Faculty Opinions recommendation of Conical electron tomography of a chemical synapse: vesicles docked to the active zone are hemi-fused.

Abstract Most vesicles in the interior of synaptic terminals are clustered in clouds close to active zone regions of the plasma membrane where exocytosis occurs. Electron-dense structures, termed bridges, have been reported between a small minority of pairs of neighboring vesicles within the clouds. Synapsin proteins have been implicated previously, but the existence of the bridges as stable structures in vivo has been questioned. Here we use electron tomography to show that the bridges are present but less frequent in synapsin knockouts compared to wildtype. An analysis of distances between neighbors in wildtype tomograms indicated that the bridges are strong enough to resist centrifugal forces likely induced by fixation with aldehydes. The results confirm that the bridges are stable structures and that synapsin proteins are involved in formation or stabilization.

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The structure and function of ‘active zone material’ at synapses

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2014.0189 ◽

2015 ◽

Vol 370 (1672) ◽

pp. 20140189 ◽

Cited By ~ 13

Author(s):

Joseph A. Szule ◽

Jae Hoon Jung ◽

Uel J. McMahan

Keyword(s):

Spatial Resolution ◽

Active Zone ◽

Synaptic Vesicles ◽

Neuromuscular Junctions ◽

Electron Tomography ◽

Structure And Function ◽

Presynaptic Membrane ◽

Axon Terminals ◽

Active Zones ◽

And Function

The docking of synaptic vesicles on the presynaptic membrane and their priming for fusion with it to mediate synaptic transmission of nerve impulses typically occur at structurally specialized regions on the membrane called active zones. Stable components of active zones include aggregates of macromolecules, ‘active zone material’ (AZM), attached to the presynaptic membrane, and aggregates of Ca 2+ -channels in the membrane, through which Ca 2+ enters the cytosol to trigger impulse-evoked vesicle fusion with the presynaptic membrane by interacting with Ca 2+ -sensors on the vesicles. This laboratory has used electron tomography to study, at macromolecular spatial resolution, the structure and function of AZM at the simply arranged active zones of axon terminals at frog neuromuscular junctions. The results support the conclusion that AZM directs the docking and priming of synaptic vesicles and essential positioning of Ca 2+ -channels relative to the vesicles' Ca 2+ -sensors. Here we review the findings and comment on their applicability to understanding mechanisms of docking, priming and Ca 2+ -triggering at other synapses, where the arrangement of active zone components differs.

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Automated electron tomography: from data collection to image processing

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100136507 ◽

1995 ◽

Vol 53 ◽

pp. 26-27

Author(s):

Weiping Liu ◽

Jennifer Fung ◽

W.J. de Ruijter ◽

Hans Chen ◽

John W. Sedat ◽

...

Keyword(s):

Image Processing ◽

Data Collection ◽

Structural Information ◽

Imaging Technique ◽

Electron Tomography ◽

Ccd Camera ◽

Automated Data Collection ◽

Full Control ◽

Transmission Electron ◽

Amorphous Structures

Electron tomography is a technique where many projections of an object are collected from the transmission electron microscope (TEM), and are then used to reconstruct the object in its entirety, allowing internal structure to be viewed. As vital as is the 3-D structural information and with no other 3-D imaging technique to compete in its resolution range, electron tomography of amorphous structures has been exercised only sporadically over the last ten years. Its general lack of popularity can be attributed to the tediousness of the entire process starting from the data collection, image processing for reconstruction, and extending to the 3-D image analysis. We have been investing effort to automate all aspects of electron tomography. Our systems of data collection and tomographic image processing will be briefly described.To date, we have developed a second generation automated data collection system based on an SGI workstation (Fig. 1) (The previous version used a micro VAX). The computer takes full control of the microscope operations with its graphical menu driven environment. This is made possible by the direct digital recording of images using the CCD camera.

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Practical electron tomography

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140087 ◽

1995 ◽

Vol 53 ◽

pp. 742-743

Author(s):

C.L. Woodcock

Keyword(s):

Electron Tomography ◽

Tomographic Reconstruction ◽

Three Dimensional ◽

3D Shape ◽

Computational Resources ◽

Shape Of Particles

Despite the potential of the technique, electron tomography has yet to be widely used by biologists. This is in part related to the rather daunting list of equipment and expertise that are required. Thanks to continuing advances in theory and instrumentation, tomography is now more feasible for the non-specialist. One barrier that has essentially disappeared is the expense of computational resources. In view of this progress, it is time to give more attention to practical issues that need to be considered when embarking on a tomographic project. The following recommendations and comments are derived from experience gained during two long-term collaborative projects.Tomographic reconstruction results in a three dimensional description of an individual EM specimen, most commonly a section, and is therefore applicable to problems in which ultrastructural details within the thickness of the specimen are obscured in single micrographs. Information that can be recovered using tomography includes the 3D shape of particles, and the arrangement and dispostion of overlapping fibrous and membranous structures.

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Three-Dimensional reconstruction of isolated centrosomes by automated electron tomography

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100169286 ◽

1994 ◽

Vol 52 ◽

pp. 310-311

Author(s):

M.B. Braunfeld ◽

M. Moritz ◽

B.M. Alberts ◽

J.W. Sedat ◽

D.A. Agard

Keyword(s):

Electron Tomography ◽

Three Dimensional ◽

Vital Role ◽

Complex Nature ◽

Animal Cells ◽

Microtubule Organizing Center ◽

Nucleation Sites ◽

Dimensional Reconstruction ◽

Organizing Center ◽

Resolution Model

In animal cells, the centrosome functions as the primary microtubule organizing center (MTOC). As such the centrosome plays a vital role in determining a cell's shape, migration, and perhaps most importantly, its division. Despite the obvious importance of this organelle little is known about centrosomal regulation, duplication, or how it nucleates microtubules. Furthermore, no high resolution model for centrosomal structure exists.We have used automated electron tomography, and reconstruction techniques in an attempt to better understand the complex nature of the centrosome. Additionally we hope to identify nucleation sites for microtubule growth.Centrosomes were isolated from early Drosophila embryos. Briefly, after large organelles and debris from homogenized embryos were pelleted, the resulting supernatant was separated on a sucrose velocity gradient. Fractions were collected and assayed for centrosome-mediated microtubule -nucleating activity by incubating with fluorescently-labeled tubulin subunits. The resulting microtubule asters were then spun onto coverslips and viewed by fluorescence microscopy.

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On the Use of POCS for Restoring the “Missing Wedge” in Electron Tomography

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010018135x ◽

1990 ◽

Vol 48 (1) ◽

pp. 520-521

Author(s):

Neng-Yu Zhang ◽

Bruce F. McEwen ◽

Joachim Frank

Keyword(s):

Function Space ◽

Convex Sets ◽

Electron Tomography ◽

Spinal Ganglion ◽

Fourier Space ◽

Missing Information ◽

Voltage Electron ◽

High Voltage Electron Microscope ◽

Energy Bound ◽

Spatial Boundedness

Reconstructions of asymmetric objects computed by electron tomography are distorted due to the absence of information, usually in an angular range from 60 to 90°, which produces a “missing wedge” in Fourier space. These distortions often interfere with the interpretation of results and thus limit biological ultrastructural information which can be obtained. We have attempted to use the Method of Projections Onto Convex Sets (POCS) for restoring the missing information. In POCS, use is made of the fact that known constraints such as positivity, spatial boundedness or an upper energy bound define convex sets in function space. Enforcement of such constraints takes place by iterating a sequence of function-space projections, starting from the original reconstruction, onto the convex sets, until a function in the intersection of all sets is found. First applications of this technique in the field of electron microscopy have been promising.To test POCS on experimental data, we have artificially reduced the range of an existing projection set of a selectively stained Golgi apparatus from ±60° to ±50°, and computed the reconstruction from the reduced set (51 projections). The specimen was prepared from a bull frog spinal ganglion as described by Lindsey and Ellisman and imaged in the high-voltage electron microscope.

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Electron tomography

AccessScience ◽

10.1036/1097-8542.801290 ◽

2015 ◽

Keyword(s):

Electron Tomography

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