Rat macrophage activity against Toxoplasma gondii studied by electron microscopy

An electron microscope model was used to study the effect of rat peritoneal macrophages on Toxoplasma gondii. 10(7) tachyzoites were injected i.p. in 30 days-old rats. After 1, 2, 4, 8 and 24 h peritoneal exudate was withdrawn and infected phagocytic cells were prepared for electronic microscope studies. Toxoplasma organisms inside of rat macrophages showed remarkable lesions such as vacuolization and organisms were totally lysed inside of macrophages of more than 8 h infection rats. The results confirm at molecular level, the importance of rat macrophages in the natural adaptation of this rodent to T. gondii.     

Mapping restriction sites on large DNAs by electron microscopy

We have developed a novel technique to map restriction sites on large duplex DNAs by electron microscopy. In this method, the sample DNA is first cut with a restriction enzyme. The resulting fragments are briefly digested with Escherichia coli exonuclease III, and treated with wheat germ RNA polymerase II to fill-in with RNA the resulting gaps. These small RNAs, complementary to sequences immediately adjacent to either side of the restriction site, are isolated from the DNA template and R-looped to the full-length DNA. When this material is prepared by the formamide-cytochrome spreading technique, small bubbles are visible wherever there is a restriction site on the DNA. Improved methods of mapping are outlined.     

A novel mirror-symmetry analysis approach for the study of macromolecular assemblies imaged by electron microscopy

Multivariate statistical symmetry analysis is widely employed in single-particle electron-microscopy studies for the detection of symmetry components within a set of noisy two-dimensional images. So far, this technique has been used to retrieve information from the analysis of end-on view oriented particles only. Here, we propose a method to detect symmetry components from side- and tilted-view oriented particles. This method is validated using a number of in silico generated as well as real datasets, can be used to analyze stoichiometrically heterogeneous datasets, and is useful for separating particle datasets with respect to their symmetry components. Additionally, translational components in lock-washer ring configurations can be detected. Most relevantly, this method represents a powerful tool for the characterisation of distinct symmetry components within multi-layered protein assemblies, and any putative symmetry mismatch between layers. Such configurations have often been postulated, though rarely observed directly, and are thought to have a crucial role in conferring dynamicity to molecular machineries like nucleic acid packaging motors, ClpAP/ClpXP proteases, flagellar motors and the F1/F0 ATPase.     

Allostery in very large molecular assemblies

In contrast to small allosteric systems (like hemoglobin) those containing very large numbers (n) of binding sites never exhibit cooperativity (as measured by the Hill coefficient, nH) even approaching the potential limit, n. The reason for this appears to be that in such macromolecules the cooperative unit always represents some sub-structure of the entire structure. On the other hand, it is frequently observed that such sub-structures, when isolated, do not exhibit cooperativity at all. This paper describes studies of some molluscan hemocyanins that explore this apparent anomaly. It is concluded that it is the higher order structure of the molecule that provides a framework within which the sub-structures may exhibit their allosteric behavior.     

Proteomic and electron microscopy study of myogenic differentiation of alveolar mucosa multipotent mesenchymal stromal cells in three-dimensional culture

Myocyte differentiation is featured by adaptation processes, including mitochondria repopulation and cytoskeleton re-organization. The difference between monolayer and spheroid cultured cells at the proteomic level is uncertain. We cultivated alveolar mucosa multipotent mesenchymal stromal cells in spheroids in a myogenic way for the proper conditioning of ECM architecture and cell morphology, which induced spontaneous myogenic differentiation of cells within spheroids. Electron microscopy analysis was used for the morphometry of mitochondria biogenesis, and proteomic was used complementary to unveil events underlying differences between two-dimensional/three-dimensional myoblasts differentiation. The prevalence of elongated mitochondria with an average area of 0.097 μm² was attributed to monolayer cells 7 days after the passage. The population of small mitochondria with a round shape and area of 0.049 μm² (p < 0.05) was observed in spheroid cells cultured under three-dimensional conditions. Cells in spheroids were quantitatively enriched in proteins of mitochondria biogenesis (DNM1L, IDH2, SSBP1), respiratory chain (ACO2, ATP5I, COX5A), extracellular proteins (COL12A1, COL6A1, COL6A2), and cytoskeleton (MYL6, MYL12B, MYH10). Most of the Rab-related transducers were inhibited in spheroid culture. The proteomic assay demonstrated delicate mechanisms of mitochondria autophagy and repopulation, cytoskeleton assembling, and biogenesis. Differences in the ultrastructure of mitochondria indicate active biogenesis under three-dimensional conditions.     

Macromolecular mass spectrometry and electron microscopy as complementary tools for investigation of the heterogeneity of bacteriophage portal assemblies

The success of electron-cryo microscopy (cryo-EM) and image reconstruction of cyclic oligomers, such as the viral and bacteriophage portals, depends on the accurate knowledge of their order of symmetry. A number of statistical methods of image analysis address this problem, but often do not provide unambiguous results. Direct measurement of the oligomeric state of multisubunit protein assemblies is difficult when the number of subunits is large and one subunit renders only a small increment to the full size of the oligomer. Moreover, when mixtures of different stochiometries are present techniques such as analytical centrifugation or size-exclusion chromatography are also less helpful. Here, we use electrospray ionization mass spectrometry to directly determine the oligomeric states of the in vitro assembled portal oligomers of the phages P22, Phi-29 and SPP1, which range in mass from 430 kDa to about 1 million Da. Our data unambiguously reveal that the oligomeric states of Phi-29 and SPP1 portals were 12 and 13, respectively, in good agreement with crystallographic and electron microscopy data. However, in vitro assembled P22 portals were a mixture of 11- and 12-mer species in an approximate ratio of 2:1, respectively. A subsequent reference-free alignment of electron microscopy images of the P22 portal confirmed this mixture of oligomeric states. We conclude that macromolecular mass spectrometry is a valuable tool in structural biology that can aide in the determination of oligomeric states and symmetry of assemblies, providing a good starting point for improved image analysis of cryo-EM data.     

Solution NMR of large molecules and assemblies

Solution NMR spectroscopy represents a powerful tool for examining the structure and function of biological macromolecules. The advent of multidimensional (2D-4D) NMR, together with the widespread use of uniform isotopic labeling of proteins and RNA with the NMR-active isotopes, 15N and 13C, opened the door to detailed analyses of macromolecular structure, dynamics, and interactions of smaller macromolecules (< approximately 25 kDa). Over the past 10 years, advances in NMR and isotope labeling methods have expanded the range of NMR-tractable targets by at least an order of magnitude. Here we briefly describe the methodological advances that allow NMR spectroscopy of large macromolecules and their complexes and provide a perspective on the wide range of applications of NMR to biochemical problems.     

Morphology of sheet-like assemblies of pN-collagen, pC-collagen and procollagen studied by scanning transmission electron microscopy mass measurements

At high concentrations, type I pN-collagen, pC-collagen and procollagen (the first 2 generated from procollagen by enzymic cleavage of C-propeptides and N-propeptides, respectively) can all be made to assemble in vitro into thin D-periodic sheets or tapes. Scanning transmission electron microscopy mass measurements show that the pN-collagen sheets and procollagen tapes have a mass per unit area corresponding to that of approximately 6.8 monolayers of close-packed molecules. pN-collagen sheets are extensive and remarkably uniform in mass thickness (fractional S.D. 0.035); procollagen tapes are neither as extensive nor as uniform in thickness. The mean thickness of pC-collagen tapes is less and the variability is greater. In pN-collagen sheets, the overlap: gap mass contrast in a D-period is increased from 5:4 (the ratio in a native collagen fibril) to 6:4, showing that the N-propeptides do not project into the gap but are folded back over the overlap zone. Assuming all N-propeptides to be constrained to the two surfaces of a sheet, their surface density can be found from the mass thickness of the sheet. In a lateral direction (i.e. normal to the axial direction where the spacing is D-periodic), the N-propeptide domains are calculated to be spaced, centre to centre, by 2.23 (+/- 0.1) nm on both surfaces. This value (approx. 1.5 x the triple-helix diameter) implies close-packing laterally with adjacent domains in contact. Sheet formation and the "surface-seeking" behaviour of propeptides can be understood in terms of the dual character of the molecules, evident from solubility data, with propeptides possessing interaction properties very different from those displayed by the rest of the molecule. The form and stability of sheets (and of first-formed fibrils assembling in vivo) could, it is suggested, depend on the partially fluid-like nature of lateral contacts between collagen molecules.     

Bridging fluorescence microscopy and electron microscopy

Development of new fluorescent probes and fluorescence microscopes has led to new ways to study cell biology. With the emergence of specialized microscopy units at most universities and research centers, the use of these techniques is well within reach for a broad research community. A major breakthrough in fluorescence microscopy in biology is the ability to follow specific targets on or in living cells, revealing dynamic localization and/or function of target molecules. One of the inherent limitations of fluorescence microscopy is the resolution. Several efforts are undertaken to overcome this limit. The traditional and most well-known way to achieve higher resolution imaging is by electron microscopy. Moreover, electron microscopy reveals organelles, membranes, macromolecules, and thus aids in the understanding of cellular complexity and localization of molecules of interest in relation to other structures. With the new probe development, a solid bridge between fluorescence microscopy and electron microscopy is being built, even leading to correlative imaging. This connection provides several benefits, both scientifically as well as practically. Here, I summarize recent developments in bridging microscopy.     

High-resolution electron cryomicroscopy of macromolecular assemblies

Electron cryomicroscopy is a high-resolution imaging technique that is particularly appropriate for the structural determination of large macromolecular assemblies, which are difficult to study by X-ray crystallography or NMR spectroscopy. For some biological molecules that form two-dimensional crystals, the application of electron cryomicroscopy and image reconstruction can help elucidate structures at atomic resolution. In instances where crystals cannot be formed, atomic-resolution information can be obtained by combining high-resolution structures of individual components determined by X-ray crystallography or NMR with image-derived reconstructions at moderate resolution. This can provide unique and crucial information on the mechanisms of these complexes. Finally, image reconstructions can be used to augment X-ray studies by providing initial models that facilitate phasing of crystals of large macromolecular machines such as ribosomes and viruses.     

A survey of 50 S ribosomal subunits by dark field electron microscopy

A survey of dark field electron micrographs of the 50 S ribosomal subunit of E. coli has been performed and supplemented, for comparative purposes, by examination of negatively stained or metal shadowed specimens in the bright field mode. Attention was directed to the so-called "crown" and "kidney" views. The elongated appendage seen in negatively stained crown profiles was not observed in unstained or positively stained samples examined in dark field; these showed only symmetrical crown profiles regardless of changes in buffer type and drying method and of the presence or absence of uranyl acetate treatment and glutaraldehyde fixation. The crown view occasionally displayed a bifurcation in one of the lateral lobes, while the kidney profile showed a groove near the base of the convex edge. Uranyl acetate treatment produced delicate stripes which may give an indication of the surface RNA distribution.     

A differential cytolocalization assay for analysis of macromolecular assemblies in the eukaryotic cytoplasm

We have developed a differential cytolocalization assay (DCLA) that allows the observation of cytoplasmic protein/protein interactions in vivo. In the DCLA, interactions are visualized as a relocalization of a green fluorescent protein-tagged "prey" by a membrane-bound "bait." This assay was tested and utilized in Caenorhabditis elegans to probe interactions among proteins involved in RNA interference (RNAi) and nonsense-mediated decay (NMD) pathways. Several previously documented interactions were confirmed with DCLA, whereas uniformly negative results were obtained in several controls in which no interaction was expected. Novel interactions were also observed, including the association of SMG-5, a protein required for NMD, to several components of the RNAi pathway. The DCLA can be readily carried out under diverse conditions, allowing a dynamic assessment of protein interactions in vivo. We used this property to test a subset of the RNAi and NMD interactions in animals in which proteins central to each mechanism were mutated; several key associations in each machinery that can occur in vivo in the absence of a functional process were identified.