[show abstract][hide abstract] ABSTRACT: Synaptic vesicles dock at active zones on the presynaptic plasma membrane of a neuron's axon terminals as a precondition for fusing with the membrane and releasing their neurotransmitter to mediate synaptic impulse transmission. Typically, docked vesicles are next to aggregates of plasma membrane-bound macromolecules called active zone material (AZM). Electron tomography on tissue sections from fixed and stained axon terminals of active and resting frog neuromuscular junctions has led to the conclusion that undocked vesicles are directed to and held at the docking sites by the successive formation of stable connections between vesicle membrane proteins and proteins in different classes of AZM macromolecules. Using the same nanometer scale 3D imaging technology on appropriately stained frog neuromuscular junctions, we found that ∼10% of a vesicle's luminal volume is occupied by a radial assembly of elongate macromolecules attached by narrow projections, nubs, to the vesicle membrane at ∼25 sites. The assembly's chiral, bilateral shape is nearly the same vesicle to vesicle, and nubs, at their sites of connection to the vesicle membrane, are linked to macromolecules that span the membrane. For docked vesicles, the orientation of the assembly's shape relative to the AZM and the presynaptic membrane is the same vesicle to vesicle, whereas for undocked vesicles it is not. The connection sites of most nubs on the membrane of docked vesicles are paired with the connection sites of the different classes of AZM macromolecules that regulate docking, and the membrane spanning macromolecules linked to these nubs are also attached to the AZM macromolecules. We conclude that the luminal assembly of macromolecules anchors in a particular arrangement vesicle membrane macromolecules, which contain the proteins that connect the vesicles to AZM macromolecules during docking. Undocked vesicles must move in a way that aligns this arrangement with the AZM macromolecules for docking to proceed.
PLoS ONE 01/2013; 8(7):e69410. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: The docking of synaptic vesicles at active zones on the presynaptic plasma membrane of axon terminals is essential for their fusion with the membrane and exocytosis of their neurotransmitter to mediate synaptic impulse transmission. Dense networks of macromolecules, called active zone material, (AZM) are attached to the presynaptic membrane next to docked vesicles. Electron tomography has shown that some AZM macromolecules are connected to docked vesicles, leading to the suggestion that AZM is somehow involved in the docking process. We used electron tomography on the simply arranged active zones at frog neuromuscular junctions to characterize the connections of AZM to docked synaptic vesicles and to search for the establishment of such connections during vesicle docking. We show that each docked vesicle is connected to 10-15 AZM macromolecules, which fall into four classes based on several criteria including their position relative to the presynaptic membrane. In activated axon terminals fixed during replacement of docked vesicles by previously undocked vesicles, undocked vesicles near vacated docking sites on the presynaptic membrane have connections to the same classes of AZM macromolecules that are connected to docked vesicles in resting terminals. The number of classes and the total number of macromolecules to which the undocked vesicles are connected are inversely proportional to the vesicles' distance from the presynaptic membrane. We conclude that vesicle movement toward and maintenance at docking sites on the presynaptic membrane are directed by an orderly succession of stable interactions between the vesicles and distinct classes of AZM macromolecules positioned at different distances from the membrane. Establishing the number, arrangement and sequence of association of AZM macromolecules involved in vesicle docking provides an anatomical basis for testing and extending concepts of docking mechanisms provided by biochemistry.
PLoS ONE 01/2012; 7(3):e33333. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Electron tomography was used to view macromolecules composing active zone material (AZM) in axon terminals at mouse neuromuscular junctions. Connections of the macromolecules to each other, to calcium channels in the presynaptic membrane, and to synaptic vesicles docked on the membrane prior to fusing with it during synaptic transmission were similar to those of AZM macromolecules at frog neuromuscular junctions previously examined by electron tomography and support the hypothesis that AZM regulates vesicle docking and fusion. A species difference in the arrangement of AZM relative to docked vesicles may help account for a greater vesicle-presynaptic membrane contact area during docking and a greater probability of fusion during synaptic transmission in mouse. Certain AZM macromolecules in mouse were connected to synaptic vesicles contacting the presynaptic membrane at sites where fusion does not occur. These secondary docked vesicles had a different relationship to the membrane and AZM macromolecules than primary docked vesicles, consistent with their having a different AZM-regulated behavior.
The Journal of Comparative Neurology 05/2009; 513(5):457-68. · 3.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Reconstructed volumes generated by tilt-image electron-microscope tomography offer the best spatial resolution currently available for studying cell structures in situ. Analysis is often accomplished by creating surface models that delineate grayscale contrast boundaries. Here, we introduce a specialized and convenient sequence of segmentation operations for making such models that greatly improves their reliability and spatial resolution as compared to current approaches, providing a basis for making accurate measurements. To assess the reliability of the surface models, we introduce a spatial uncertainty measurement based on grayscale gradient scale length. The model generation and measurement methods are validated by applying them to synthetic data, and their utility is demonstrated by using them to characterize macromolecular architecture of active zone material at the frog's neuromuscular junction.
[show abstract][hide abstract] ABSTRACT: Electron microscope tomography produces reconstructed biological tissue volumes containing structural data with few-nanometer spatial resolution. Frequently, surface models are constructed to visualize and analyze the structural data. These models are usually constructed to approximate the contrast boundaries of various structural components within the volume. An automatic method is presented for obtaining isodensity surface models that correspond most closely to the steepest contrast boundary at the edge of each component. We validate our method using simulated volumetric data, and demonstrate its application to a reconstructed volume obtained from the neuromuscular junction of the frog.
Engineering in Medicine and Biology Society, 2003. Proceedings of the 25th Annual International Conference of the IEEE; 10/2003
[show abstract][hide abstract] ABSTRACT: Active zone material at the nervous system's synapses is situated next to synaptic vesicles that are docked at the presynaptic plasma membrane, and calcium channels that are anchored in the membrane. Here we use electron microscope tomography to show the arrangement and associations of structural components of this compact organelle at a model synapse, the frog's neuromuscular junction. Our findings indicate that the active zone material helps to dock the vesicles and anchor the channels, and that its architecture provides both a particular spatial relationship and a structural linkage between them. The structural linkage may include proteins that mediate the calcium-triggered exocytosis of neurotransmitter by the synaptic vesicles during synaptic transmission.
[show abstract][hide abstract] ABSTRACT: Three-dimensional reconstruction of a section of biological tissue by electron tomography requires precise alignment of a series of two-dimensional images of the section made at numerous successive tilt angles. Gold beads on or in the section serve as fiducial markers. A scheme is described that automatically detects the position of these markers and indexes them from image to image. The resulting set of position vectors are arranged in a matrix representation of the tilt geometry and, by inversion, alignment information is obtained. The scheme is convenient, requires little operator time and provides an accuracy of < 2 pixels RMS. A tilt series of 60-70 images can be aligned in approximately 30 min on any modern desktop computer.
Journal of Electron Microscopy 02/1999; 48(3):277-87. · 1.44 Impact Factor
[show abstract][hide abstract] ABSTRACT: We used EM tomography to examine the fine structure of the apparently amorphous electron dense material that is seen at active zones of axon terminals when viewed by conventional 2D electron microscopy. Serial 1-nm optical slices from 3D reconstructions of individual thin tissue sections reveal that the material is composed of an interconnecting network of elongate components directly linked to synaptic vesicles and the presynaptic membrane. Each vesicle at the active zone that lies adjacent to the presynaptic plasma membrane has several such connections. Information provided by reconstruction data may be useful in generating experiments aimed at understanding the mechanisms involved in the docking of synaptic vesicles and their exocytosis during synaptic transmission.
Journal of Physiology-Paris 05/1998; 92(2):75-8. · 0.82 Impact Factor