High resolution microscopy reveals an unusual architecture of the Plasmodium berghei endoplasmic reticulum

To fuel the tremendously fast replication of Plasmodium liver stage parasites, the endoplasmic reticulum (ER) must play a critical role as a major site of protein and lipid biosynthesis. In this study, we analysed the parasite's ER morphology and function. Previous studies exploring the parasite ER have mainly focused on the blood stage. Visualizing the Plasmodium berghei ER during liver stage development, we found that the ER forms an interconnected network throughout the parasite with perinuclear and peripheral localizations. Surprisingly, we observed that the ER additionally generates huge accumulations. Using stimulated emission depletion microscopy and serial block‐face scanning electron microscopy, we defined ER accumulations as intricate dense networks of ER tubules. We provide evidence that these accumulations are functional subdivisions of the parasite ER, presumably generated in response to elevated demands of the parasite, potentially consistent with ER stress. Compared to higher eukaryotes, Plasmodium parasites have a fundamentally reduced unfolded protein response machinery for reacting to ER stress. Accordingly, parasite development is greatly impaired when ER stress is applied. As parasites appear to be more sensitive to ER stress than are host cells, induction of ER stress could potentially be used for interference with parasite development.     

Towards a molecular architecture of centriole assembly

The centriole is an evolutionarily conserved macromolecular structure that is crucial for the formation of flagella, cilia and centrosomes. The ultrastructure of the centriole was first characterized decades ago with the advent of electron microscopy, revealing a striking ninefold radial arrangement of microtubules. However, it is only recently that the molecular mechanisms governing centriole assembly have begun to emerge, including the elucidation of the crucial role of spindle assembly abnormal 6 (SAS-6) proteins in imparting the ninefold symmetry. These advances have brought the field to an exciting era in which architecture meets function.     

Single-molecule analysis of the microtubule cross-linking protein MAP65-1 reveals a molecular mechanism for contact-angle-dependent microtubule bundling

Bundling of microtubules (MTs) is critical for the formation of complex MT arrays. In land plants, the interphase cortical MTs form bundles specifically following shallow-angle encounters between them. To investigate how cells select particular MT contact angles for bundling, we used an in vitro reconstitution approach consisting of dynamic MTs and the MT-cross-linking protein MAP65-1. We found that MAP65-1 binds to MTs as monomers and inherently targets antiparallel MTs for bundling. Dwell-time analysis showed that the affinity of MAP65-1 for antiparallel overlapping MTs is about three times higher than its affinity for single MTs and parallel overlapping MTs. We also found that purified MAP65-1 exclusively selects shallow-angle MT encounters for bundling, indicating that this activity is an intrinsic property of MAP65-1. Reconstitution experiments with mutant MAP65-1 proteins with different numbers of spectrin repeats within the N-terminal rod domain showed that the length of the rod domain is a major determinant of the range of MT bundling angles. The length of the rod domain also determined the distance between MTs within a bundle. Together, our data show that the rod domain of MAP65-1 acts both as a spacer and as a structural element that specifies the MT encounter angles that are conducive for bundling.     

Structure of photosystem II and molecular architecture of the oxygen-evolving centre

Photosynthesis utilizes light energy to oxidize water molecules to molecular oxygen at the oxygen-evolving centre of photosystem II. The structure of photosystem II from the cyanobacterium Thermosynechococcus elongatus has been reported at 3.5A resolution and, for the first time, the complete molecular structure of this 650 kDa complex, including the oxygen-evolving centre, has been revealed.     

A comprehensive Plasmodium falciparum protein interaction map reveals a distinct architecture of a core interactome

We derive a map of protein interactions in the parasite Plasmodium falciparum from conserved interactions in Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and Escherichia coli and pool them with experimental interaction data. The application of a clique‐percolation algorithm allows us to find overlapping clusters, strongly correlated with yeast specific conserved protein complexes. Such clusters contain core activities that govern gene expression, largely dominated by components of protein production and degradation processes as well as RNA metabolism. A critical role of protein hubs in the interactome of P. falciparum is supported by their appearance in multiple clusters and the tendencies of their interactions to reach into many distinct protein clusters. Parasite proteins with a human ortholog tend to appear in single complexes. Annotating each protein with the stage where it is maximally expressed we observe a high level of cluster integrity in the ring stage. While we find no signal in the trophozoite phase, expression patterns are reversed in the schizont phase, implying a preponderance of parasite specific functions in this late, invasive schizont stage. As such, the inference of potential protein interactions and their analysis contributes to our understanding of the parasite, indicating basic pathways and processes as unique targets for therapeutic intervention.     

Building a nuclear envelope at the end of mitosis: coordinating membrane reorganization, nuclear pore complex assembly, and chromatin de-condensation

The metazoan nucleus is disassembled and re-built at every mitotic cell division. The nuclear envelope, including nuclear pore complexes, breaks down at the beginning of mitosis to accommodate the capture of massively condensed chromosomes by the spindle apparatus. At the end of mitosis, a nuclear envelope is newly formed around each set of segregating and de-condensing chromatin. We review the current understanding of the membrane restructuring events involved in the formation of the nuclear membrane sheets of the envelope, the mechanisms governing nuclear pore complex assembly and integration in the nascent nuclear membranes, and the regulated coordination of these events with chromatin de-condensation.     

Taxol reveals cortical sites of microtubule assembly in Xenopus oocytes. Role of the nucleus

A binding colchicine assay together with an immunostaining study with an anti-tubulin antibody showed that taxol, when added to the incubation medium, induces the formation of new microtubules in the Xenopus oocyte cortex. The capacity of the tubulin assembly in the submembranous cytoplasm decreases in progesterone-matured oocytes. In contrast, in enucleated matured oocytes this change does not occur. Altogether, these results show that taxol provokes tubulin assembly exclusively in the cortex of prophase oocyte, whereas in normal matured oocytes both cortical and cytoplasmic cytaster microtubules can be induced by taxol. The swelling of the oocyte nucleus therefore controls the spatial distribution of nucleation centers for tubulin assembly during meiotic maturation.     

Protein localisation by electron microscopy reveals the architecture of the yeast spliceosomal B complex

The spliceosome assembles on a pre‐mRNA intron by binding of five snRNPs and numerous proteins, leading to the formation of the pre‐catalytic B complex. While the general morphology of the B complex is known, the spatial arrangement of proteins and snRNP subunits within it remain to be elucidated. To shed light on the architecture of the yeast B complex, we immuno‐labelled selected proteins and located them by negative‐stain electron microscopy. The B complex exhibited a triangular shape with main body, head and neck domains. We located the U5 snRNP components Brr2 at the top and Prp8 and Snu114 in the centre of the main body. We found several U2 SF3a (Prp9 and Prp11) and SF3b (Hsh155 and Cus1) proteins in the head domain and two U4/U6 snRNP proteins (Prp3 and Lsm4) in the neck domain that connects the main body with the head. Thus, we could assign distinct domains of the B complex to the respective snRNPs and provide the first detailed picture of the subunit architecture and protein arrangements of the B complex.     

Atomic force microscopy reveals the architecture of the epithelial sodium channel (ENaC)

The epithelial sodium channel (ENaC) is a member of the ENaC/degenerin superfamily. ENaC is a heteromultimer containing three homologous subunits (α, β, and γ); however, the subunit stoichiometry is still controversial. Here, we addressed this issue using atomic force microscopy imaging of complexes between isolated ENaC and antibodies/Fab fragments directed against specific epitope tags on the α-, β- and γ-subunits. We show that for α-, β- and γ-ENaC alone, pairs of antibodies decorate the channel at an angle of 120°, indicating that the individual subunits assemble as homotrimers. A similar approach demonstrates that αβγ-ENaC assembles as a heterotrimer containing one copy of each subunit. Intriguingly, all four subunit combinations also produce higher-order structures containing two or three individual trimers. The trimer-of-trimers organization would account for earlier reports that ENaC contains eight to nine subunits.     

Atomic force microscopy reveals the assembly of potential DNA "nanocarriers" by poly-L-ornithine

Atomic force microscopy (AFM) has been used to visualize the process of condensation of plasmid DNA by poly-L-ornithine on mica surface. AFM images reveal that the transition of negatively charged DNA to condensed nanoparticles on addition of increasing amounts of positively charged poly-L-ornithine (charge ratio (Z+/Z-) varied between 0.1 and 1) at a wide range of DNA concentrations (3-20 ng/microl) occurs through formation of several distinct morphologies. The nature of the complexes is strongly dependent on both the charge ratio and the DNA concentration. Initiation of condensation when the concentration of DNA is low (approximately 3-7 ng/microl) occurs possibly through formation of monomolecular complexes which are thick rod-like in shape. On the contrary, when condensation is carried out at DNA concentrations of 13-20 ng/microl, multimolecular structures are also formed even at low charge ratios. This difference in pathway seems to result in differences in the extent of condensation as well as size and aggregation of the nanoparticles formed at the high charge ratios. To the best of our knowledge, this is the first direct single molecule elucidation of the mechanism of DNA condensation by poly-L-ornithine. Cationic poly-aminoacids like poly-L-ornithine are known to be efficient in delivery of plasmid DNA containing therapeutic genes in a variety of mammalian cell lines by forming condensed "nanocarriers" with DNA. Single molecule insight into the mechanism by which such nanocarriers are packaged during the condensation process could be helpful in predicting efficacy of intracellular delivery and release of DNA from them and also provide important inputs for design of new gene delivery vectors.     

Polo-like kinase-1 is a pivotal regulator of microtubule assembly during mouse oocyte meiotic maturation,fertilization, and early embryonic mitosis

Polo-like kinases (Plks) are a family of serine/threonine protein kinases that have been activated through phosphorylation. The activity of these kinases has been shown to be required for regulating multiple stages of mitotic progression in somatic cells. In this experiment, the changes in Plk1 expression were detected in mouse oocytes through Western blotting. The subcellular localization of Plk1 during oocyte meiotic maturation, fertilization, and early cleavage as well as after antibody microinjection or microtubule assembly disturbance was studied by confocal microscopy. The quantity of Plk1 protein remained stable during meiotic maturation and decreased gradually after fertilization. Plk1 was localized to the spindle poles of both meiotic and mitotic spindles at the early M phase and then translocated to the middle region. At anaphase and telophase, Plk1 was concentrated at the midbody of cytoplasmic cleavages. Plk1 was concentrated between the male and female pronuclei after fertilization. Plk1 disappeared at the spindle region when microtubule formation was inhibited by colchicine or staurosporine, while it was concentrated as several dots in the cytoplasm after taxol treatment. Plk1 antibody injection decreased the germinal vesicle breakdown rate and distorted MI spindle organization. Our results indicate that Plk1 is a pivotal regulator of microtubule organization during mouse oocyte meiosis, fertilization, and cleavage and that its functions may be regulated by other kinases, such as staurosporine-sensitive kinases.     

The clamp-loader-helicase interaction in Bacillus. Atomic force microscopy reveals the structural organisation of the DnaB-tau complex in Bacillus

The clamp-loader-helicase interaction is an important feature of the replisome. Although significant biochemical and structural work has been carried out on the clamp-loader-clamp-DNA polymerase alpha interactions in Escherichia coli, the clamp-loader-helicase interaction is poorly understood by comparison. The tau subunit of the clamp-loader mediates the interaction with DnaB. We have recently characterised this interaction in the Bacillus system and established a tau(5)-DnaB(6) stoichiometry. Here, we have obtained atomic force microscopy images of the tau-DnaB complex that reveal the first structural insight into its architecture. We show that despite the reported absence of the shorter gamma version in Bacillus, tau has a domain organisation similar to its E.coli counterpart and possesses an equivalent C-terminal domain that interacts with DnaB. The interaction interface of DnaB is also localised in its C-terminal domain. The combined data contribute towards our understanding of the bacterial replisome.     

Visualizing the 3D architecture of multiple erythrocytes infected with Plasmodium at nanoscale by focused ion beam-scanning electron microscopy

Different methods for three-dimensional visualization of biological structures have been developed and extensively applied by different research groups. In the field of electron microscopy, a new technique that has emerged is the use of a focused ion beam and scanning electron microscopy for 3D reconstruction at nanoscale resolution. The higher extent of volume that can be reconstructed with this instrument represent one of the main benefits of this technique, which can provide statistically relevant 3D morphometrical data. As the life cycle of Plasmodium species is a process that involves several structurally complex developmental stages that are responsible for a series of modifications in the erythrocyte surface and cytoplasm, a high number of features within the parasites and the host cells has to be sampled for the correct interpretation of their 3D organization. Here, we used FIB-SEM to visualize the 3D architecture of multiple erythrocytes infected with Plasmodium chabaudi and analyzed their morphometrical parameters in a 3D space. We analyzed and quantified alterations on the host cells, such as the variety of shapes and sizes of their membrane profiles and parasite internal structures such as a polymorphic organization of hemoglobin-filled tubules. The results show the complex 3D organization of Plasmodium and infected erythrocyte, and demonstrate the contribution of FIB-SEM for the obtainment of statistical data for an accurate interpretation of complex biological structures.     

In situ visualization of a simple bipartite kinetochore with a single microtubule attachment in Giardia intestinalis (Metamonada)

To understand general features in evolution of kinetochore organization, investigating a wide range of mitotic mechanisms in various non-model eukaryotes is necessary. A binucleate flagellate Giardia intestinalis is a representative of highly divergent eukaryotic lineage of Metamonads. FIB/SEM tomography was used to investigate ultrastructural details of its mitotic architecture, including kinetochores. Giardia undergoes semi-open mitosis, with the nuclear envelope remaining intact except for polar fenestrae, allowing microtubules to enter the nucleoplasm. At the onset of mitosis, the nuclear envelope bends inward, forming a concave depression at the spindle poles. Spindle microtubules emanate from a cytoplasmic fuzzy microtubule organizing center near the flagellar basal bodies. Kinetochoral microtubules enter the nucleoplasm and bind to kinetochores. A small bipartite kinetochore composed of a dense inner disk, approximately 46 nm in diameter, and a two-armed outer fork, is attached to just one microtubule. To our knowledge, this is the first in situ evidence of a one-microtubule attachment to a kinetochore, which could represent a basic eukaryotic situation.     

On the relationship between nucleotide hydrolysis and microtubule assembly: studies with a GTP-regenerating system

The assembly of pure tubulin dimer has been studied in two buffer systems (containing low and high glycerol/Mg), using a regeneration system protocol to assess the amount of GDP-tubulin in the assembling polymer. For both assembly systems studied, the GDP content is effectively stoichiometric with tubulin throughout assembly. This indicates a high degree of coupling between assembly and GTP-hydrolysis, giving a hydrolysis rate at least 10-fold faster than previously deduced. The steady state GTP hydrolysis rate is quantitatively consistent with this finding. We conclude that the extent of any GTP-tubulin cap is below the detectable limit, both during elongation and at steady state.     

Comparison of MukB homodimer versus MukBEF complex molecular architectures by electron microscopy reveals a higher-order multimerization

The complex of MukF, MukE, and MukB proteins participates in organization of sister chromosomes and partitioning into both daughter cells in Escherichia coli. We purified the MukB homodimer and the MukBEF complex and analyzed them by electron microscopy to compare both structures. A MukB homodimer shows a long rod-hinge-rod v-shape with small globular domains at both ends. The MukBEF complex shows a similar structure having larger globular domains than those of the MukB homodimer. These results suggest that MukF and MukE bind to the globular domains of a MukB homodimer. The globular domains of the MukBEF complex frequently associate with each other in an intramolecular fashion, forming a ring. In addition, MukBEF complex molecules tend to form multimers by the end-to-end joining with other MukBEF molecules in an intermolecular fashion, resulting in fibers and rosette-form structures in the absence of ATP and DNA in vitro.     

Control of shape and pattern during the assembly of a large microtubule bundle

Microtubules are packed and linked together in a well defined hexagonal arrangement in the cytopharyngeal microtubule bundles of the ciliate Nassula. Early stages in the morphogenesis of these bundles have been examined. Elements which nucleate assembly of bundle microtubules are apparently closely associated before tubule assembly commences. These nucleating elements seem to be bound together in highly ordered arrays to form microtubule-nucleating-templetes. Each array of elements is attached to the proximal end of a basal body and appears to establish the pattern of tubule packing and cross-sectional shape of a tubule bundle. A self-assembly procedure which accounts for the anisometric growth and shaping of a template and its microtubule bundle is proposed.     

Erythrocyte microtubule assembly in vitro. Determination of the effects of erythrocyte tau, tubulin isoforms, and tubulin oligomers on erythrocyte tubulin assembly, and comparison with brain microtubule assembly

Two tubulin variants, isolated from chicken brain and erythrocytes and known to have different peptide maps and electrophoretic properties, are demonstrated to exhibit different assembly properties in vitro: 1) erythrocyte tubulin assembles with greater efficiency (lower critical concentration, greater elongation rate) but exhibits a lower nucleation rate than brain tubulin, and 2) erythrocyte tubulin readily forms oligomers whose presence significantly retards the rate of elongation, suggesting that tubulin oligomers may also be important for determining the rate of assembly and the length of microtubules in erythrocytes. Erythrocyte tubulin isolated by cycles of in vitro assembly-disassembly is also demonstrated to contain a 67-kDa tau factor that greatly enhances microtubule nucleation but has little effect on elongation rates or critical concentration. Immunofluorescence microscopy with tau antibody indicates that tau is specifically associated with marginal band microtubules, suggesting that it may be important for determining microtubule function in vivo.