We have used thin sectioning and conical electron tomography to determine the three-dimensional structure of synaptic vesicles that were associated (docked) at release sites of the presynaptic membrane, called active-zones. Vesicles docked at the active zone occupied a strategic location: they formed regions of contact with the plasma membrane on one side and with that of one or more vesicles located deeper within the presynaptic terminal on the other side. The region of contact with the active zone measured approximately 15 nm in diameter ( approximately 2% of the vesicle's surface) and contained a smaller approximately 6 nm region where the proximal leaflets merged (hemi-fused). Hemi-fusion was only observed on the side of vesicles in contact with the active zone; at the side of contact between neighboring vesicles, the membranes were not hemi-fused. Approximately three-fourths of the docked vesicles contained hemi-fused regions. Vesicles fully fused to the active zone (exhibiting pores that appeared as interruptions of a single membrane) were less frequently observed ( approximately 1 of 10 hemi-fused vesicles). In conclusion, our observations in cortical synapses strengthen the hypothesis that hemi-fusion is a stable intermediary that precedes full fusion and release.
In this study, we tested the hypothesis that the structure of the active zone of chemical synapses has remained uncertain because of limitations of conventional electron microscopy. To resolve these limitations, we reconstructed chemical synapses of rat neocortex, the archetypical "average" synapse, by conical electron tomography, a method that exhibits an isotropic in plane resolution of ϳ3 nm and eliminates the need to impose symmetry or use averaging methods to increase signal-to-noise ratios. Analysis of 17 reconstructions by semiautomatic density segmentation indicated that the active zone was constructed of a variable number of distinct "synaptic units" comprising a polyhedral cage and a corona of approximately seven vesicles. The polyhedral cages measured ϳ60 nm in diameter, with a density of ϳ44/m 2 and were associated with vesicles at the active zone ("first tier"). Vesicles in this first-tier position represented ϳ7.5% of the total number of vesicles in the terminal and were contiguous, hemifused (ϳ4% of total), or fully fused (ϳ0.5% of total) to the plasma membrane. Our study supports the hypothesis that rat neocortical synapses are constructed of variable numbers of distinct synaptic units that facilitate the docking of vesicles to the active zone and determine the number of vesicles available for immediate release.
We are presenting a program for interactive segmentation of tomographic maps, based on objective criteria so as to yield reproducible results. The strategy starts with the automatic segmentation of the entire volume with the watershed algorithm in 3D. The watershed regions are clustered successively by supervised classification, allowing the segmentation of known organelles, such as membranes, vesicles and microtubules. These organelles are processed with topological models and input parameters manually derived from the tomograms. After known organelles are extracted from the volume, all other watershed regions can be organized into homogeneous assemblies on the basis of their densities. To complete the process, all voxels in the volume are assigned either to the background or individual structures, which can then be extracted for visualization with any rendering technique.The user interface of the program is written in Java, and computational routines are written in C. For some operations, involving the visualization of the tomogram, we refer to existing software, either open or commercial. While the program runs, a history file is created, that allows all parameters and other data to be saved for the purposes of comparison or exchange. Initially, the program was developed for the segmentation of synapses, and organelles belonging to these structures have thus far been the principal targets modeled with JUST. Since each organelle is clustered independently from the rest of the volume, however, the program can accommodate new models of different organelles as well as tomograms of other types of preparations of tissue, such as citoskeletal components in vitreous ice.
Studies using conventional electron microscopy describe the cytoplasm of lens fiber cells as having essentially an amorphous structure. We hypothesized that significant structural detail might have been lost as a result of projecting the entire thickness of the section (50–100 nm) onto a single plane (the “projection artifact”). To test this hypothesis, we studied the 3D-structure of rat lens cortical fibers before and after extracting the “soluble” crystallins with low ionic strength buffers to make “ghosts.” Tomographic series in conical geometry were collected at 55° tilts and by 5° rotations until completing a 360° turn by low dose methods. They were aligned using fiduciary points, reconstructed with the weighted back projection algorithm and refined by projection matching. Analysis of the 3D-maps included semiautomatic density segmentation using a computer program based on the watershed algorithm. We found that the cytoplasm of cortical fibers, though appearing amorphous in regions of the highest density, was in fact comprised of an ordered structure resembling a “clustered matrix.” The matrix was comprised of thin (~6 nm diameter) filaments bent sharply at 110–120° angles and studded with cubeshaped particles (the “beaded” filaments). In cortical fibers, the particles measured a = 14 ± 2, b = 13 ± 2 and c = 10 ± 2.4 nm (n = 30, mean ± SD) and were spaced at distances measuring 27.5 ± 2.4 nm apart (n = 8, mean ± SD), center-to-center. The matrix was formed as “beaded” filaments, bound to clusters of “soluble” proteins, crossed each other at nearly perpendicular angles. The matrix also made contact with the plasma membrane at a large number of distinct regions. We thus concluded that the cytoplasm of cortical lens fibers is comprised of a cytoskeletal matrix of “beaded” filaments that organize the “soluble” crystallins in separate regions. The association of this matrix with the plasma membrane allows the lens to maintain its structural integrity, while its association with crystallins yields its long-term transparency. Loss of either function likely would play a significant role in cataract formation.
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