Journal of Structural Biology

Autophagy and endocytosis in the amnion.

Publication Date: 2008 May PMID: 18413288
Authors: Shen, Z. Y. - Xu, L. Y. - Li, E. M. - Zhuang, B. R. - Lu, X. F. - Shen, J. - Wu, X. Y. - Li, Q. S. - Lin, Y. J. - Chen, Y. W. - Tan, L. J.
Journal: J Struct Biol

To search for the origin of nutrition in the amnion, we focused attention on both endocytotic and autophagic pathways. Using ultrastructural and biochemical methods, we examined 20 human amnions at term gestation. The uptake of horseradish peroxidase (HRP) was used for the detection of endocytosis. Transfection of the LC3-GFP plasmid and staining with monodansylcadaverine (MDC) and LysoTracker red (LTR) were used to demonstrate the formation of autophagic vacuoles. In addition, two autophagic genes, beclin 1 and Atg5, were assayed by RT-PCR. Within the amniotic epithelial (AE) cells, autophagic vacuoles contained organelles and cytoplasmic components and were enclosed by a double membrane. They contained autophagosomes with transfected LC3-GFP that stained positive for MDC and autolysosomes that stained positive for LTR. Endocytosis was an extremely active process in the cellular uptake of fluid and fluid contents and led to formation of vesicles and endosomes, which were found to be positive by HRP test. Many uniform vesicles were collected in the multivesicular bodies (MVBs). Finally, both endosomes and autophagosomes were fused and degraded by lysosomes. The data also demonstrated that large autophagosomes engulfed some endosomes or MVBs. Transcription of beclin 1 and Atg5 occurred in the amnion at term gestation. Taken together, these results show that AE cells have active endocytotic and autophagic capacities and that lysosomes are involved in the intracellular degradation of endosomes and autophagosomes. Sometimes the autophagic and endocytotic pathways converge. This study suggests that of endocytosis and autophagy activities in AE cells can be induced by nutrient limitation and are probably also evoked in response to some hormones in the amniotic fluid. Activation of both endocytotic and autophagic pathways plays different roles in the ability of the cell to acquire nutrients needed for its survival.

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Fast projection matching for cryo-electron microscopy image reconstruction.

Publication Date: 2008 May PMID: 18353677
Authors: Estrozi, L. F. - Navaza, J.
Journal: J Struct Biol

A new FFT-accelerated projection matching method is presented and tested. The electron microscopy images are represented by their Fourier-Bessel transforms and the 3D model by its expansion in spherical harmonics, or more specifically in terms of symmetry-adapted functions. The rotational and translational properties of these representations are used to quickly access all the possible 2D projections of the 3D model, which allow an exhaustive inspection of the whole five-dimensional domain of parameters associated to each particle.

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Spina cortica and Tapetum spinosus, two new microstructures of flight feathers: description, function and distribution in modern birds.

Publication Date: 2008 May PMID: 18343684
Authors: Straker, L. C. - Raposo, M. A. - Attias, M.
Journal: J Struct Biol

The importance of feathers for the avian group has made them one of the most studied epidermal structures both from the morphological and evolutionary point of view. Surprisingly, our observations by Scanning Electron Microscopy detected the presence of two structures widely distributed within different avian groups and not yet described. In this paper we describe these two new structures (Spina cortica and Tapetum spinosus) and map their distribution within modern birds. The S. cortica is a thorn-like microstructure that grows on the barb cortex and the T. spinosus is the assemblage of these thorns. The distribution of these new structures among birds and their morphological diversity could be of great interest to taxonomists and evolutionary biologists interested in the origin of bird flight.

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Intra-cellular storage, transport and exocytosis of halogenated compounds in marine red alga Laurencia obtusa.

Publication Date: 2008 May PMID: 18337120
Authors: Salgado, L. T. - Viana, N. B. - Andrade, L. R. - Leal, R. N. - da Gama, B. A. - Attias, M. - Pereira, R. C. - Amado Filho, G. M.
Journal: J Struct Biol

The production of secondary metabolites in seaweed have been related to a capability to partition compounds into cellular specialized storage structures, like gland cells and the corps en cerise (CC) or cherry bodies. The possible mechanisms that bring these compounds to the thallus surface remain poorly understood. Therefore, the aim of this work is perform a characterization of the CC and determine the intra-cellular dynamics of halogenated compounds in Laurencia obtusa. The dynamics of CC and the mechanisms related to the intra-cellular transport of halogenated compounds were evaluated by using optical tweezers and time-lapse video microscopy. The CC were isolated and its elemental composition was characterized using X-ray microanalysis. The cellular distribution of halogenated compounds was also demonstrated by fluorescence microscopy. Three-dimensional reconstruction technique was used to provide a visualization of the structures that connect CC to cell periphery. As main findings, we confirmed that the halogenated compounds are mainly found in CC and also in vesicles distributed along the cytoplasm and within the chloroplasts. We demonstrated that CC is mechanically fixed to cell periphery by a stalk-like connection. A vesicle transport though membranous tubular connections was seen occurring from CC to cell wall region. We also demonstrated a process of cortical cell death event, resulting in degradation of CC. We suggested that the vesicle transportation along membranous tubular connections and cell death events are related to the mechanisms of halogenated compounds exudation to the thallus surface and consequently with defensive role against herbivores and fouling.

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Forming nacreous layer of the shells of the bivalves Atrina rigida and Pinctada margaritifera: an environmental- and cryo-scanning electron microscopy study.

Publication Date: 2008 May PMID: 18328730
Authors: Nudelman, F. - Shimoni, E. - Klein, E. - Rousseau, M. - Bourrat, X. - Lopez, E. - Addadi, L. - Weiner, S.
Journal: J Struct Biol

A key to understanding control over mineral formation in mollusk shells is the microenvironment inside the pre-formed 3-dimensional organic matrix framework where mineral forms. Much of what is known about nacre formation is from observations of the mature tissue. Although these studies have elucidated several important aspects of this process, the structure of the organic matrix and the microenvironment where the crystal nucleates and grows are very difficult to infer from observations of the mature nacre. Here, we use environmental- and cryo-scanning electron microscopy to investigate the organic matrix structure at the onset of mineralization in the nacre of two mollusk species: the bivalves Atrina rigida and Pinctada margaritifera. These two techniques allow the visualization of hydrated biological materials coupled with the preservation of the organic matrix close to physiological conditions. We identified a hydrated gel-like protein phase filling the space between two interlamellar sheets prior to mineral formation. The results are consistent with this phase being the silk-like proteins, and show that mineral formation does not occur in an aqueous solution, but in a hydrated gel-like medium. As the tablets grow, the silk-fibroin is pushed aside and becomes sandwiched between the mineral and the chitin layer.

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Distinct horizontal patterns in the spatial organization of superficial zone chondrocytes of human joints.

Publication Date: 2008 May PMID: 18325787
Authors: Rolauffs, B. - Williams, J. M. - Grodzinsky, A. J. - Kuettner, K. E. - Cole, A. A.
Journal: J Struct Biol

A better understanding of the unique cellular and functional properties of the superficial zone of articular cartilage may aid current strategies in tissue engineering which attempts a layered design for the repair of cartilage lesions to avert or postpone the onset of osteoarthritis. However, data pertaining to the cellular organization of non-degenerated superficial zone of articular cartilage is not available for most human joints. The present study analyzed the arrangement of chondrocytes of non-degenerated human joints (shoulder, elbow, knee, and ankle) by using fluorescence microscopy of the superficial zone in a top-down view. The resulting horizontal chondrocyte arrangements were tested for randomness, homogeneity or a significant grouping via point pattern analysis and were correlated with the joint type in which they occurred. The present study demonstrated that human superficial chondrocytes occurred in four distinct patterns of strings, clusters, pairs or single chondrocytes. Those patterns represented a significant grouping (p < 0.0001) with horizontal alignment. Each articular joint surface was dominated by only one of these four patterns (p < 0.001). Specific patterns correlated with specific diarthrodial joint types (p < 0.001). Further studies need to establish whether these organizational patterns are a consequence of their surrounding environment or whether they are linked to a functional purpose.

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Structural and kinetic contributions of the oxyanion binding site to the catalytic activity of acylaminoacyl peptidase.

Publication Date: 2008 May PMID: 18325786
Authors: Kiss, A. L. - Pallo, A. - Naray-Szabo, G. - Harmat, V. - Polgar, L.
Journal: J Struct Biol

It is widely accepted that the catalytic activity of serine proteases depends primarily on the Asp-His-Ser catalytic triad and other residues within the vicinity of this motif. Some of these residues form the oxyanion binding site that stabilizes the tetrahedral intermediate by hydrogen bonding to the negatively charged oxyanion. In acylaminoacyl peptidase from the thermophile Aeropyrum pernix, the main chain NH group of Gly369 is one of the hydrogen bond donors forming the oxyanion binding site. The side chain of His367, a conserved residue in acylaminoacyl peptidases across all species, fastens the loop holding Gly369. Determination of the crystal structure of the H367A mutant revealed that this loop, including Gly369, moves away considerably, accounting for the observed three orders of magnitude decrease in the specificity rate constant. For the wild-type enzyme ln(k(cat)/K(m)) vs. 1/T deviates from linearity indicating greater rate enhancement with increasing temperature for the dissociation of the enzyme-substrate complex compared with its decomposition to product. In contrast, the H367A variant provided a linear Arrhenius plot, and its reaction was associated with unfavourable entropy of activation. These results show that a residue relatively distant from the active site can significantly affect the catalytic activity of acylaminoacyl peptidase without changing the overall structure of the enzyme.

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Conformational changes accompany activation of reovirus RNA-dependent RNA transcription.

Publication Date: 2008 May PMID: 18321727
Authors: Mendez, I. I. - Weiner, S. G. - She, Y. M. - Yeager, M. - Coombs, K. M.
Journal: J Struct Biol

Many critical biologic processes involve dynamic interactions between proteins and nucleic acids. Such dynamic processes are often difficult to delineate by conventional static methods. For example, while a variety of nucleic acid polymerase structures have been determined at atomic resolution, the details of how some multi-protein transcriptase complexes actively produce mRNA, as well as conformational changes associated with activation of such complexes, remain poorly understood. The mammalian reovirus innermost capsid (core) manifests all enzymatic activities necessary to produce mRNA from each of the 10 encased double-stranded RNA genes. We used rapid freezing and electron cryo-microscopy to trap and visualize transcriptionally active reovirus core particles and compared them to inactive core images. Rod-like density centered within actively transcribing core spike channels was attributed to exiting nascent mRNA. Comparative radial density plots of active and inactive core particles identified several structural changes in both internal and external regions of the icosahedral core capsid. Inactive and transcriptionally active cores were partially digested with trypsin and identities of initial tryptic peptides determined by mass spectrometry. Differentially-digested peptides, which also suggest transcription-associated conformational changes, were placed within the known three-dimensional structures of major core proteins.

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Zernike phase contrast electron microscopy of ice-embedded influenza A virus.

Publication Date: 2008 May PMID: 18313941
Authors: Yamaguchi, M. - Danev, R. - Nishiyama, K. - Sugawara, K. - Nagayama, K.
Journal: J Struct Biol

The ultrastructure of the frozen-hydrated influenza A virus was examined by Zernike phase contrast electron microscopy. Using this new microscopy, not only lipid bilayers but also individual glycoprotein spikes on viral envelopes were clearly resolved with high contrast in micrographs taken in focus. In addition to spherical and elongated virions, three other classes of virions were distinguished on the basis of the features of their viral envelope: virions with a complete matrix layer, which were the most predominant, virions with a partial matrix layer, and virions with no matrix layer under the lipid bilayer. About 450 glycoprotein spikes were present in an average-sized spherical virion. Eight ribonucleoprotein complexes, that is, a central one surrounded by seven others, were distinguished in one viral particle. Thus, Zernike phase contrast electron microscopy is a powerful tool for resolving the ultrastructure of viruses, because it enables high-contrast images of ice-embedded particles free of contrast transfer function artifacts that can be a problem in conventional cryo-electron microscopy.

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Cell cycle-dependent kinetochore localization of condensin complex in Saccharomyces cerevisiae.

Publication Date: 2008 May PMID: 18296067
Authors: Bachellier-Bassi, S. - Gadal, O. - Bourout, G. - Nehrbass, U.
Journal: J Struct Biol

In budding yeast mitosis is endonuclear and associated with a very limited condensation of the chromosomes. Despite this partial chromosomal condensation, condensin is conserved and essential for the Saccharomyces cerevisiae mitotic cycle. Here, we investigate the localization of condensin during the mitotic cycle. In addition to a constitutive association with rDNA, we have discovered that condensin is localized to the kinetochore in a cell cycle-dependent manner. Shortly after duplication of the spindle pole body, the yeast equivalent of the centrosome, we observed a local enrichment of condensin colocalizing with kinetochore components. This specific association is consistent with mutant phenotypes of chromosome loss and defective sister chromatid separation at anaphase. During a short period of the cell cycle, we observed, at the single cell level, a spatial proximity of condensin and a cohesin rosette, without colocalization. Furthermore, using a genetic screen we demonstrated that condensin localization at kinetochores is specifically impaired in a mutant for ulp2/smt4, an abundant SUMO protease. In conclusion, during chromosome segregation, we established a SUMO-dependent cell cycle-specific condensin concentration colocalizing with kinetochores.

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Crystal structure of human transgelin.

Publication Date: 2008 May PMID: 18291675
Authors: Li, M. - Li, S. - Lou, Z. - Liao, X. - Zhao, X. - Meng, Z. - Bartlam, M. - Rao, Z.
Journal: J Struct Biol

Transgelin (TAGLN), also known as smooth muscle protein 22 (SM22), is a highly conserved protein found in smooth muscle tissues of adult vertebrates. Abolition of transgelin gene expression by the oncogenic Ras may be an important early event in tumor progression and a diagnostic marker for breast and colon cancer development. Transgelin contains a single calponin homology (CH) domain. However, the question of whether this single CH domain can bind actin remains open. Here we report the 2.3 A resolution crystal structure of full length human transgelin, whose main structural feature is confirmed to be a CH domain. Secondary structures of CH domains from different proteins were analyzed and conserved residues were identified that maintain similar tertiary structures.

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Anatomy of the E2 ligase fold: implications for enzymology and evolution of ubiquitin/Ub-like protein conjugation.

Publication Date: 2008 May PMID: 18276160
Authors: Burroughs, A. M. - Jaffee, M. - Iyer, L. M. - Aravind, L.
Journal: J Struct Biol

The configuration of the active site of E2 ligases, central enzymes in the ubiquitin/ubiquitin-like protein (Ub/Ubl) conjugation systems, has long puzzled researchers. Taking advantage of the wealth of newly available structures and sequences of E2s from diverse organisms, we performed a large-scale comparative analysis of these proteins. As a result we identified a previously under-appreciated diversity in the active site of these enzymes, in particular, the spatial location of the catalytic cysteine and a constellation of associated conserved residues that potentially contributes to catalysis. We observed structural innovations of differing magnitudes occurring in various families across the E2 fold that might correlate in part with differences in target interaction. A key finding was the independent emergence on multiple occasions of a polar residue, often a histidine, in the vicinity of the catalytic cysteine in different E2 families. We propose that these convergently emerging polar residues have a common function, such as in the stabilization of oxyanion holes during Ub/Ubl transfer and spatial localization of the Ub/Ubl tails in the active site. Thus, the E2 ligases represent a rare example in enzyme evolution of high structural diversity of the active site and position of the catalytic residue despite all characterized members catalyzing a similar reaction. Our studies also indicated certain evolutionarily conserved features in all active members of the E2 superfamily that stabilize the unusual flap-like structure in the fold. These features are likely to form a critical mechanical element of the fold required for catalysis. The results presented here could aid in new experiments to understand E2 catalysis.

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Structural analysis of 2D crystals of gastric H+,K+-ATPase in different states of the transport cycle.

Publication Date: 2008 May PMID: 18276159
Authors: Nishizawa, T. - Abe, K. - Tani, K. - Fujiyoshi, Y.
Journal: J Struct Biol

The H+,K+-ATPase uses ATP to pump protons across the gastric membrane. We used electron crystallography and limited trypsin proteolysis to study conformational changes in the H+,K+-ATPase. Well-ordered 2D crystals were obtained with detergent-solubilized H+,K+-ATPase at low pH in the absence of nucleotides, E1 state, and in the presence of fluoroaluminate and ADP, mimicking the E1PADP state. Projection maps obtained with frozen-hydrated two-dimensional crystals of the H+,K+-ATPase in these two states looked very similar, suggesting only small conformational changes during the transition from the E1 to the E1P x ADP state. This result differs from the X-ray crystal structures of the related ATPase SERCA, which revealed substantially different conformations in the E1 and E1P x ADP states. To further characterize the conformational changes in the H+,K+-ATPase during its transport cycle, we performed limited proteolysis with trypsin. All examined states of the H+,K+-ATPase, including the E1 and E1P x ADP states present in the 2D crystals,showed characteristic differences in the digestion patterns. While the results from the limited proteolysis experiments thus show that the H+,K+-ATPase adopts distinct conformations during different stages of the transport cycle, the projection maps indicate that the structural rearrangements in the H+,K+-ATPase are much smaller than those observed in the related SERCA ATPase.

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Angle determination for side views in single particle electron microscopy.

Publication Date: 2008 May PMID: 18272396
Authors: Baker, L. A. - Rubinstein, J. L.
Journal: J Struct Biol

In order to build a first model in single particle electron microscopy the relative angular orientation of each image of a protein complex must be determined. These orientations can be described by three Eulerian angles. Images of complexes that present the same view can be aligned in two-dimensions and averaged in order to increase their signal-to-noise ratio. Based on these averaged images, several standard approaches exist for determining Euler angles for randomly oriented projection images. The common lines and angular reconstitution methods work well for particles with symmetry while the random conical tilting and related orthogonal tilt reconstruction methods work in most cases but require the acquisition of tilt pairs of images. For the situation where views of particles can be identified that are rotations about a single axis parallel to the grid, an alternative algorithm to determine the orientations of class averages without the need to acquire tilt pairs can be applied. This type of view of a complex is usually called a side view. This paper describes the detailed workings and characterization of an algorithm, named rotational analysis, which uses real-space fiducial markers derived from the averages themselves to determine the Euler angles for side views. We demonstrate how this algorithm works in practice by applying it to a data set of images of affinity-purified bovine mitochondrial ATP synthase.

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Packing of transmembrane helices in bacteriorhodopsin folding: structure and thermodynamics.

Publication Date: 2008 May PMID: 18262435
Authors: Chen, C. C. - Wei, C. C. - Sun, Y. C. - Chen, C. M.
Journal: J Struct Biol

We propose a coarse-grained (CG) model to study the native structure and physical properties of helical membrane proteins (HMPs) using off-lattice computer simulations. Instead of considering sequence heterogeneity explicitly, we model its effect on the packing of helices by employing a mean packing parameter r(0), which is calculated from an all-atom (AA) model. Specifically, this CG model is applied to investigate the packing of helices in bacteriorhodopsin (BR), and predicts the seven helix bundle structure of BR with a root mean square deviation (RMSD) in coordinates of helix backbone atoms (N, C, C(alpha)) of 3.99 A from its crystal structure. This predicted structure is further refined in an AA model by Amber and the refined structure has a RMSD (in coordinates of helix backbone atoms) of 2.64 A. The predicted packing position, tilting angle, and orientation angle of each helix in the refined structure are consistent with experimental data and their physical origins can be well understood in our model. Our results show that a reasonably good structure of BR can be predicted by using such a dual-scale approach, provided that its secondary structure is known. Starting from a random initial configuration, the folded structure can be obtained in days using a regular desktop computer. Various thermodynamic properties of helix packing of BR are also investigated in this CG model.

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A combinatorial approach to crystallization of PX-BAR unit of the human Sorting Nexin 9.

Publication Date: 2008 May PMID: 18065239
Authors: Pylypenko, O. - Ignatev, A. - Lundmark, R. - Rasmuson, E. - Carlsson, S. R. - Rak, A.
Journal: J Struct Biol

Sorting nexins (SNXs) form a family of proteins known to interact with endosomal vesicles and to regulate various steps of vesicle transport. Sorting Nexin 9 (SNX9) is involved in the interface of endocytic, actin polymerizing, and signal transduction events in the cell. Here we report crystallization of the SNX9 PX-BAR domain protein. Initially we used an ordinary protein construct design, and protein crystallization approaches resulted in obtaining granular crystal-like precipitation. SDS-PAGE and MS analysis of the crystal-like precipitation followed by protein construct optimization and using of macro seeding technique resulted in X-ray quality diffracting crystals. The crystals belonged to P2(1)2(1)2(1) space group (a=65.6 A, b=117.5 A, c=145.8 A) with two protein molecules per asymmetric unit. A complete SAD data set from Se-Methionine derived crystal (3.2 A) has been collected to solve the structure.

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