Native Immunogold Labeling of Cell Surface Proteins and Viral Glycoproteins for Cryo-Electron Microscopy and Cryo-Electron Tomography Applications

Numerous methods have been developed for immunogold labeling of thick, cryo-preserved biological specimens. However, most of the methods are permutations of chemical fixation and sample sectioning, which select and isolate the immunolabeled region of interest. We describe a method for combining immunogold labeling with cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) of the surface proteins of intact mammalian cells or the surface glycoproteins of assembling and budding viruses in the context of virus-infected mammalian cells cultured on EM grids. In this method, the cells were maintained in culture media at physiologically relevant temperatures while sequentially incubated with the primary and secondary antibodies. Subsequently, the immunogold-labeled specimens were vitrified and observed under cryo-conditions in the transmission electron microscope. Cryo-EM and cryo-ET examination of the immunogold-labeled cells revealed the association of immunogold particles with the target antigens. Additionally, the cellular structure was unaltered by pre-immunolabeling chemical fixation and retained well-preserved plasma membranes, cytoskeletal elements, and macromolecular complexes. We think this technique will be of interest to cell biologists for cryo-EM and conventional studies of native cells and pathogen-infected cells.     

Protein Secondary Structure Determination by Constrained Single-Particle Cryo-Electron Tomography

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Highlights? Efficient extraction of high-resolution information from cryo-EM tilt series ? Successful CTF correction strategy despite low-contrast and quality of tilted images ? Use of geometric constraints in refinement improves orientational accuracy of images ? Enforcement of tomographic constraints reduces model bias and overfitting artifacts     

Automated Correlation and Averaging of Three-Dimensional Reconstructions Obtained by Electron Tomography

We have developed a least-squares refinement procedure that in an automated way performs three-dimensional alignment and averaging of objects from multiple reconstructions. The computer implementation aligns the three-dimensional structures by a two-step procedure that maximizes the density overlap for all objects. First, an initial average density is built by successive incorporation of individual objects, after a global search for their optimal three-dimensional orientations. Second, the initial average is subsequently refined by excluding individual objects one at a time, realigning them with the reduced average containing all other objects and including them into the average again. The refinement is repeated until no further change of the average occurs. The resulting average model is therefore minimally biased by the order in which the individual reconstructions are incorporated into the average. The performance of the procedure was tested using a synthetic data set of randomly oriented objects with Poisson-distributed noise added. The program managed well to align and average the objects at the signal/noise ratio 1.0. The increase in signal/noise ratio was in all investigated cases almost equal to the expected square root of the number of objects. The program was also successfully tested on a set of authentic three-dimensional reconstructions from anin situspecimen containingEscherichia coli70S ribosomes, where the immediate environment of the reconstructed objects may also contain variable amounts of other structures.     

分子生物学技术在风疹病毒包膜糖蛋白研究中的应用

风疹病毒(RV)是最重要的人类致畸病毒。对RV包膜糖蛋白的研究,有助于构建与表达RVRNA病毒样颗粒,用于核酸检测质控品的研究与开发;并且有助于理解RV产生免疫的机理,以研制基因工程疫苗、亚单位疫苗、多肽疫苗等新型疫苗。本文对分子生物学技术在RV包膜糖蛋白研究中的应用进展进行了综述,包括风疹病毒RNA病毒样颗粒的构建与表达、建立风疹病毒免疫学诊断方法以及制备基因工程疫苗和亚单位疫苗的研究等。     

Computational Methods Toward Unbiased Pattern Mining and Structure Determination in Cryo-Electron Tomography Data

Cryo-electron tomography can uniquely probe the native cellular environment for macromolecular structures. Tomograms feature complex data with densities of diverse, densely crowded macromolecular complexes, low signal-to-noise, and artifacts such as the missing wedge effect. Post-processing of this data generally involves isolating regions or particles of interest from tomograms, organizing them into related groups, and rendering final structures through subtomogram averaging. Template-matching and reference-based structure determination are popular analysis methods but are vulnerable to biases and can often require significant user input. Most importantly, these approaches cannot identify novel complexes that reside within the imaged cellular environment. To reliably extract and resolve structures of interest, efficient and unbiased approaches are therefore of great value. This review highlights notable computational software and discusses how they contribute to making automated structural pattern discovery a possibility. Perspectives emphasizing the importance of features for user-friendliness and accessibility are also presented.     

Cryo-Electron Tomography Elucidates the Molecular Architecture of Treponema pallidum,the Syphilis Spirochete

Cryo-electron tomography (CET) was used to examine the native cellular organization of Treponema pallidum, the syphilis spirochete. T. pallidum cells appeared to form flat waves, did not contain an outer coat and, except for bulges over the basal bodies and widening in the vicinity of flagellar filaments, displayed a uniform periplasmic space. Although the outer membrane (OM) generally was smooth in contour, OM extrusions and blebs frequently were observed, highlighting the structure''s fluidity and lack of attachment to underlying periplasmic constituents. Cytoplasmic filaments converged from their attachment points opposite the basal bodies to form arrays that ran roughly parallel to the flagellar filaments along the inner surface of the cytoplasmic membrane (CM). Motile treponemes stably attached to rabbit epithelial cells predominantly via their tips. CET revealed that T. pallidum cell ends have a complex morphology and assume at least four distinct morphotypes. Images of dividing treponemes and organisms shedding cell envelope-derived blebs provided evidence for the spirochete''s complex membrane biology. In the regions without flagellar filaments, peptidoglycan (PG) was visualized as a thin layer that divided the periplasmic space into zones of higher and lower electron densities adjacent to the CM and OM, respectively. Flagellar filaments were observed overlying the PG layer, while image modeling placed the PG-basal body contact site in the vicinity of the stator-P-collar junction. Bioinformatics and homology modeling indicated that the MotB proteins of T. pallidum, Treponema denticola, and Borrelia burgdorferi have membrane topologies and PG binding sites highly similar to those of their well-characterized Escherichia coli and Helicobacter pylori orthologs. Collectively, our results help to clarify fundamental differences in cell envelope ultrastructure between spirochetes and gram-negative bacteria. They also confirm that PG stabilizes the flagellar motor and enable us to propose that in most spirochetes motility results from rotation of the flagellar filaments against the PG.Spirochetes are an ancient and extremely successful eubacterial phylum characterized by distinctive helical or planar wave-form morphology and flagellar filaments confined to the periplasmic space (55, 87). Spirochetes from the genera Leptospira, Treponema, and Borrelia are highly invasive pathogens that pose public health problems of global dimensions (1, 6, 57, 109). Treponema denticola and numerous other treponemal species, most of which remain uncultivated, are major components of the polymicrobial biofilms that cause periodontal disease (34, 56) and also have been implicated as risk factors for atherosclerosis (4, 125). The treponemal symbionts that dwell in the hindguts of termites, where they provide their insect host with essential nutrients (10), are one of the most striking examples of the extraordinary biodiversity achieved by spirochetes. It is readily apparent, therefore, that in the course of their complex evolution, spirochetes have exploited a basic ultrastructural plan to accommodate an immense spectrum of metabolic activities and lifestyles, both commensal and pathogenic.Venereal syphilis is a multistage, sexually transmitted disease caused by the noncultivatable spirochete Treponema pallidum. Following inoculation, usually in the genital region, T. pallidum disseminates via lymphatics and blood to diverse organs, where it can establish persistent, even life-long, infection (68, 97). Over the years there has been great interest in defining ultrastructural features of the syphilis spirochete that might contribute to syphilis pathogenesis (58, 64, 84, 120, 121). Classic electron microscopy studies established that T. pallidum possesses a characteristic spirochete ultrastructure consisting of outer and cytoplasmic membranes and periplasmic flagellar filaments originating from cytoplasmic membrane-associated, subterminal basal bodies (55, 58). Hovind-Hougen (58) identified a putative peptidoglycan (PG) layer surrounding the cytoplasmic membrane (CM), and she noted that the end of the bacterium contains a distinct structural entity which she speculated mediates polar attachment to mammalian cells and extracellular matrix components. Freeze-fracture analysis has shown that the T. pallidum outer membrane (OM) contains a lower density of membrane-spanning proteins than its counterparts in either gram-negative bacteria or cultivatable spirochetes (99, 118), and it is thought that the paucity of surface-exposed antigenic targets resulting from this unusual OM ultrastructure is an important element of the spirochete''s strategy for immune evasion (14, 93, 97).In the more than 10 years since the publication of the T. pallidum genomic sequence made available a much-needed parts list for the bacterium (44), we have learned comparatively little about how these components are organized to create this extremely virulent and immunoevasive pathogen. Cryo-electron tomography (CET) has emerged as a powerful methodology for bridging the gap between protein-protein interactions and cellular architecture (70, 71). With this technique, thin films of cells are vitreously frozen to preserve cell structure in a close-to-native state, thereby avoiding chemical fixation, dehydration, and staining artifacts typically associated with conventional electron microscopy (EM). A series of images acquired as the sample is progressively tilted in an electron microscope are used to generate a three-dimensional (3D) reconstruction of the intact cell. In recent years, investigators have used CET to examine a variety of eukaryotic and prokaryotic cell types (70, 73, 77). With respect to spirochetes, CET has been used to visualize the intact flagellar motors of Treponema primitia (79) and Borrelia burgdorferi (67, 72); novel internal and external structural features of T. denticola (60); Treponema primitia (80), B. burgdorferi (66), and Leptospira interrogans (74); the flat ribbon configuration of B. burgdorferi periplasmic flagella (18); and the defects created in B. burgdorferi OMs when organisms are incubated with a borreliacidal monoclonal antibody (69). In the present study, we used CET to examine the native cellular organization of T. pallidum. These analyses demonstrated, not surprisingly, that T. pallidum shares many structural features with T. denticola while, at the same time, calling attention to the fluidity and dynamism of the syphilis spirochete''s cell envelope. Our study also revealed that T. pallidum cell ends possess an unexpected degree of structural complexity and diversity compared to those of other spirochetes examined to date by CET. Lastly, our work has clarified the location of the PG layer within the periplasmic space and its spatial relationship to the motility apparatus, which are prerequisites for understanding spirochete movement and, by extension, invasiveness. As a whole, the information obtained underscores and clarifies fundamental differences in cell envelope composition and organization between T. pallidum, as well as other pathogenic spirochetes, and the model gram-negative bacterium, Escherichia coli.     

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein ‘spikes’ is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 Å and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.     

The Synthesis and Localization of Envelope Glycoproteins in Oocytes of Xenopus laevis using Immunocytochemical Methods

The site of origin and the mode of differentiation of the coelomic envelope (CE) in growing oocytes of Xenopus laevis were studied using the rabbit antiserum raised against the isolated envelope from oviposited eggs. The antiserum preabsorbed with egg extracts reacted with most components of CE glycoproteins detectable by SDS-PAGE, and stained specifically the CE of full-grown (st. VI) oocytes using indirect immunofluorescence methods. Transmission electron microscopy employing IgG-conjugated colloidal gold demonstrated that the CE antigens were distributed throughout the whole cytoplasm of st. I oocytes, and began to be deposited around the oocyte surface at late st. I. During st. II to VI the density of CE antigens in the oocyte cytoplasm decreased markedly, while the deposition of extracellular CE antigens increased gradually in association with the formation of a fibrillar network. The CE antigens were observed in and around the highly extended oocyte microvilli during st. II to IV, but were never found in follicle cells at any stages of oocyte growth. On western blot analyses, the extracellular CE components appeared first at st. II, and increased both in amount and number of bands during st. III - V to attain a typical electrophoretic profiles of well-developed CE. The cytosols of growing oocytes, however, possessed several antigenic components which were distinct from those of extracellular CE, suggesting the occurrence of intracellular precursor molecules for CE. The CEs of st. IV oocytes defolliculated and cultured in [³H] leucine contained certain CE components that expressed the radiolabel on fluorography. These results indicate that in Xenopus laevis the oocyte is directly involved in the synthesis and assembly, as well as secretion of CE with least contribution by the follicle cells.     

分子伴侣与病毒糖蛋白

分子伴侣(molecular chaperone)的概念由Lasky于1978年首先提出[1],真核细胞内质网中的分子伴侣是由多种蛋白质组成,它们可以介导新合成蛋白质的正确折叠与装配,并在真核生物的细胞中广泛存在.     

Analysis of the Intact Surface Layer of Caulobacter crescentus by Cryo-Electron Tomography

The surface layers (S layers) of those bacteria and archaea that elaborate these crystalline structures have been studied for 40 years. However, most structural analysis has been based on electron microscopy of negatively stained S-layer fragments separated from cells, which can introduce staining artifacts and allow rearrangement of structures prone to self-assemble. We present a quantitative analysis of the structure and organization of the S layer on intact growing cells of the Gram-negative bacterium Caulobacter crescentus using cryo-electron tomography (CET) and statistical image processing. Instead of the expected long-range order, we observed different regions with hexagonally organized subunits exhibiting short-range order and a broad distribution of periodicities. Also, areas of stacked double layers were found, and these increased in extent when the S-layer protein (RsaA) expression level was elevated by addition of multiple rsaA copies. Finally, we combined high-resolution amino acid residue-specific Nanogold labeling and subtomogram averaging of CET volumes to improve our understanding of the correlation between the linear protein sequence and the structure at the 2-nm level of resolution that is presently available. The results support the view that the U-shaped RsaA monomer predicted from negative-stain tomography proceeds from the N terminus at one vertex, corresponding to the axis of 3-fold symmetry, to the C terminus at the opposite vertex, which forms the prominent 6-fold symmetry axis. Such information will help future efforts to analyze subunit interactions and guide selection of internal sites for display of heterologous protein segments.Surface layers (S layers) are the outermost cell wall component in many archaea and bacteria (6, 44). Most S layers are composed of a single protein or glycoprotein species that self-organizes into two-dimensional (2D) lattices of various sizes, usually with square or hexagonal symmetry (7, 14, 43). This geometrical arrangement is almost the only commonality among species, since sequence homology between S-layer proteins is low and functionality differs in many cases. In many archaea, the S layer is the only cell wall component, so it may have a role in shape determination. However, in bacteria such as Caulobacter crescentus, the role is more likely related to protection against a variety of predatorial assaults (8).One interest in understanding S layers comes from their potential applications in nanotechnology (46) and therapeutic applications, such as anti-HIV microbicide development (37) and cancer therapy (9). The concept is to display heterologous proteins from within the S-layer structure in order to create dense arrays of foreign insertions. Resolving the S-layer organization and structure at high resolution in cells as close to a native state as possible is crucial to understand or predict where proteins are displayed in the array, particularly when more than one foreign peptide is being displayed simultaneously.A significant limitation has been the difficulty in obtaining an atomic resolution structural analysis for any S layer with standard structural methods, such as X-ray crystallography. It has been assumed that the difficulty in obtaining three-dimensional crystals is the consequence of the propensity for two-dimensional assembly, which prevents the proteins from being sufficiently well behaved for crystallization. Despite that, there are a few examples of limited success for portions of S layers (38, 39). Moreover, many studies have been conducted on isolated in vitro S-layer sheets using negative-stain electron microscopy. This approach removes the interaction of the S layer with other cell wall components, which makes it more difficult to understand how a crystalline structure develops on a growing bacterium. Defects in structure that occur during the introduction of newly secreted subunits or to accommodate covering areas of strong curvature may well not be appreciated in isolated fragments, where rearrangements of the two-dimensional array are likely to occur. This may result in a more regular structure and even assist image analysis methods but does not represent what is occurring on the dynamic cell surface. Quoting Engelhardt (17): “Functional aspects have usually been investigated with isolated S-layer sheets or proteins, which disregards the interactions between S-layers and the underlying cell envelope components.”Imaging technologies such as freeze-etch and negative-stain microscopy of whole cells (48) can obtain quality images of the S layer directly on the cell. However, each of these techniques presents drawbacks, which would make impossible to extract the conclusions summarized in this paper. For example, it is very difficult to combine freeze-etching with site-specific labeling methods and is impossible with any label in the size range of Nanogold (NG). Moreover, freeze-etch images do not contain three-dimensional (3D) information. Negative staining of S layers on intact cells is difficult and generally can be imaged only on lysed (eviscerated) cells (48). In this case, resolution is hampered by overlying double S layers, membrane debris, and the stain itself. Labeling with the typical 5- to 10-nm colloidal gold probes with either approach is not amenable to averaging techniques designed to localize the labels.Prior work has shown that C. crescentus, a Gram-negative bacterium, has an S-layer subunit composed of a single highly expressed (27) protein (RsaA), secreted by a type I mechanism, such that there is no cleaved N-terminal signal leader but there is an uncleaved C-terminal secretion signal (4, 11). Six RsaA monomers (12) form the characteristic hexagonal core with p6 symmetry seen by image analysis (47, 48), and the 2D lattice is completed by hexagonal cores connected at junction points with p3 symmetry (Fig. ​(Fig.11 A). Secretion and subsequent self-assembly require calcium (35), and it is assumed that the RTX repeat domain (characteristic of type I secreted proteins) that is located adjacent to the C-terminal secretion signal is responsible for at least some of the calcium interaction; whether there is a second domain to complete subunit-subunit interactions is unknown. The S layer is attached to the outer membrane (OM) surface by interaction of an N-terminal attachment domain of approximately 200 amino acids (18) with the fraction of lipopolysaccharide (LPS) that is modified with an O-polysaccharide (3, 52).Open in a separate windowFIG. 1.(A) Schematic from a previous publication (12) showing how six RsaA monomers build a hexagonal S-layer subunit in C. crescentus. The six-point star shows the center of 6-fold symmetry, and the triangle indicates the center of 3-fold symmetry. The figure is based on results presented previously (35). (B) Cross section of a C. crescentus tomogram to show the cell wall components. The effect of the missing wedge blurring the features on the top and bottom of the cell is obvious.In this paper, we present a quantitative analysis of the C. crescentus S layer that sheds light on the overall S-layer organization as well as improves our understanding of the structure within the RsaA monomer, in advance of achieving true atomic resolution, by combining cryo-electron tomography (CET) of intact cells with statistical image-processing algorithms. CET is an ideal imaging technology with which to obtain a view of intact prokaryotic cells at molecular resolution (28, 29, 30). This technology allows the visualization of S-layer architecture directly as it exists on the dynamic, growing cell surface. The sample in the microscope is kept close to the native state without staining artifacts, while projections are obtained from different tilt angles to reconstruct a 3D density map of the sample. However, due to low contrast and a generally high sample thickness, CET images have a low signal-to-noise-ratio (SNR). In particular, C. crescentus has a diameter of approximately 600 nm, which is considered close to the thickness limit of CET imaging for these kinds of samples. Statistical image-processing techniques and target samples as thin as possible are needed to overcome the low SNR and to perform quantitative analysis of the S-layer characteristics in situ.To obtain structural information from within the RsaA monomer, we introduced unique cysteine residues at locations ranging from the N terminus to as close as possible to the C terminus (without disrupting the secretion signal), followed by labeling with maleimide-coupled Nanogold particles. At 1 to 2 nm in size, the Nanogold is not visible in individual CET images of intact cells. However, it is possible to extract thousands of small volumes containing S-layer subunits from CET images and combine them using subtomogram averaging techniques to produce higher-resolution structures. In these averages, we identified locations of high-density areas representing the Nanogold, effectively using the regularity of the S-layer structure to increase the resolution of CET imaging.In short, we found that the long-range order is substantially lower than we had expected and that there were areas of double layers, especially when RsaA was overexpressed. By comparing the site-specific Nanogold labeling to the 2-nm-resolution structure that is available (47), we have begun to correlate the primary sequence to positions within the averaged hexagonal structure, which represents a significant step toward having a rational basis for site selection for heterologous protein insertions in nanotechnology applications.     

Evidence that Envelope and Thylakoid Membranes from Pea Chloroplasts Lack Glycoproteins

Envelope and thylakoid membranes from pea (Pisum sativum var. Laxton's Progress No. 9) chloroplasts were analyzed for the presence of glycoproteins using two different approaches. First, the sugar composition of delipidated membrane polypeptides was measured directly using gas chromatographic analysis. The virtual absence of sugars suggests that plastid membranes lack glycoproteins. Second, membrane polypeptides separated by sodium dodecyl sulfate gel electrophoresis were tested for reactivity toward three different lectins: Concanavalin A, Ricinus communis agglutinin, and wheat germ agglutinin. In each case, there was no reactivity between any of the lectins and the plastid polypeptides. Microsomal membranes from pea tissues were used as a positive control. Glycoproteins were readily detectable in microsomal membranes using either of the two techniques. From these results it was concluded that pea chloroplast membranes do not contain glycosylated polypeptides.     

The Transmembrane Domains of Sindbis Virus Envelope Glycoproteins Induce Cell Death

Sindbis virus, the prototype alphavirus, kills cells by inducing apoptosis. To investigate potential mechanisms by which Sindbis virus induces apoptosis, we examined whether specific viral gene products were able to induce cell death. Genes encoding the three structural proteins—capsid, the precursor E1 (6K plus E1), and the precursor E2 (P62 or E3 plus E2)—were cotransfected with a β-galactosidase reporter plasmid in transient-transfection assays in rat prostate adenocarcinoma AT3 cells. Cell death, as determined by measuring the loss of blue cells, was observed in AT3 cells transfected with 6K plus E1 and with P62 but not in cells transfected with capsid. Deletion mutagenesis of P62 indicated that large regions of the cytoplasmic domain and extracellular domain were not essential for the induction of cell death. However, constructs containing the minimal E3 signal sequence fused to the E2 transmembrane domain and the minimal E3 signal sequence fused to the E1 transmembrane domain induced death as efficiently as full-length P62 and 6K plus E1, whereas no cell death was observed after transfection with a control construct containing the E3 signal sequence linked to the transmembrane domain of murine CD4. These data demonstrate that intracellular expression of the transmembrane domains of the Sindbis virus envelope glycoproteins can kill AT3 cells.