high-pass filtered and smoothed for clarity.
- supplemental material - Movie 2.� Z-series of cone pedicle labeled with ribbon-label.� Same cell as in Fig. 4B
- supplemental material - Movie 3.� Rotated 3D projection of ribbon from same cone as in Movie 2.
- supplemental material - Movie 4.� Rotated 3D projection of retinal bipolar cell with ribbon label.� Same cell as in Fig. 4A.
