Biomedical subjects

Achilleas S Frangakis

Publications and source records attributed to Achilleas S Frangakis.

11 recordsLinked to original sources

PubMed2007

Visualization of cell microtubules in their native state.

BACKGROUND INFORMATION: Over the past decades, cryo-electron microscopy of vitrified specimens has yielded a detailed understanding of the tubulin and microtubule structures of samples reassembled in vitro from purified components. However, our knowledge of microtubule structure in vivo remains limited by the chemical treatments commonly used to observe cellular architecture using electron microscopy. RESULTS: We used cryo-electron microscopy and cryo-electron tomography of vitreous sections to investigate the ultrastructure of microtubules in their cellular context. Vitreous sections were obtained from organotypic slices of rat hippocampus and from Chinese-hamster ovary cells in culture. Microtubules revealed their protofilament ultrastructure, polarity and, in the most favourable cases, molecular details comparable with those visualized in three-dimensional reconstructions of microtubules reassembled in vitro from purified tubulin. The resolution of the tomograms was estimated to be approx. 4 nm, which enabled the detection of luminal particles of approx. 6 nm in diameter inside microtubules. CONCLUSIONS: The present study provides a first step towards a description of microtubules, in addition to other macromolecular assemblies, in an unperturbed cellular context at the molecular level. As the resolution appears to be similar to that obtainable with plunge-frozen samples, it should allow for the in vivo identification of larger macromolecular assemblies in vitreous sections of whole cells and tissues.

Animals↗

PubMed2006

Plastic-embedded protein crystals.

Rapid vitrification followed by the replacement of the vitrified water by a solvent (freeze substitution) and then resin is a widely used procedure for preparing biological samples for electron microscopy. The resulting plastic-embedded samples permit convenient room-temperature sectioning (microtomy) and can yield well preserved cellular structures. Here this procedure has been applied to crystalline protein samples, and it is shown that it is possible to freeze-substitute vitrified crystals while preserving some of their original diffraction properties. The plastic-embedded crystals were used to collect a series of complete room-temperature data sets at a powerful macromolecular crystallography synchrotron beamline. Whereas one normally observes specific damage to disulfide bonds upon X-ray radiation, no such damage was seen for the plastic-embedded sample. The X-ray diffraction data allowed an initial atomic analysis to be made of the effects of freeze-substitution and plastic embedding on biological samples.

Acrylic Resins↗

PubMed2006

Implementation and performance evaluation of reconstruction algorithms on graphics processors.

The high-throughput needs in electron tomography and in single particle analysis have driven the parallel implementation of several reconstruction algorithms and software packages on computing clusters. Here, we report on the implementation of popular reconstruction algorithms as weighted backprojection, simultaneous iterative reconstruction technique (SIRT) and simultaneous algebraic reconstruction technique (SART) on common graphics processors (GPUs). The speed gain achieved on the GPUs is in the order of sixty (60x) to eighty (80x) times, compared to the performance of a single central processing unit (CPU), which is comparable to the acceleration achieved on a medium-range computing cluster. This acceleration of the reconstruction is caused by the highly specialized architecture of the GPU. Further, we show that the quality of the reconstruction on the GPU is comparable to the CPU. We present detailed flow-chart diagrams of the implementation. The reconstruction software does not require special hardware apart from the commercially available graphics cards and could be easily integrated in software packages like SPIDER, XMIPP, TOM-Package and others.

Algorithms↗

PubMed2006

Structural analysis of Mycoplasma pneumoniae by cryo-electron tomography.

Bacteria of the genus Mycoplasma lack obvious homologs of prokaryotic or eukaryotic cytoskeletal, as well as motility-related genes (except FtsZ). Nevertheless, they maintain characteristic cell shapes and show adhesion and gliding abilities on both artificial surfaces and cells. Earlier genetic, biochemical, and electron microscopic analyses have shown that the tip structure, located at the tapered end of gliding mycoplasmas, is indispensable for this behavior. In this study, we have analyzed the fine structure of the Mycoplasma pneumoniae tip by cryo-electron tomography. We show that the central rod is surrounded by quasi-periodical electron-dense macromolecular complexes. Additional complexes are located at the distal end of the rod which connect the rod to the cytoplasmic membrane. Furthermore, we detect a structure at the proximal end of the rod that attaches the rod to the cell membrane. The surface protein complexes have been mapped in detail and their distribution on the cell surface has been visualized. Since the rod structures were detected at a close to native state of the cells, they allow us to build a hypothesis describing the motility mechanism of M. pneumoniae. Finally, we have evaluated the ribosome density of the organism by a template matching approach, whereby the reliability of the detection was supported by a comparative bioinformatics analysis.

Antigens, Surface↗

PubMed2006

Tilt-series and electron microscope alignment for the correction of the non-perpendicularity of beam and tilt-axis.

In electron tomography the sample is tilted in the electron microscope and projections are recorded at different viewing angles. In the correct geometric setting, the tilt-axis of the object under scrutiny is perpendicular to the beam direction. However, we will demonstrate that this does not necessarily apply to all electron microscopes equipped with the default column alignment. The resulting effect is that a conical tilt is performed, which has to be considered in the reconstruction to avoid artifacts and to improve the resolution. A novel solution, with significantly improved convergence properties, will be introduced for calculating the three-dimensional marker model, which is necessary for the alignment of the tilt-series. Thereby, the angle between the beam direction and the tilt-axis is calculated, together with other geometrical distortions, like magnification and rotation changes, and incorporated in the reconstruction. Hereby, artifacts can be eliminated at the image processing basis, and the resolution can be significantly improved at the medium to high range frequencies. Synthetical and real data are used to demonstrate the obstructions caused by this effect and the quality improvement of the reconstructions. Finally, we also present a way to align the hardware of the microscope to correct for the non-perpendicularity between the beam direction and the tilt-axis, which is specifically tailored for tomographic applications.

Image Processing, Computer-Assisted↗

PubMed2006

Multiple large filament bundles observed in Caulobacter crescentus by electron cryotomography.

While the absence of any cytoskeleton was once recognized as a distinguishing feature of prokaryotes, it is now clear that a number of different bacterial proteins do form filaments in vivo. Despite the critical roles these proteins play in cell shape, genome segregation and cell division, molecular mechanisms have remained obscure in part for lack of electron microscopy-resolution images where these filaments can be seen acting within their cellular context. Here, electron cryotomography was used to image the widely studied model prokaryote Caulobacter crescentus in an intact, near-native state, producing three-dimensional reconstructions of these cells with unprecedented clarity and fidelity. We observed many instances of large filament bundles in various locations throughout the cell and at different stages of the cell cycle. The bundles appear to fall into four major classes based on shape and location, referred to here as 'inner curvature', 'cytoplasmic', 'polar' and 'ring-like'. In an attempt to identify at least some of the filaments, we imaged cells where crescentin and MreB filaments would not be present. The inner curvature and cytoplasmic bundles persisted, which together with their localization patterns, suggest that they are composed of as-yet unidentified cytoskeletal proteins. Thus bacterial filaments are frequently found as bundles, and their variety and abundance is greater than previously suspected.

Actin Cytoskeleton↗

PubMed2005

Cryo-electron tomography reveals the cytoskeletal structure of Spiroplasma melliferum.

Evidence has accumulated recently that not only eukaryotes but also bacteria can have a cytoskeleton. We used cryo-electron tomography to study the three-dimensional structure of Spiroplasma melliferum cells in a close-to-native state at approximately 4-nanometer resolution. We showed that these cells possess two types of filaments arranged in three parallel ribbons underneath the cell membrane. These two filamentous structures are built of the fibril protein and possibly the actin-like protein MreB. On the basis of our structural data, we could model the motility modes of these cells and explain how helical Mollicutes can propel themselves by means of coordinated length changes of their cytoskeletal ribbons.

Bacterial Proteins↗

PubMed2004

Computational exploration of structural information from cryo-electron tomograms.

Cryo-electron tomography aims to act as an interface between in vivo cell imaging and techniques achieving atomic resolution. This attempt to bridge the resolution gap is facilitated by recent software and hardware advances. Information provided by atomically resolved macromolecules and molecular interaction data need to be put into a common framework in order to create a hybrid multidimensional cellular image. A major partner in this enterprise is the development of regularization and pattern recognition techniques, which try to identify macromolecular complexes as a function of their structural signature in cryo-electron tomograms of living cells.

Animals↗

PubMed2002

Macromolecular architecture in eukaryotic cells visualized by cryoelectron tomography.

Electron tomography of vitrified cells is a noninvasive three-dimensional imaging technique that opens up new vistas for exploring the supramolecular organization of the cytoplasm. We applied this technique to Dictyostelium cells, focusing on the actin cytoskeleton. In actin networks reconstructed without prior removal of membranes or extraction of soluble proteins, the cross-linking of individual microfilaments, their branching angles, and membrane attachment sites can be analyzed. At a resolution of 5 to 6 nanometers, single macromolecules with distinct shapes, such as the 26S proteasome, can be identified in an unperturbed cellular environment.

Actin Cytoskeleton↗

PubMed2002

Identification of macromolecular complexes in cryoelectron tomograms of phantom cells.

Electron tomograms of intact frozen-hydrated cells are essentially three-dimensional images of the entire proteome of the cell, and they depict the whole network of macromolecular interactions. However, this information is not easily accessible because of the poor signal-to-noise ratio of the tomograms and the crowded nature of the cytoplasm. Here, we describe a template matching algorithm that is capable of detecting and identifying macromolecules in tomographic volumes in a fully automated manner. The algorithm is based on nonlinear cross correlation and incorporates elements of multivariate statistical analysis. Phantom cells, i.e., lipid vesicles filled with macromolecules, provide a realistic experimental scenario for an assessment of the fidelity of this approach. At the current resolution of approximately 4 nm, macromolecules in the size range of 0.5-1 MDa can be identified with good fidelity.

Algorithms↗

PubMedDate not supplied

Segmentation of two- and three-dimensional data from electron microscopy using eigenvector analysis.

An automatic image segmentation method is used to improve processing and visualization of data obtained by electron microscopy. Exploiting affinity criteria between pixels, e.g., proximity and gray level similarity, in conjunction with an eigenvector analysis, the image is subdivided into areas which correspond to objects or meaningful regions. Extending a proposal by Shi and Malik (1997, Proceedings of the IEEE conference on Computer Vision and Pattern Recognition, pp. 731-737) the approach was adapted to the field of electron microscopy, especially to three-dimensional application as needed by electron tomography. Theory, implementation, parameter setting, and results obtained with a variety of data are presented and discussed. The method turns out to be a powerful tool for visualization with the potential for further improvement by developing and tuning new affinity.

Algorithms↗