The supramolecular organization of fibrillin-rich microfibrils.

We propose a new model for the alignment of fibrillin molecules within fibrillin microfibrils. Automated electron tomography was used to generate three-dimensional microfibril reconstructions to 18.6-A resolution, which revealed many new organizational details of untensioned microfibrils, including heart-shaped beads from which two arms emerge, and interbead diameter variation. Antibody epitope mapping of untensioned microfibrils revealed the juxtaposition of epitopes at the COOH terminus and near the proline-rich region, and of two internal epitopes that would be 42-nm apart in unfolded molecules, which infers intramolecular folding. Colloidal gold binds microfibrils in the absence of antibody. Comparison of colloidal gold and antibody binding sites in untensioned microfibrils and those extended in vitro, and immunofluorescence studies of fibrillin deposition in cell layers, indicate conformation changes and intramolecular folding. Mass mapping shows that, in solution, microfibrils with periodicities of <70 and >140 nm are stable, but periodicities of approximately 100 nm are rare. Microfibrils comprise two in-register filaments with a longitudinal symmetry axis, with eight fibrillin molecules in cross section. We present a model of fibrillin alignment that fits all the data and indicates that microfibril extensibility follows conformation-dependent maturation from an initial head-to-tail alignment to a stable approximately one-third staggered arrangement.

The C-terminal domain of the Pseudomonas secretin XcpQ forms oligomeric rings with pore activity.

The Pseudomonas secretin XcpQ forms an oligomeric complex, which is involved in the translocation of proteins across the outer membrane via the type II secretion pathway. Pseudomonas aeruginosa produces only small amounts of this complex, 50 to 100 copies per bacterium, and overexpression is lethal to these cells. However, overexpression of Pseudomonas alcaligenes XcpQ could be achieved in the P. alcaligenes mutant strain 537. Protease protection experiments with P. alcaligenes XcpQ showed that the C-terminal domain of XcpQ, which is conserved in all the different members of the secretin family, is largely resistant to proteinase K. This protease-resistant fragment is embedded in the membrane and remains a stable complex, indicating that this domain is involved in complex formation. Both the intact and the protease-protected XcpQ complex showed a tendency to form two-dimensional crystal-like structures. Electron microscopic analysis of these structures showed that the overall oligomeric rings of the intact and of the protease-resistant complex are highly similar. The central cavity of the intact XcpQ complex contains structured mass. Both the intact and the protease-protected XcpQ complex showed pore-forming activity in planar lipid bilayers, consistent with their role as a translocation channel. However, the single-channel conductances observed were not uniform. Together, these results demonstrate that the C-terminal secretin homology domain of XcpQ is the structural domain that forms the channel through which macromolecules are being transported.

A novel virus family, the Rudiviridae: Structure, virus-host interactions and genome variability of the sulfolobus viruses SIRV1 and SIRV2.

The unenveloped, stiff-rod-shaped, linear double-stranded DNA viruses SIRV1 and SIRV2 from Icelandic Sulfolobus isolates form a novel virus family, the Rudiviridae. The sizes of the genomes are 32. 3 kbp for SIRV1 and 35.8 kbp for SIRV2. The virions consist of a tube-like superhelix formed by the DNA and a single basic 15.8-kD DNA-binding protein. The tube carries a plug and three tail fibers at each end. One turn of the DNA-protein superhelix measures 4.3 nm and comprises 16.5 turns of B DNA. The linear DNA molecules appear to have covalently closed hairpin ends. The viruses are not lytic and are present in their original hosts in carrier states. Both viruses are quite stable in these carrier states. In several laboratory hosts SIRV2 was invariant, but SIRV1 formed many different variants that completely replaced the wild-type virus. Some of these variants were still variable, whereas others were stable. Up to 10% nucleotide substitution was found between corresponding genome fragments of three variants. Some variants showed deletions. Wild-type SIRV1, but not SIRV2, induces an SOS-like response in Sulfolobus. We propose that wild-type SIRV1 is unable to propagate in some hosts but surmounts this host range barrier by inducing a host response effecting extensive variation of the viral genome.

Influence of charged surfaces on the morphology of DNA condensed with multivalent ions.

DNA in solution can be condensed into dense aggregates by multivalent counterions. Here we investigate the effect of a nearby surface on the morphology of DNA condensates. We show that, contrary to what has often been assumed, interactions between DNA condensates and the surface can strongly influence the observed morphology. This limits the usefulness of surface probes such as atomic force microscopy for studying the morphology of condensates in bulk solution. Surprisingly, we find that the most negatively charged surface disturbs the condensate morphology most, suggesting that the microscopic mechanism resulting in DNA condensation is also responsible for the attractive force between DNA and the surface.

Automatic TEM image alignment by trifocal geometry.

Here we propose a novel method for automatic, markerless, feature-based alignment of TEM images suitable for electron tomography. The proposed method, termed trifocal alignment, is more accurate than the previous markerless methods. The key components developed are: (1) a reliable multi-resolution algorithm for matching feature points between images; (2) a robust, maximum-likelihood-based estimator for determining the geometry of three views--the trifocal constraint--required for validating the correctness of the matches; and (3) a robust, large-scale optimization framework to compute the alignment parameters from hundreds of thousands of feature point measurements from a few hundred images. The ability to utilize such a large number of measurements successfully compensates for point localization errors. The method was experimentally confirmed with electron tomography tilt series of biological and material sciences samples, consisting of from 40 to 150 images. The results show that, with this feature-based alignment approach, a level of accuracy comparable with fiducial marker alignment can be achieved.

SNDV, a novel virus of the extremely thermophilic and acidophilic archaeon Sulfolobus.

We describe a novel virus, SNDV (Sulfolobus neozealandicus droplet-shaped virus), of the crenarchaeotal archaeon Sulfolobus, which was found in a carrier state in a Sulfolobus strain isolated from a field sample from New Zealand. SNDV particles are droplet-shaped and densely covered by thin tail fibers at their pointed ends. The virion consists of a core and a coat. The latter has the appearance of a beehive and has a surface that is either helically ribbed or a stack of hoops. The genome is cccDNA of 20 kb, which is modified by dam-like methylation. It is cleaved by only a few type II restriction enzymes e.g., DpnI but not MboI, demonstrating an N(6)-methylation of the adenine residue in GATC sequences. The DNA-modifying system differentiates between virus and host. We postulate a virus-encoded methylase that is active on hemimethylated DNA. The host range of SNDV is confined to few Sulfolobus strains from New Zealand. The virus persists in an unstable carrier state rather than as a prophage. Due to its uniqueness we propose to assign it to a novel virus family termed Guttaviridae.

Correction of autofocusing errors due to specimen tilt for automated electron tomography.

Transmission electron microscopy images acquired under tilted-beam conditions experience an image shift as a function of defocus settings - a fact that is exploited as a method for defocus determination in most of the automated tomography data collection systems. Although the method was shown to be highly accurate for a large variety of specimens, we point out that in its original design it can strictly only be applied to images of untilted samples. The application to tilted samples and thus in automated electron tomography is impaired mainly due to a defocus change across the images, resulting in reduced accuracy. In this communication we present a method that can be used to improve the accuracy of the basic autofocusing procedures currently used in systems for automated electron tomography.

Corneal collagen fibril structure in three dimensions: Structural insights into fibril assembly, mechanical properties, and tissue organization.

The ability of the cornea to transmit light while being mechanically resilient is directly attributable to the formation of an extracellular matrix containing orthogonal sheets of collagen fibrils. The detailed structure of the fibrils and how this structure underpins the mechanical properties and organization of the cornea is understood poorly. In this study, we used automated electron tomography to study the three-dimensional organization of molecules in corneal collagen fibrils. The reconstructions show that the collagen molecules in the 36-nm diameter collagen fibrils are organized into microfibrils (approximately 4-nm diameter) that are tilted by approximately 15 degrees to the fibril long axis in a right-handed helix. An unexpected finding was that the microfibrils exhibit a constant-tilt angle independent of radial position within the fibril. This feature suggests that microfibrils in concentric layers are not always parallel to each other and cannot retain the same neighbors between layers. Analysis of the lateral structure shows that the microfibrils exhibit regions of order and disorder within the 67-nm axial repeat of collagen fibrils. Furthermore, the microfibrils are ordered at three specific regions of the axial repeat of collagen fibrils that correspond to the N- and C-telopeptides and the d-band of the gap zone. The reconstructions also show macromolecules binding to the fibril surface at sites that correspond precisely to where the microfibrils are most orderly.

Automated high-throughput electron tomography by pre-calibration of image shifts.

Electron tomography is a versatile method for obtaining three-dimensional (3D) images with transmission electron microscopy. The technique is suitable to investigate cell organelles and tissue sections (100-500 nm thick) with 4-20 nm resolution. 3D reconstructions are obtained by processing a series of images acquired with the samples tilted over different angles. While tilting the sample, image shifts and defocus changes of several microm can occur. The current generation of automated acquisition software detects and corrects for these changes with a procedure that incorporates switching the electron optical magnification. We developed a novel method for data collection based on the measurement of shifts prior to data acquisition, which results in a five-fold increase in speed, enabling the acquisition of 151 images in less than 20 min. The method will enhance the quality of a tilt series by minimizing the amount of required focus-change compensation by aligning the optical axis to the tilt axis of the specimen stage. The alignment is achieved by invoking an amount of image shift as deduced from the mathematical model describing the effect of specimen tilt. As examples for application in biological and materials sciences 3D reconstructions of a mitochondrion and a zeolite crystal are presented.

Three-dimensional reconstructions of extracellular matrix polymers using automated electron tomography.

The extracellular matrix is an intricate network of macromolecules which provides support for cells and a framework for tissues. The detailed structure and organisation of most matrix polymers is poorly understood. These polymers have a complex ultrastructure, and it has proved a major challenge both to define their structural organisation and to relate this to their biological function. However, new approaches using automated electron tomography are beginning to reveal important insights into the molecular assembly and structural organisation of two of the most abundant polymer systems in the extracellular matrix. We have generated three-dimensional reconstructions of collagen fibrils from bovine cornea and fibrillin microfibrils from ciliary zonules. Analysis of these data has provided new insights into the organisation and function of these large macromolecular assemblies.

Automated electron tomography of the septal pore cap in Rhizoctonia solani.

Dolipore septa and septal pore caps (SPCs) in filamentous basidiomycetes may play an important role in maintaining the integrity of hyphal cells. We have investigated the ultrastructure of the dolipore septum and the SPC in Rhizoctonia solani hyphal cells after high-pressure freezing, freeze substitution, and Spurr embedding. We visualized the SPC with associated cell ultrastructures in three dimensions by automated electron tomography of thick-sectioned cells, followed by 3D tomographic reconstructions. Using these methods we were able to document the passage of mitochondria through the SPC, small tubular membranous structures at the entrance of the septal pore channel, filamentous structures connecting the inner side of the SPC with pore-plugging material, thin filaments anchoring the pore-plugging material with the plasma membrane, small vesicles attached to the plugging material, and tubular endoplasmic reticulum continuous with the base of the SPC. We hypothesize that the SPC, the filamentous structures, the plugging material, and the endoplasmic reticulum act in a coordinated fashion to maintain cellular integrity, intercellular communication, and the transport of solutes and cell organelles in the filamentous fungus R. solani.

A novel lipothrixvirus, SIFV, of the extremely thermophilic crenarchaeon Sulfolobus.

We describe a novel lipothrixvirus, SIFV, of the crenarchaeotal archaeon Sulfolobus islandicus. SIFV (S. islandicus filamentous virus) has a linear virion with a linear double-stranded DNA genome. These two features coincide in several crenarchaeotal but not in any other viruses. The SIFV core is formed by a zipper-like array of DNA-associated protein subunits and is covered by a lipid envelope containing host lipids. We sequenced approximately 96% of the virus genome excepting the DNA termini, which were modified in an unusual, yet uncharacterized, manner. Both, the 5' and the 3' DNA termini were insensitive to enzymatic degradation and labelling. Two open reading frames (ORFs) of the SIFV genome are likely to encode helicases and resemble uncharacterized ORFs from other archaea in sequence. Three ORFs showed sequence similarity with each other and each contained a glycosyl transferase motif. Another ORF of the SIFV genome showed significant sequence similarity to the ORF a291 from the well characterized, spindle-shaped Sulfolobus virus SSV1. Due to its structure, SIFV is classified as a lipothrixvirus.

Endosomal compartmentalization in three dimensions: implications for membrane fusion.

Endosomes are major sorting stations in the endocytic route that send proteins and lipids to multiple destinations in the cell, including the cell surface, Golgi complex, and lysosomes. They have an intricate architecture of internal membrane structures enclosed by an outer membrane. Recycling proteins remain on the outer membrane, whereas proteins that are destined for degradation in the lysosome are sorted to the interior. Recently, a retrograde pathway was discovered whereby molecules, like MHC class II of the immune system, return from the internal structures to the outer membrane, allowing their further transport to the cell surface for T cell activation. Whether this return involves back fusion of free vesicles with the outer membrane, or occurs via the continuity of the two membrane domains, is an unanswered question. By electron tomography of cryo-immobilized cells we now demonstrate that, in multivesicular endosomes of B-lymphocytes and dendritic cells, the inner membranes are free vesicles. Hence, protein transport from inner to outer membranes cannot occur laterally in the plane of the membrane, but requires fusion between the two membrane domains. This implies the existence of an intracellular machinery that mediates fusion between the exoplasmic leaflets of the membranes involved, which is opposite to regular intracellular fusion between cytoplasmic leaflets. In addition, our 3D reconstructions reveal the presence of clathrin-coated areas at the cytoplasmic face of the outer membrane, known to participate in protein sorting to the endosomal interior. Interestingly, profiles reminiscent of inward budding vesicles were often in close proximity to the coats.