Conical tomography : a simple method to study proteins in cells at high resolution

Despite advances in molecular biology and genetics, the location of thousand of proteins in cells remains undetermined. The principal problems are their small dimensions and their capacity to form large assemblies by associating either with themselves or other proteins. We took advantage of the tendency to form aggregates and developed a simple method that describe the three-dimensional structure of these assemblies in cells at high resolution (2-3nm) and in three-dimensions. As a proof of principle, we studied the alphaA-crystalline, a small chaperone that plays an important role in lens transparency and cataract formation. To identify the assemblies containing the chaperone, lens tissues labeled with primary anti-alphaA-crystalline and probed with both 2nm and 5nm diameter gold particle conjugates were reconstructed by conical tomography. First, we determined the location of all gold particles contained within the reconstructed volume. From maps of their 3D-distribution, we determined that gold particles formed files that repeated at 6-7nm center-to-center apart and bent at angles measuring ~90\ub0 or ~120\ub0. Second, we identified the tethers that linked each gold particle to the assemblies containing the chaperone. Independent of the diameter of the gold particle, tethers formed by the association of primary and secondary antibodies measured ~14nm in length. Finally, by applying the constraints represented by the repeat period, the angles and the structure of the assemblies, we identified the chaperone in unlabeled tissues as small globules spaced 6-7nm apart decorating thin filaments of the cytoskeleton. In conclusion, the high resolution in three-dimensions, the reliance on geometric constraints instead of exogenous probes and the technical simplicity are unrivaled properties of our method for studying the contribution that proteins made to normal cell homeostasis and pathological conditions. Supported by NIH EY-0411

Conical tomography II: A method for the study of cellular organelles in thin sections

We have used conical electron tomography in order to reconstruct neuronal organelles in thin sections of plastic embedded rat somato-sensory cortical tissue. The conical tilt series were collected at a 55 degrees tilt and at 5 degrees rotations, aligned using gold. particles as fiduciary markers, and reconstructed using the weighted back projection algorithm. After a refinement process based on projection matching, the 3D maps showed the "unit membrane pattern" along the entire reconstructed volume. This pattern is indicative of the bilayer arrangement of phospholipids in biological membranes. Based on Fourier correlation methods as well as the visualization of the "unit membrane" pattern, we estimated resolutions of similar to 4 nm. To illustrate the prospective advantages of conical tomography, we segmented "coated" vesicles in the reconstructed volumes. These vesicles were comprised of a central core enclosing a small lumen, and a protein "coating" extending into the cytoplasm. The "coated" vesicle was attached to the plasma membrane through a complex structure shaped as an arch where the ends are attached to the membrane and the crook is connected to the vesicle. We concluded that conical electron tomography of thin-sectioned specimens provides a powerful experimental approach for studying thin-sectioned neuronal organelles at resolution levels of similar to 4 nm. (C) 2005 Elsevier Inc. All rights reserved

Conical Electron Tomography of a Chemical Synapse: Polyhedral Cages Dock Vesicles to the Active Zone

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 \u201caverage\u201d synapse, by conical electron tomography, a method that exhibits an isotropic in plane resolution of 3 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 \u201csynaptic units\u201d comprising a polyhedral cage and a corona of approximately seven vesicles. The polyhedral cages measured 60 nm in diameter, with a density of 44/ m2 and were associated with vesicles at the active zone (\u201cfirst tier\u201d). Vesicles in this first-tier position represented 7.5% of the total number of vesicles in the terminal and were contiguous, hemifused ( 4% of total), or fully fused ( 0.5% 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

Conical tomography of freeze-fracture replicas : a method for the study of integral membrane proteins inserted in phospholipid bilayers

We have used conical tomography to study the structure of integral proteins in their phospholipid bilayer environments. Complete conical series were collected from replicas of the water channel aquaporin-0 (AQP0), a 6.6 nm side tetramer with a molecular weight of similar to120 kDa that was purified and reconstituted in liposomes. The replicas were tilted at 38degrees, 50degrees or 55degrees and rotated by 2.5degrees, 4degrees, or 5degrees increments until completing 360degrees turns. The elliptical paths of between 6 and 12 freeze-fracture particles aligned the images to a common coordinate system. Using the weighted back projection algorithm, small volumes of the replicas were independently reconstructed to reconstitute the field. Using the Fourier Shell Correlation computed from reconstructions of even and odd projections of the series. we estimated a resolution of 2-3 nm, a value that was close to the thickness of the replica (similar to1.5 nm). The 3D reconstructions exhibited isotropic resolution along the x-y plane, which simplified the analysis of particles, oriented randomly in the membrane plane. In contrast to reconstruct ions from single particles imaged using random conical tilt [J Mol. Biol. 325 (2003) 210], the reconstructions using conical tomography allowed the size and shape of individual particles representing the AQP0 channel to be identified without averaging or imposing symmetry. In conclusion. the reconstruction of freeze-fracture replicas with electron tomography has provided a novel experimental approach for the study of integral proteins inserted in phospholipid bilayers. (C) 2004 Elsevier Inc. All rights reserved

Conical Electron Tomography of a Chemical Synapse: Vesicles Docked to the Active Zone are Hemi-Fused

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 ∼15 nm in diameter (∼2% of the vesicle's surface) and contained a smaller ∼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 (∼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

From Membrane Pores to Aquaporins: 50 Years Measuring Water Fluxes

This review focuses on studies of water movement across biological membranes performed over the last 50 years. Different scientific approaches had tried to elucidate such intriguing mechanism, from hypotheses emphasizing the role of the lipid bilayer to the cloning of aquaporins, the ubiquitous proteins described as specific water channels. Pioneering and clarifying biophysical work are reviewed beside results obtained with the help of recent sophisticated techniques, to conclude that great advances in the subject live together with old questions without definitive answers