The Journal of Neuroscience, January 10, 2007, ():
Intersectin Is a Negative Regulator of Dynamin Recruitment to the Synaptic Endocytic Zone in the Central Synapse
J. Neurosci. Evergren et al.
27: 379
Supplemental Data
Files in this Data Supplement:
- supplemental material
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FIGURE S1: Serial section analysis of synapses microinjected with GST-SH3C and LIS-AC antibodies.
(A) Electron micrographs of five ultrathin sections from an uninjected reticulospinal synapse stimulated at 5 Hz. The synapse was cut in serial sections. Images show a part of this series. Every other section is shown. (B) Electron micrographs of five ultrathin sections from a synapse microinjected with GST-SH3C domain, analyzed as in A. (C) Electron micrographs of five ultrathin sections from a synapse microinjected with LIS-AC antibodies, analyzed as in A. Designations as in Figs. 3, 4. Scale bar 0.4 µm.
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FIGURE S2: Microinjection of intersectin GST-SH3B in reticulospinal synapses does not result in an accumulation of coated pits or membrane invaginations.
(A, B) Electron micrographs of a reticulospinal synapse microinjected with intersectin GST-SH3B domain and stimulated at 5 Hz for 30 min. Note that a significant reduction in the number of vesicles was observed compared to uninjected control synapses (C). This reduction was likely due to effects downstream of fission since single synaptic vesicles (thin arrows) and free clathrin-coated vesicles (ccv) were observed in the axoplasmic matrix outside the periactive zone. Inset in B shows the boxed area at higher magnification. (D) Bar graph representing the relative average number of clathrin-coated intermediates per active zone length. No significant difference was found between injected and uninjected synapses (p>0.05; two-tailed Student’s t-test). (E) Bar graph representing the average length of membrane structures at endocytic zones (ez). No significant difference was found between injected and uninjected synapses (p>0.05; two-tailed Student’s t-test). (F) Bar graph showing the relative abundance of different stages of coated intermediates in synapses injected with GST-SH3B compared to uninjected synapses (open bars). 100 coated intermediates were randomly collected. Stages 1-5 represent sequential stages, while ‘d’ represents intermediates with a distorted morphology. Error bars represent the standard error for each group, n represents the number of middle sections analyzed in each group. *** p<0.0001; two-tailed Student’s t-test. Designations as in Figs. 3, 4. Scale bars, A: 0.5 µm; B: 1 µm.
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Fig S3: Microinjection of intersectin GST-SH3C or intersectin antibodies results in a reorganization of the actin matrix in the periactive zone.
(A-C) Confocal images of phalloidin-Alexa488 fluorescence at synaptic sites in reticulospinal axons microinjected with GST-SH3C (A1-3), LIS-AC antibodies (B1-3), and uninjected control (C1-2). Axons were injected with GST-SH3C or LIS-AC antibodies, stimulated for 20 min at 5 Hz extracellularly as described in Methods, microinjected with phalloidin during stimulation, and imaged in a confocal microscope. Neighboring axons from the same preparation were microinjected with phalloidin only and are referred to as control. The average diameter of ‘phalloidin-rings’ was significantly larger in SH3C injected axons compared to control (3.0 ± 0.1 µm; 2.5 ± 0.1 µm, respectively; mean ± SEM; n=21; p<0.01; two-tailed Student’s t-test). The same was true for LIS-AC injected axons (3.0 ± 0.1 µm; 2.5 ± 0.1 µm, respectively; mean ± SEM; n=21; p<0.01; two-tailed Student’s t-test). ‘Phalloidin-rings’ in axons microinjected with GST-SH3C or LIS-AC antibodies were fragmented compared to control. Microinjection of GST or unspecific IgG did not perturb the shape of ‘phalloidin-rings’ (unpublished observations). Comparison of fluorescence profile plots using ImageJ showed a higher variability in the fluorescence pattern. See examples of 10 randomly selected profiles normalized to the diameter of phalloidin rings. Statistical analysis of the variance of fluorescence intensity demonstrated a significant difference in the center (0.5) of the ‘phalloidin-ring’ (GST-SH3C: 1215, n = 16 profiles; LIS-AC: 1205, n = 40 profiles; control: 44, n = 10 (for both cases); p(F<=f)<0.0001, respectively; one-tailed F-test). The mean fluorescence intensity in the center (0.5) was not significantly different to control (GST-SH3C: 37.4; LIS-AC: 50.5; control: 32.3; p>0.05, respectively, two-tailed Student’s t-test).
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FIGURE S4: Graphs illustrating the distribution of clathrin-coated intermediates after microinjection of GST and IgG.
(A, B) Bar graph showing the relative abundance of different stages of coated intermediates in stimulated synapses injected with GST (A) and unspecific IgG (B) compared to uninjected synapses (open bars). 100 coated intermediates were collected randomly. Stages 1-5 represent sequential stages and ‘d’ represents intermediates with a distorted morphology.
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FIGURE S5: Disruption of periactive zone organization by microinjection of intersectin antibodies and subsequent high-frequency stimulation.
Stimulation of reticulospinal synapses at 20 Hz results in a partial depletion of vesicles at active zones (C, E), while simultaneously, an accumulation of synaptic vesicle membrane at periactive zones occurs. These effects are interpreted as being the consequence of the inability of endocytosis to keep pace with exocytosis. This stimulation protocol is therefore useful to ascertain the function of an endocytic protein while membrane reuptake is severely challenged. (A, B) Electron micrographs of synapses in reticulospinal axons stimulated at 20 Hz for 20 min after microinjection of LIS-AC antibodies. Note the lack of vesicles clustered at active zones (thick arrows) and the connections between the plasma membrane and the membrane infolds (asterisk; boxed area in B is shown at higher magnification in B’). (C) A control synapse located outside of the site of injection in the same axon. Curved arrows indicate “membrane pockets”. The inset shows a membrane pocket filled with filamentous cytoskeletal matrix (double arrow) marked with the rectangle in C. (D) Micrograph showing a reticulospinal synapse from an axon microinjected with synapsin G304 antibodies, stimulated in the same way as axons microinjected with intersectin antibodies. (E) Bar graph showing the average relative reduction in the number of synaptic vesicles in 20 Hz stimulated control synapses compared to synapses injected with LIS-AC antibodies. Open bars represent uninjected, stimulated synapses and hatched bars represent injected, stimulated synapses. (F) Bar graph illustrating the average length of membrane structures at the endocytic zone (ez) in the same group of synapses. (G) Changes in the average number of coated intermediates induced by antibody injection in stimulated synapses. Bars represent averaged numbers derived from the groups of synapses analyzed in E. (H) High-power micrograph from a synapse after injection of LIS-AC antibodies and 20 Hz stimulation showing spherical membrane structures connected by thin tubules (thin arrows). In some spheres, clathrin-coated pits were found on the inner membrane. g: glia, m: mitochondrion. Other designations as in Figures 3, 4. Error bars represent the standard errors for each group. Two-tailed Student’s t-test was applied: *** p<0.0001 compared to the stimulated control; Scale bars, A, C, D: 0.5 µm; B: 0.5 µm; B’: 0.1 µm; H: 0.1 µm.
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FIGURE S6: Schematic representation of the experimental setup for pre-embedding immunocytochemistry of reticulospinal axons in combination with microinjections.
(A) A schematic representation of sequential steps of the labeling procedure of microinjected axons. Microinjected axons were first divided into two pieces (dashed lines) at the injection site and then cut longitudinally to open the injected regions. This resulted in four pieces, which were labeled with four different antibodies. Two adjacent pieces were incubated with dynamin and amphiphysin antibodies. The other two were incubated with dynamin antibodies or without primary antibodies. A’ shows an example of five reticulospinal axons injected with fluorescently tagged LIS-AC antibodies. (B) Electron micrograph showing a clathrin-coated pit from a LIS-AC microinjected axon labeled for dynamin. Scale bar 0.05 µm.
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FIGURE S7: Quantification of immmunolabeling at clathrin-coated pits.
A schematic representation of a clathrin-coated pit that shows the areas that were used for quantification of immunogold labeling. Dashed lines mark the total area and the gray box indicates the neck area.
