Light and electron microscopy on the sporulation of the oocysts of Eimeria brunetti. II. Development into the sporocyst and formation of the sporozoite

The later stages of sporulation in oocysts of Eimeria brunetti were examined in samples which had been allowed to sporulate at 27 degrees C for 24, 36 and 48 hours. It was observed that the sporoblasts became ellipsoidal and the nucleus underwent the final division. A nucleus with associated Golgi bodies was not observed at either end of the organism. The cytoplasm was limited by two unit membranes and contained rough endoplasmic reticulum, dense bodies, electron translucent vacuoles and mitochondria. The first evidence of sporozoite formation was the appearance of a dense plaque at either end of the organism. This appeared in the vicinity of the nuclei, and adjacent to the limiting membrane of the soroblast. At this stage the sporocyst wall was still unformed. Then the two sporozoites were formed from opposite ends of the organism by growth of the dense plaques and invaginations of the plasmalemma which thus formed the pellicles of the developing sporozoites. A conoid and subpellicular microtubules were observed at this stage as development continued, a number of vacuoles were found between the nucleus and the conoid. These vacuoles constituted the precursors of the rhoptries and micronemes. At the same stage a large dense body had appeared within the forming sporozoite. As the sporozoite developed, this body, anterior refractile body, is followed by the nucleus and another dense body which formed the posterior refractile body. During this period, the thin sporocyst wall was formed and Stieda and sub-Stieda bodies were now present at one end of the sporocyst. Each mature sporocyst contained two sporozoites.     

脂蛋白电子显微结构研究的优化负染方法

人类心血管等疾病与胆固醇含量的高低有着十分密切的关系．人体内胆固醇主要通过血液中的脂蛋白来调节和运载．因此,新一代调节胆固醇的药物设计迫切地需要揭示脂蛋白分子结构与功能之间的关系．由于脂蛋白分子的成分高度复杂、结构特征灵活、尺寸微小,传统的蛋白质结构标定技术,如X射线衍射、核磁共振谱、质谱和冷冻电子显微镜等手段都面临着巨大的困难．针对这一问题,任罡小组最近提出一种优化的电镜负染制备方法,可以观测到单个脂蛋白分子的空间结构．此方法应用的成功,使得对单个脂蛋白分子的结构研究成为可能．同时也证明该负染色技术作为一种普遍的实验手段,可以用于对蛋白质小分子的结构研究,从而将促进新一代蛋白质分子药物的研发．     

Proteomics analysis and protein expression during sporozoite excystation of Cryptosporidium parvum (Coccidia, Apicomplexa)

Cryptosporidiosis, caused by coccidian parasites of the genus Cryptosporidium, is a major cause of human gastrointestinal infections and poses a significant health risk especially to immunocompromised patients. Despite intensive efforts for more than 20 years, there is currently no effective drug treatment against these protozoa. This study examined the zoonotic species Cryptosporidium parvum at two important stages of its life cycle: the non-excysted (transmissive) and excysted (infective) forms. To increase our understanding of the molecular basis of sporozoite excystation, LC-MS/MS coupling with a stable isotope N-terminal labeling strategy using iTRAQ reagents was used on soluble fractions of both non-excysted and excysted sporozoites, i.e. sporozoites both inside and outside oocysts were examined. Sporozoites are the infective stage that penetrates small intestinal enterocytes. Also to increase our knowledge of the C. parvum proteome, shotgun sequencing was performed on insoluble fractions from both non-excysted and excysted sporozoites. In total 303 C. parvum proteins were identified, 56 of which, hitherto described as being only hypothetical proteins, are expressed in both excysted and non-excysted sporozoites. Importantly we demonstrated that the expression of 26 proteins increases significantly during excystation. These excystation-induced proteins included ribosomal proteins, metabolic enzymes, and heat shock proteins. Interestingly three Apicomplexa-specific proteins and five Cryptosporidium-specific proteins augmented in excysted invasive sporozoites. These eight proteins represent promising targets for developing vaccines or chemotherapies that could block parasite entry into host cells.     

Light and electron microscopy on the sporulation of the oocysts of Eimeria brunetti. I. Development of the zygote and formation of the sporoblasts

The initial stages of sporulation in oocysts of Eimeria brunetti were examined in samples sporulated at 27 degrees C for 0, 12 and 24 hours. The initial zygote was found to be roughly spherical and to contain a number of polysaccharide granules which were congregated in one region of the organism. The cytoplasm also contained some strands of rough endoplasmic reticulum together with a number of mitochondria, some Golgi bodies, and some electron translucent vacuoles. The nucleus was large, with amorphous nucleoplasm and a nucleous. The cytoplasmic mass of the zygote was limited by a single unit membrane which possessed some micropores. After initiation of the sporulation, the metabolic activity of the organism appeared to increase as evidenced by the augmentation in the cytoplasm of the amounts of rough endoplasmic reticulum, number of Golgi bodies, and the appearance of polyribosomes. However, at this stage, the presence of large spherical bodies (anlagen of the refractile bodies of the sporozoites) constituted the most obvious change in the cytoplasm of the organism. After nuclear division the daughter nuclei were situated well separated in the cytoplasm and the polysaccharide granules were evenly distributed throughout the cytoplasm of the zygote. Eventually four sporoblasts were formed by invaginations of the limiting membrane. Each sporoblast was limited by a unit membrane and contained a nucleus, and the same cytoplasmic organelles as found in the zygote. The development of the sporoblast was initially accompanied by the appearance of a second limiting membrane.     

Optimized flow cytometry protocol for analysis of surface expression of interleukin-1 receptor types I and II

The biological effects of interleukin (IL)-1 are realized through binding to specific membrane-bound receptors. The efficiency of IL-1 action depends on the number of receptors on the cell. We determined the percentage of cells that express IL-1 receptor type I (IL-1RI) and IL-1 receptor type II (IL-1RII) by flow cytometry using phycoerythrin (PE)-labelled antibodies to the IL-1Rs, and the mean absolute number of membrane-bound IL-1Rs per cell using QuantiBRITE PE calibration beads. We showed that different subpopulations of immunocompetent cells expressed different numbers of molecules of membrane-bound IL-1RI and IL-1RII. We also established that when cells were stimulated with bacterial lipopolysaccharide, there was a significant increase in the number of IL-1RI expressed, and a significant decrease in the mean number of IL-1RII molecules per cell. Determination of the mean number of membrane-bound IL-1R molecules using this protocol enables us to obtain precise and reproducible data that are necessary for full evaluation of expression levels.     

A computer-based protocol for semiautomated assignments and 3D structure determination of proteins

Summary A strategy is presented for the semiautomated assignment and 3D structure determination of proteins from heteronuclear multidimensional Nuclear Magnetic Resonance (NMR) data. This approach involves the computer-based assignment of the NMR signals, identification of distance restraints from nuclear Overhauser effects, and generation of 3D structures by using the NMR-derived restraints. The protocol is described in detail and illustrated on a resonance assignment and structure determination of the FK506 binding protein (FKBP, 107 amino acids) complexed to the immunosuppressant, ascomycin. The 3D structures produced from this automated protocol attained backbone and heavy atom rmsd of 1.17 and 1.69 Å, respectively. Although more highly resolved structures of the complex have been obtained by standard interpretation of NMR data (Meadows et al. (1993) Biochemistry, 32, 754–765), the structures generated with this automated protocol required minimal manual intervention during the spectral assignment and 3D structure calculations stages. Thus, the protocol may yield an approximate order of magnitude reduction in the time required for the generation of 3D structures of proteins from NMR data.     

Geometric morphometric 3D shape analysis and covariation of human mandibular and maxillary first molars

Dental casts of 160 Greek subjects (80 males, 80 females) were scanned by a structured‐light scanner. The upper and lower right first molar occlusal surface 3D meshes were processed using geometric morphometric methods. A total of 265 and 274 curve and surface sliding semilandmarks were placed on the upper and lower molar surfaces, respectively. Principal component analysis and partial least square analysis were performed to assess shape parameters. Molars tended to vary between an elongated and a more square form. The first two principal components (PCs), comprising almost 1/3 of molar shape variation, were related to mesiodistal–buccolingual ratios and relative cusp position. Distal cusps displayed the greatest shape variability. Molars of males were larger than those of females (2.8 and 3.2% for upper and lower molars respectively), but no shape dimorphism was observed. Upper and lower molar sizes were significantly correlated (r² = 0.689). Allometry was observed for both teeth. Larger lower molars were associated with shorter cusps, expansion of the distal cusp, and constriction of the mesial cusps (predicted variance 3.25%). Upper molars displayed weaker allometry (predicted variance 1.59%). Upper and lower molar shape covariation proved significant (RV = 17.26%, P < 0.0001). The main parameter of molar covariation in partial least square axis 1, contributing to 30% of total covariation, was cusp height, in contrast to the primary variability traits exhibited by PC1 and PC2. The aim of this study was to evaluate shape variation and covariation, including allometry and sexual dimorphism, of maxillary and mandibular first permanent molar occlusal surfaces. Am J Phys Anthropol 152:186–196, 2013. © 2013 Wiley Periodicals, Inc.     

FT3D: three-dimensional Fourier analysis on small Unix workstations for electron microscopy and tomo graphic studies

FT3D is a self-contained package of tools for three-dimensionalFourier analysis, written in the C language for Unix workstations.It can evaluate direct transforms of three-dimensional realfunctions, inverse transforms, auto-and cross-correlations andspectra. The library has been developed to support three-dimensionalreconstructions of biological structures from projections obtainedin the electron microscope. This paper discusses some fraturesof the library, which has been implemented in such a way asto profit from the resources of modern workstations. A tableof elapsed times for jobs of different dimensions with differentRAM buffers is reported for the particular hardware used inthe authors' laboratory.     

Characterization of chromatin domains by 3D fluorescence microscopy: An automated methodology for quantitative analysis and nuclei screening

Fluorescence microscopy has provided a route to qualitatively analyze features of nuclear structures and chromatin domains with increasing resolution. However, it is becoming increasingly important to develop tools for quantitative analysis. Here, we present an automated method to quantitatively determine the enrichment of several endogenous factors, immunostained in pericentric heterochromatin domains in mouse cells. We show that this method permits an unbiased characterization of changes in the enrichment of several factors with statistical significance from a large number of nuclei. Furthermore, the nuclei can be sorted according to the enrichment value of these factors. This method should prove useful to monitor events related to changes in the amount, rather than the presence or absence, of any factor. By adapting a few parameters, it could be extended to other nuclear structures and the benefit of using available software will permit its use in many biological labs.     

3D structured illumination microscopy of mammalian embryos and spermatozoa

Background

Super-resolution fluorescence microscopy performed via 3D structured illumination microscopy (3D-SIM) is well established on flat, adherent cells. However, blastomeres of mammalian embryos are non-adherent, round and large. Scanning whole mount mammalian embryos with 3D-SIM is prone to failure due to the movement during scanning and the large distance to the cover glass.

Results

Here we present a highly detailed protocol that allows performing 3D-SIM on blastomeres of mammalian embryos with an image quality comparable to scans in adherent cells. This protocol was successfully tested on mouse, rabbit and cattle embryos and on rabbit spermatozoa.

Conclusions

Our protocol provides detailed instructions on embryo staining, blastomere isolation, blastomere attachment, embedding, correct oil predictions, scanning conditions, and oil correction choices after the first scan. Finally, the most common problems are documented and solutions are suggested. To our knowledge, this protocol presents for the first time a highly detailed and practical way to perform 3D-SIM on mammalian embryos and spermatozoa.

     

3D map of the plant photosystem II supercomplex obtained by cryoelectron microscopy and single particle analysis

Here we describe the first 3D structure of the photosystem II (PSII) supercomplex of higher plants, constructed by single particle analysis of images obtained by cryoelectron microscopy. This large multisubunit membrane protein complex functions to absorb light energy and catalyze the oxidation of water and reduction of plastoquinone. The resolution of the 3D structure is 24 A and emphasizes the dimeric nature of the supercomplex. The extrinsic proteins of the oxygen-evolving complex (OEC) are readily observed as a tetrameric cluster bound to the lumenal surface. By considering higher resolution data, obtained from electron crystallography, it has been possible to relate the binding sites of the OEC proteins with the underlying intrinsic membrane subunits of the photochemical reaction center core. The model suggests that the 33 kDa OEC protein is located towards the CP47/D2 side of the reaction center but is also positioned over the C-terminal helices of the D1 protein including its CD lumenal loop. In contrast, the model predicts that the 23/17 kDa OEC proteins are positioned at the N-terminus of the D1 protein incorporating the AB lumenal loop of this protein and two other unidentified transmembrane helices. Overall the 3D model represents a significant step forward in revealing the structure of the photosynthetic OEC whose activity is required to sustain the aerobic atmosphere on our planet.     

The potential of 3D-FISH and super-resolution structured illumination microscopy for studies of 3D nuclear architecture: 3D structured illumination microscopy of defined chromosomal structures visualized by 3D (immuno)-FISH opens new perspectives for studies of nuclear architecture

Three-dimensional structured illumination microscopy (3D-SIM) has opened up new possibilities to study nuclear architecture at the ultrastructural level down to the ~100 nm range. We present first results and assess the potential using 3D-SIM in combination with 3D fluorescence in situ hybridization (3D-FISH) for the topographical analysis of defined nuclear targets. Our study also deals with the concern that artifacts produced by FISH may counteract the gain in resolution. We address the topography of DAPI-stained DNA in nuclei before and after 3D-FISH, nuclear pores and the lamina, chromosome territories, chromatin domains, and individual gene loci. We also look at the replication patterns of chromocenters and the topographical relationship of Xist-RNA within the inactive X-territory. These examples demonstrate that an appropriately adapted 3D-FISH/3D-SIM approach preserves key characteristics of the nuclear ultrastructure and that the gain in information obtained by 3D-SIM yields new insights into the functional nuclear organization.     

Light and electron microscopy continuum-resolution imaging of 3D cell cultures

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3D electron microscopy of biological nanomachines: principles and applications

Transmission electron microscopy is a powerful technique for studying the three-dimensional (3D) structure of a wide range of biological specimens. Knowledge of this structure is crucial for fully understanding complex relationships among macromolecular complexes and organelles in living cells. In this paper, we present the principles and main application domains of 3D transmission electron microscopy in structural biology. Moreover, we survey current developments needed in this field, and discuss the close relationship of 3D transmission electron microscopy with other experimental techniques aimed at obtaining structural and dynamical information from the scale of whole living cells to atomic structure of macromolecular complexes. Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet France, 14–19 October, 2006.     

A quantitative 3D motility analysis of Trypanosoma brucei by use of digital in-line holographic microscopy

We present a quantitative 3D analysis of the motility of the blood parasite Trypanosoma brucei. Digital in-line holographic microscopy has been used to track single cells with high temporal and spatial accuracy to obtain quantitative data on their behavior. Comparing bloodstream form and insect form trypanosomes as well as mutant and wildtype cells under varying external conditions we were able to derive a general two-state-run-and-tumble-model for trypanosome motility. Differences in the motility of distinct strains indicate that adaption of the trypanosomes to their natural environments involves a change in their mode of swimming.