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Tritrichomonas murisis a flagellated protist isolated from the cecum of wild mice in the Czech Republic. This commensal protist has been shown previously to alter immune phenotypes in laboratory mice. Other trichomonads, previously referred to asTritrichomonas musculisandTritrichomonas rainier, also naturally colonize laboratory mice and cause immune alterations. This report formally describes two new trichomonads,Tritrichomonas musculusn. sp., andTritrichomonas casperin. sp., at the ultrastructural and molecular level. These two protists were isolated from laboratory mice, and were differentiated by their size and the structure of their undulating membrane and posterior flagellum. Analysis at the18S rRNAand trans-ITSgenetic loci supported their designation as distinct species, related toT. muris. To further assess the true extent of parabasalid diversity infecting laboratory mice, 135 mice were screened at the NIH using pan-parabasalid primers that amplify the trans-ITSregion. Forty-four percent of mice were positive for parabasalids, encompassing a total of 8 distinct sequence types.Tritrichomonas casperiandTrichomitus-like protists were dominant.T. musculusandT. rainierwere also detected, butT. muriswas not. Our work establishes a previously underappreciated diversity of commensal trichomonad protists that naturally colonize the enteric cavity of laboratory mice.

Little is known about the structural basis of prion strains. Here we provide a high (3.0 Å) resolution cryo-electron microscopy-based structure of infectious brain-derived fibrils of the mouse anchorless RML scrapie strain which, like the recently determined hamster 263K strain, has a parallel in-register β-sheet-based core. Several structural motifs are shared between these ex vivo prion strains, including an amino-proximal steric zipper and three β-arches. However, detailed comparisons reveal variations in these shared structural topologies and other features. Unlike 263K and wildtype RML prions, the anchorless RML prions lack glycophosphatidylinositol anchors and are severely deficient in N-linked glycans. Nonetheless, the similarity of our anchorless RML structure to one reported for wildtype RML prion fibrils in an accompanying paper indicates that these post-translational modifications do not substantially alter the amyloid core conformation. This work demonstrates both common and divergent structural features of prion strains at the near-atomic level.

Accumulation of misfolded host proteins is central to neuropathogenesis of numerous human brain diseases including prion and prion-like diseases. Neurons of retina are also affected by these diseases. Previously, our group and others found that prion-induced retinal damage to photoreceptor cells in mice and humans resembled pathology of human retinitis pigmentosa caused by mutations in retinal proteins. Here, using confocal, epifluorescent and electron microscopy we followed deposition of disease-associated prion protein (PrPSc) and its association with damage to critical retinal structures following intracerebral prion inoculation. The earliest time and place of retinal PrPSc deposition was 67 days post-inoculation (dpi) on the inner segment (IS) of cone photoreceptors. At 104 and 118 dpi, PrPSc was associated with the base of cilia and swollen cone inner segments, suggesting ciliopathy as a pathogenic mechanism. By 118 dpi, PrPSc was deposited in both rods and cones which showed rootlet damage in the IS, and photoreceptor cell death was indicated by thinning of the outer nuclear layer. In the outer plexiform layer (OPL) in uninfected mice, normal host PrP (PrPC) was mainly associated with cone bipolar cell processes, but in infected mice, at 118 dpi, PrPSc was detected on cone and rod bipolar cell dendrites extending into ribbon synapses. Loss of ribbon synapses in cone pedicles and rod spherules in the OPL was observed to precede destruction of most rods and cones over the next 2–3 weeks. However, bipolar cells and horizontal cells were less damaged, indicating high selectivity among neurons for injury by prions. PrPSc deposition in cone and rod inner segments and on the bipolar cell processes participating in ribbon synapses appear to be critical early events leading to damage and death of photoreceptors after prion infection. These mechanisms may also occur in human retinitis pigmentosa and prion-like diseases, such as AD.

Little is known about the structural basis of prion strains. Here we provide a high (3.0 Å) resolution cryo-electron microscopy-based structure of brain-derived fibrils of the mouse anchorless RML scrapie strain which, like the recently determined hamster 263K strain, has a parallel in-register β-sheet-based core. However, detailed comparisons reveal that variations in shared structural motifs provide a basis for prion strain determination.One-sentence summaryCryo-electron microscopy reveals a near-atomic structure of an infectious, brain-derived murine prion fibril and strain differences.

Classical mammalian prions are assemblies of prion protein molecules that are extraordinarily transmissible, with a microgram of protein containing up to 108 lethal doses of infectivity1,2. Unlike most other pathologic and amyloidogenic proteins, prions typically contain glycolipid anchors 3 and abundant asparagine‐linked glycans4‐6. The infectious nature, complexity, and biophysical properties of prions have complicated structural analyses and stymied any prior elucidation of 3D conformation at the polypeptide backbone level7. Here we have determined the structure of the core of a fully infectious, brain‐derived prion by cryo‐electron microscopy with ∼3.1 Å resolution. The purified prions are amyloid fibrils comprised of monomers assembled with parallel in‐register intermolecular beta sheets and connecting chains. Residues ∼95‐227 of each monomer provide one rung of the ordered fibril core, with the glycans and glycolipid anchor projecting from the lateral surfaces of the fibril. The fibril ends, where prion growth occurs, are formed by single monomers in an extended serpentine combination of β‐ arches, a Greek key, and loops that presumably template the refolding of incoming monomers. Our results describe an atomic model to underpin detailed molecular hypotheses of how pathologic prion proteins can propagate as infectious agents, and how such propagation and associated pathogenesis might be impeded.

Dysregulated inflammation dominated by chemokine expression is a key feature of disease following infection with the globally important human pathogens, Zika virus (ZIKV) and dengue virus, but a mechanistic understanding of how pro-inflammatory responses are initiated is lacking. Mitophagy is a quality control mechanism that regulates innate immune signaling and cytokine production through selective degradation of damaged mitochondria. Here, we demonstrate that ZIKV NS5 antagonizes mitophagy by binding to the host protein Ajuba and preventing its translocation to depolarized mitochondria where it is required for PINK1 activation and downstream signaling. Consequent mitophagy suppression amplified the production of pro-inflammatory chemokines through PKR sensing of mitochondrial RNA. In Ajuba−/− mice, ZIKV induced early expression of pro-inflammatory chemokines associated with significantly enhanced dissemination to tissues. This work identifies Ajuba as a critical regulator of mitophagy, and demonstrates a role for mitophagy in limiting systemic inflammation following infection by globally important human viruses.

Dysregulated inflammation dominated by chemokine expression is a key feature of disease following infection with the globally important human pathogens, Zika virus (ZIKV) and dengue virus, but a mechanistic understanding of how pro-inflammatory responses are initiated is lacking. Mitophagy is a quality control mechanism that regulates innate immune signaling and cytokine production through selective degradation of damaged mitochondria. Here, we demonstrate that ZIKV NS5 antagonizes mitophagy by binding to the host protein Ajuba and preventing its translocation to depolarized mitochondria where it is required for PINK1 activation and downstream signaling. Consequent mitophagy suppression amplified the production of pro-inflammatory chemokines through PKR sensing of mitochondrial RNA. In Ajuba -/- mice, ZIKV induced early expression of pro-inflammatory chemokines associated with significantly enhanced dissemination to tissues. This work identifies Ajuba as a critical regulator of mitophagy, and demonstrates a role for mitophagy in limiting systemic inflammation following infection by globally important human viruses.

Leptospires and other members of the evolutionarily ancient phylum of Spirochaetes are bacteria often characterized by long, highly motile spiral- or wave-shaped cells. Morphology and motility are critical factors in spirochete physiology, contributing to the ability of these bacteria to successfully colonize diverse environments. However, the mechanisms conferring the helical structure of Leptospira spp. have yet to be fully elucidated. We have identified five Leptospira biflexa bactofilin proteins, a recently characterized protein family with cytoskeletal properties. These five bactofilins are conserved in all species of the Leptospiraceae, indicating that these proteins arose early in the evolution of this family. One member of this protein family, LbbD, confers the optimal pitch distance in the helical structure of L. biflexa. Mutants lacking lbbD display a unique compressed helical morphology, a reduced motility and a decreased ability to tolerate cell wall stressors. The change in the helical spacing, combined with the motility and cell wall integrity defects, showcases the intimate relationship and coevolution between shape and motility in these spirochetes.

Eukaryotic flagella are complex cellular extensions involved in many human diseases gathered under the term ciliopathies. Currently, detailed insights on flagellar structure come mostly from studies on protists. Here, cryo-electron tomography (cryo-ET) was performed on intact human spermatozoon tails and showed a variable number of microtubules in the singlet region (inside the end-piece). Inside the microtubule plus end, a novel left-handed interrupted helix which extends several micrometers was discovered. This structure was named Tail Axoneme Intra-Lumenal Spiral (TAILS) and binds directly to 11 protofilaments on the internal microtubule wall, in a coaxial fashion with the surrounding microtubule lattice. It leaves a gap over the microtubule seam, which was directly visualized in both singlet and doublet microtubules. We speculate that TAILS may stabilize microtubules, enable rapid swimming or play a role in controlling the swimming direction of spermatozoa.

Within the extensive range of self-propagating pathologic protein aggregates of mammals, prions are the most clearly infectious (e.g., ∼109 lethal doses per milligram). The structures of such lethal assemblies of PrP molecules have been poorly understood. Here we report a near-atomic core structure of a brain-derived, fully infectious prion (263K strain). Cryo-electron microscopy showed amyloid fibrils assembled with parallel in-register intermolecular β sheets. Each monomer provides one rung of the ordered fibril core, with N-linked glycans and glycolipid anchors projecting outward. Thus, single monomers form the templating surface for incoming monomers at fibril ends, where prion growth occurs. Comparison to another prion strain (aRML) revealed major differences in fibril morphology but, like 263K, an asymmetric fibril cross-section without paired protofilaments. These findings provide structural insights into prion propagation, strains, species barriers, and membrane pathogenesis. This structure also helps frame considerations of factors influencing the relative transmissibility of other pathologic amyloids.

Pseudomonas moraviensis stanleyae was recently isolated from the roots of the selenium (Se) hyperaccumulator plant Stanleya pinnata. This bacterium tolerates normally lethal concentrations of SeO3(2-) in liquid culture, where it also produces Se nanoparticles. Structure and cellular ultrastructure of the Se nanoparticles as determined by cellular electron tomography shows the nanoparticles as intracellular, of narrow dispersity, symmetrically irregular and without any observable membrane or structured protein shell. Protein mass spectrometry of a fractionated soluble cytosolic material with selenite reducing capability identified nitrite reductase and glutathione reductase homologues as NADPH dependent candidate enzymes for the reduction of selenite to zerovalent Se nanoparticles. In vitro experiments with commercially sourced glutathione reductase revealed that the enzyme can reduce SeO3(2-) (selenite) to Se nanoparticles in an NADPH-dependent process. The disappearance of the enzyme as determined by protein assay during nanoparticle formation suggests that glutathione reductase is associated with or possibly entombed in the nanoparticles whose formation it catalyzes. Chemically dissolving the nanoparticles releases the enzyme. The size of the nanoparticles varies with SeO3(2-) concentration, varying in size form 5 nm diameter when formed at 1.0 μM [SeO3(2-)] to 50 nm maximum diameter when formed at 100 μM [SeO3(2-)]. In aggregate, we suggest that glutathione reductase possesses the key attributes of a clonable nanoparticle system: ion reduction, nanoparticle retention and size control of the nanoparticle at the enzyme site.

Varicella-zoster virus (VZV) is highly cell associated when grown in culture and has a much higher (4,000- to 20,000-fold increased) particle-to-PFU ratio in vitro than herpes simplex virus (HSV). In contrast, VZV is highly infectious in vivo by airborne transmission. Neurons are major targets for VZV in vivo ; in neurons, the virus can establish latency and reactivate to produce infectious virus. Using neurons derived from human embryonic stem cells (hESC) and cell-free wild-type (WT) VZV, we demonstrated that neurons are nearly 100 times more permissive for WT VZV infection than very-early-passage human embryonic lung cells or MRC-5 diploid human fibroblasts, the cells used for vaccine production or virus isolation. The peak titers achieved after infection were ∼10-fold higher in human neurons than in MRC-5 cells, and the viral genome copy number-to-PFU ratio for VZV in human neurons was 500, compared with 50,000 for MRC-5 cells. Thus, VZV may not necessarily have a higher particle-to-PFU ratio than other herpesviruses; instead, the cells previously used to propagate virus in vitro may have been suboptimal. Furthermore, based on electron microscopy, neurons infected with VZV produced fewer defective or incomplete viral particles than MRC-5 cells. Our data suggest that neurons derived from hESC may have advantages compared to other cells for studies of VZV pathogenesis, for obtaining stocks of virus with high titers, and for isolating VZV from clinical specimens. IMPORTANCE Varicella-zoster virus (VZV) causes chickenpox and shingles. Cell-free VZV has been difficult to obtain, both for in vitro studies and for vaccine production. While numerous cells lines have been tested for their ability to produce high titers of VZV, the number of total virus particles relative to the number of viral particles that can form plaques in culture has been reported to be extremely high relative to that in other viruses. We show that VZV grows to much higher titers in human neurons than in other cell types in vitro and that the number of total virus genomes relative to the number of viral particles that can form plaques in culture is much lower in human neurons than other cultured cells. These findings indicate that human neurons may be useful for studying VZV in vitro , for growing preparations of virus with high titers, and for isolating the virus from human samples.

The long-standing inability to visualize connections between poxvirus membranes and cellular organelles has led to uncertainty regarding the origin of the viral membrane. Indeed, there has been speculation that viral membranes form de novo in cytoplasmic factories. Another possibility, that the connections are too short-lived to be captured by microscopy during a normal infection, motivated us to identify and characterize virus mutants that are arrested in assembly. Five conserved vaccinia virus proteins, referred to as Viral Membrane Assembly Proteins (VMAPs), that are necessary for formation of immature virions were found. Transmission electron microscopy studies of two VMAP deletion mutants had suggested retention of connections between viral membranes and the endoplasmic reticulum (ER). We now analyzed cells infected with each of the five VMAP deletion mutants by electron tomography, which is necessary to validate membrane continuity, in addition to conventional transmission electron microscopy. In all cases, connections between the ER and viral membranes were demonstrated by 3D reconstructions, supporting a role for the VMAPs in creating and/or stabilizing membrane scissions. Furthermore, coexpression of the viral reticulon-like transmembrane protein A17 and the capsid-like scaffold protein D13 was sufficient to form similar ER-associated viral structures in the absence of other major virion proteins. Determination of the mechanism of ER disruption during a normal VACV infection and the likely participation of both viral and cell proteins in this process may provide important insights into membrane dynamics.

Eukaryotic flagella are complex cellular extensions involved in many human diseases gathered under the term ciliopathies. Currently, detailed insights on flagellar structure come mostly from studies on protists. Here, cryo-electron tomography (cryo-ET) was performed on intact human spermatozoon tails and showed a variable number of microtubules in the singlet region (inside the end-piece). Inside the microtubule plus end, a novel left-handed interrupted helix which extends several micrometers was discovered. This structure was named Tail Axoneme Intra-Lumenal Spiral (TAILS) and binds directly to 11 protofilaments on the internal microtubule wall, in a coaxial fashion with the surrounding microtubule lattice. It leaves a gap over the microtubule seam, which was directly visualized in both singlet and doublet microtubules. We speculate that TAILS may stabilize microtubules, enable rapid swimming or play a role in controlling the swimming direction of spermatozoa.

Influenza A virus haemagglutinin conformational change drives the membrane fusion of viral and endosomal membranes at low pH. Membrane fusion proceeds through an intermediate called hemifusion(1,2). For viral fusion, the hemifusion structures are not determined(3). Here, influenza virus-like particles(4) carrying wild-type haemagglutinin or haemagglutinin hemifusion mutant G1S(5) and liposome mixtures were studied at low pH by Volta phase plate cryo-electron tomography, which improves the signal-to-noise ratio close to focus. We determined two distinct hemifusion structures: a hemifusion diaphragm and a novel structure termed a 'lipidic junction'. Liposomes with lipidic junctions were ruptured with membrane edges stabilized by haemagglutinin. The rupture frequency and hemifusion diaphragm diameter were not affected by G1S mutation, but decreased when the cholesterol level in the liposomes was close to physiological concentrations. We propose that haemagglutinin induces a merger between the viral and target membranes by one of two independent pathways: a rupture-insertion pathway leading to the lipidic junction and a hemifusion-stalk pathway leading to a fusion pore. The latter is relevant under the conditions of influenza virus infection of cells. Cholesterol concentration functions as a pathway switch because of its negative spontaneous curvature in the target bilayer, as determined by continuum analysis.

Giardia lamblia is a protistan parasite that infects and colonizes the small intestine of mammals. It is a widespread and particularly endemic in the developing world. Here we present a detailed structural study by 3-D negative staining and cryo-electron tomography of a unique Giardia organelle, the ventral disc. The disc is composed of a regular array of microtubules and associated sheets, called microribbons that form a large spiral, held together by a myriad of mostly unknown associated proteins. In a previous study we analyzed by cryo-electron tomography the central microtubule portion (here called disc body) of the ventral disc and found a large portion of microtubule associated inner (MIPs) and outer proteins (MAPs) that render these microtubules hyper-stable. With this follow-up study we expanded our 3-D analysis to different parts of the disc such as the ventral and dorsal areas of the overlap zone, as well as the outer disc margin. There are intrinsic location-specific characteristics in the composition of microtubule-associated proteins between these regions, as well as large differences between the overall architecture of microtubules and microribbons. The lateral packing of microtubule-microribbon complexes varies substantially, and closer packing often comes with contracted lateral tethers that seem to hold the disc together. It appears that the marginal microtubule-microribbon complexes function as outer, laterally contractible lids that may help the cell to clamp onto the intestinal microvilli. Furthermore, we analyzed length, quantity, curvature and distribution between different zones of the disc, which we found to differ from previous publications.

The resolution of cryo-electron tomography can be limited by the first zero of the microscope’s contrast transfer function (CTF). To achieve higher resolution, it is critical to determine the CTF and correct its phase inversions. However, the extremely low signal-to-noise ratio (SNR) and the defocus gradient in the projections of tilted specimens make this process challenging. Two programs, CTFPLOTTER and CTFPHASEFLIP, have been developed to address these issues. CTFPLOTTER obtains a 1D power spectrum by periodogram averaging and rotational averaging and it estimates the noise background with a novel approach, which uses images taken with no specimen. The background-subtracted 1D power spectra from image regions at different defocus values are then shifted to align their first zeros and averaged together. This averaging improves the SNR sufficiently that it becomes possible to determine the defocus for subsets of the tilt series rather than just the entire series. CTFPHASEFLIP corrects images line-by-line by inverting phases appropriately in thin strips of the image at nearly constant defocus. CTF correction by these methods is shown to improve the resolution of aligned, averaged particles extracted from tomograms. However, some restoration of Fourier amplitudes at high frequencies is important for seeing the benefits from CTF correction.

SummaryPolarized cells, such as neuronal, epithelial, and fungal cells, all display a specialized organization of their microtubules (MTs). The interphase MT cytoskeleton of the rod-shaped fission yeast, Schizosaccharomyces pombe, has been extensively described by fluorescence microscopy. Here, we describe a large-scale, electron tomography investigation of S. pombe, including a 3D reconstruction of a complete eukaryotic cell volume at sufficient resolution to show both how many MTs there are in a bundle and their detailed architecture. Most cytoplasmic MTs are open at one end and capped at the other, providing evidence about their polarity. Electron-dense bridges between the MTs themselves and between MTs and the nuclear envelope were frequently observed. Finally, we have investigated structure/function relationships between MTs and both mitochondria and vesicles. Our analysis shows that electron tomography of well-preserved cells is ideally suited for describing fine ultrastructural details that were not visible with previous techniques.

Many proposed biomedical applications for engineered gold nanoparticles require their incorporation by mammalian cells in specific numbers and locations. Here, the number of gold nanoparticles inside of individual mammalian stem cells was characterized using fast focused ion beam-scanning electron microscopy based tomography. Enhanced optical microscopy was used to provide a multiscale map of the in vitro sample, which allows cells of interest to be identified within their local environment. Cells were then serially sectioned using a gallium ion beam and imaged using a scanning electron beam. To confirm the accuracy of single cross sections, nanoparticles in similar cross sections were imaged using transmission electron microscopy and scanning helium ion microscopy. Complete tomographic series were then used to count the nanoparticles inside of each cell and measure their spatial distribution. We investigated the influence of slice thickness on counting single particles and clusters as well as nanoparticle packing within clusters. For 60 nm citrate stabilized particles, the nanoparticle cluster packing volume is 2.15 ± 0.20 times the volume of the bare gold nanoparticles.

We have developed a model system in Caenorhabditis elegans to perform genetic and molecular analysis of peptidergic neurotransmission using green fluorescent protein (GFP)-tagged IDA-1. IDA-1 represents the nematode ortholog of the transmembrane proteins ICA512 and phogrin that are localized to dense core secretory vesicles (DCVs) of mammalian neuroendocrine tissues. IDA-1::GFP was expressed in a small subset of neurons and present in both axonal and dendritic extensions, where it was localized to small mobile vesicular elements that at the ultrastructural level corresponded to 50 nm electron-dense objects in the neuronal processes. The post-translational processing of IDA-1::GFP in transgenic worms was dependent on the neuropeptide proprotein convertase EGL-3, indicating that the protein was efficiently targeted to the peptidergic secretory pathway. Time-lapse epifluorescence microscopy of IDA-1::GFP revealed that DCVs moved in a saltatory and bidirectional manner. DCV velocity profiles exhibited multiple distinct peaks, suggesting the participation of multiple molecular motors with distinct properties. Differences between velocity profiles for axonal and dendritic processes furthermore suggested a polarized distribution of the molecular transport machinery. Study of a number of candidate mutants identified the kinesin UNC-104 (KIF1A) as the microtubule motor that is specifically responsible for anterograde axonal transport of DCVs at velocities of 1.6 μm/s−2.7 μm/s.

Enveloped viruses feature metastable fusion proteins, which upon their conformational change transfer configurative energy into membrane fusion. To overcome the initial and substantial energetic barrier due to hydration repulsion of phospholipids, membrane fusion is thought to proceed through a low energy intermediate called hemifusion; yet for viral fusion the hemifusion structure has never been characterized. Variations of membrane curvature theory predict hemifusion diaphragms of varying dimensions. We study the hemifusion intermediate catalyzed by wild-type and G1S hemifusion mutant influenza hemagglutinin (HA) incorporated into virus-like particles (VLP) using Volta phase-plate cryo-electron tomography. We found “Y” shaped lipidic junctions with liposomal membrane inserted into the VLP membrane as suggested previously (Lee K. K., EMBO, 2010). Lipidic junctions and hemifusion diaphragms (HD) were typical membrane structures in both mutated and wild-type HA mediated fusion. Dimensions of HD and lipidic junctions ranged widely with their variance controlled by the rigidity imposed by the matrix layer, in agreement with our continuum mechanics model. We observed HA in close proximity to free membrane edges of ruptured liposomal membrane and lipidic junctions, suggesting that HA is capable of rupture, stabilization and resealing of membrane compartments in a step-wise manner permitting HD formation by lateral merging of lipidic junctions. Unambiguous determination of a lipidic junction consisting of the edge of a liposomal bilayer merged in the outer leaflet of a VLP opens the possibility of an alternate HA-mediated membrane fusion pathway.Lee, K. K. Architecture of a nascent viral fusion pore. The EMBO journal 29, 1299-1311, http://dx.doi.org/10.1038/emboj.2010.13 (2010).

A comprehensive, cross-species electron tomography analysis of kinetochore–microtubule interfaces has provided insight into shared structural features and their likely functional consequences., Interfaces between spindle microtubules and kinetochores were examined in diverse species by electron tomography and image analysis. Overall structures were conserved in a mammal, an alga, a nematode, and two kinds of yeasts; all lacked dense outer plates, and most kinetochore microtubule ends flared into curved protofilaments that were connected to chromatin by slender fibrils. Analyses of curvature on >8,500 protofilaments showed that all classes of spindle microtubules displayed some flaring protofilaments, including those growing in the anaphase interzone. Curved protofilaments on anaphase kinetochore microtubules were no more flared than their metaphase counterparts, but they were longer. Flaring protofilaments in budding yeasts were linked by fibrils to densities that resembled nucleosomes; these are probably the yeast kinetochores. Analogous densities in fission yeast were larger and less well-defined, but both yeasts showed ring- or partial ring-shaped structures girding their kinetochore microtubules. Flaring protofilaments linked to chromatin are well placed to exert force on chromosomes, assuring stable attachment and reliable anaphase segregation.

Giardia lamblia is a flagellated, unicellular parasite of mammals infecting over one billion people worldwide. Giardia's two-stage life cycle includes a motile trophozoite stage that colonizes the host small intestine and an infectious cyst form that can persist in the environment. Similar to many eukaryotic cells, Giardia contains several complex microtubule arrays that are involved in motility, chromosome segregation, organelle transport, maintenance of cell shape and transformation between the two life cycle stages. Giardia trophozoites also possess a unique spiral microtubule array, the ventral disc, made of approximately 50 parallel microtubules and associated microribbons, as well as a variety of associated proteins. The ventral disc maintains trophozoite attachment to the host intestinal epithelium. With the help of a combined SEM/microtome based slice and view method called 3View® (Gatan Inc., Pleasanton, CA), we present an entire trophozoite cell reconstruction and describe the arrangement of the major cytoskeletal elements. To aid in future analyses of disc-mediated attachment, we used electron-tomography of freeze-substituted, plastic-embedded trophozoites to explore the detailed architecture of ventral disc microtubules and their associated components. Lastly, we examined the disc microtubule array in three dimensions in unprecedented detail using cryo-electron tomography combined with internal sub-tomogram volume averaging of repetitive domains. We discovered details of protein complexes stabilizing microtubules by attachment to their inner and outer wall. A unique tri-laminar microribbon structure is attached vertically to the disc microtubules and is connected to neighboring microribbons via crossbridges. This work provides novel insight into the structure of the ventral disc microtubules, microribbons and associated proteins. Knowledge of the components comprising these structures and their three-dimensional organization is crucial toward understanding how attachment via the ventral disc occurs in vivo.

Giardia intestinalis is a ubiquitous parasitic protist that is the causative agent of giardiasis, one of the most common protozoan diarrheal diseases in the world. Giardia trophozoites attach to the intestinal epithelium using a specialized and elaborate microtubule structure, the ventral disc. Surrounding the ventral disc is a less characterized putatively contractile structure, the lateral crest, which forms a continuous perimeter seal with the substrate. A better understanding of ventral disc and lateral crest structure, conformational dynamics, and biogenesis is critical for understanding the mechanism of giardial attachment to the host. To determine the components comprising the ventral disc and lateral crest, we used shotgun proteomics to identify proteins in a preparation of isolated ventral discs. Candidate disc-associated proteins, or DAPs, were GFP-tagged using a ligation-independent high-throughput cloning method. Based on disc localization, we identified eighteen novel DAPs, which more than doubles the number of known disc-associated proteins. Ten of the novel DAPs are associated with the lateral crest or outer edge of the disc, and are the first confirmed components of this structure. Using Fluorescence Recovery After Photobleaching (FRAP) with representative novel DAP::GFP strains we found that the newly identified DAPs tested did not recover after photobleaching and are therefore structural components of the ventral disc or lateral crest. Functional analyses of the novel DAPs will be central toward understanding the mechanism of ventral disc-mediated attachment and the mechanism of disc biogenesis during cell division. Since attachment of Giardia to the intestine via the ventral disc is essential for pathogenesis, it is possible that some proteins comprising the disc could be potential drug targets if their loss or disruption interfered with disc biogenesis or function, preventing attachment., Author Summary Giardia is a unicellular intestinal parasite that infects millions of people worldwide each year. Colonization of the small intestine is a critical stage in Giardia infection. Giardia colonizes the intestinal wall using a specialized suction cup-like structure, the ventral disc. Little is known about the protein composition of the disc or about how the disc functions during attachment. We identified and confirmed eighteen new ventral disc proteins in a preparation of isolated discs using modern genomic methods for analyzing protein composition. We imaged these disc proteins in Giardia cells by labeling the proteins with fluorescent tags. A number of these proteins were present on the rim of the ventral disc, a region that appears necessary for the disc to form a seal during attachment to the host. These new ventral disc proteins form the building blocks of the ventral disc structure. Future studies of the roles of the ventral disc proteins either in the assembly of the ventral disc during cell division, or in the functioning of the disc during attachment will enable a better understanding of Giardia's colonization of the host.

Light and electron cryo-microscopy have each proven to be powerful tools to study biological structures in a near-native state. Light microscopy provides important localization information, while electron microscopy provides the resolution necessary to resolve fine structural details. Imaging the same sample by both light and electron cryo-microscopy is a powerful new approach that combines the strengths of both techniques to provide novel insights into cellular ultrastructure. In this chapter, the methods and instrumentation currently used to correlate light and electron cryo-microscopy are described in detail.

Spore formation in Saccharomyces cerevisiae is driven by de novo assembly of new membranes termed prospore membranes. A vesicle-docking complex called the meiosis II outer plaque (MOP) forms on the cytoplasmic faces of the spindle-pole bodies at the onset of meiosis II and serves as the initiation site for membrane formation. In this study, a fluorescence-recovery assay was used to demonstrate that the dynamics of the MOP proteins change coincident with the coalescence of precursor vesicles into a membrane. Proteins within the MOP exchange freely with a soluble pool prior to membrane assembly, but after membranes are formed they remain stably within the MOP. By contrast, constitutive spindle-pole-body proteins display low exchange in both conditions. The MOP component Ady4p plays a role in maintaining the integrity of the MOP complex, but this role differs depending on whether the MOP is associated with docked vesicles or a fully formed membrane. These results suggest an architectural rearrangement of the MOP coincident with vesicle fusion.

Light and electron cryo-microscopy have each proven to be powerful tools to study biological structures in a near-native state. Light microscopy provides important localization information, while electron microscopy provides the resolution necessary to resolve fine structural details. Imaging the same sample by both light and electron cryo-microscopy is a powerful new approach that combines the strengths of both techniques to provide novel insights into cellular ultrastructure. In this chapter, the methods and instrumentation currently used to correlate light and electron cryo-microscopy are described in detail.

As a long-term goal we want to use cryoelectron tomography to understand how non-enveloped viruses, such as picornaviruses, enter cells and translocate their genomes across membranes. To this end, we developed new image-processing tools using an in vitro system to model viral interactions with membranes. The complex of poliovirus with its membrane-bound receptors was reconstructed by averaging multiple sub-tomograms, thereby producing three-dimensional maps of surprisingly high-resolution (30 A). Recognizable images of the complex could be produced by averaging as few as 20 copies. Additionally, model-free reconstructions of free poliovirus particles, clearly showing the major surface features, could be calculated from 60 virions. All calculations were designed to avoid artifacts caused by missing information typical for tomographic data ("missing wedge"). To investigate structural and conformational variability we applied a principal component analysis classification to specific regions. We show that the missing wedge causes a bias in classification, and that this bias can be minimized by supplementation with data from the Fourier transform of the averaged structure. After classifying images of the receptor into groups with high similarity, we were able to see differences in receptor density consistent with the known variability in receptor glycosylation.

Summary Fluorescence light microscopy (LM) has many advantages for the study of cell organization. Specimen preparation is easy and relatively inexpensive, and the use of appropriate tags gives scientists the ability to visualize specific proteins of interest. LM is, however, limited in resolution, so when one is interested in ultrastructure, one must turn to electron microscopy (EM), even though this method presents problems of its own. The biggest difficulty with cellular EM is its limited utility in localizing macromolecules of interest while retaining good structural preservation. We have built a cryo-light microscope stage that allows us to generate LM images of vitreous samples prepared for cryo-EM. Correlative LM and EM allows one to find areas of particular interest by using fluorescent proteins or vital dyes as markers within vitrified samples. Once located, the sample can be placed in the EM for further study at higher resolution. An additional benefit of the cryo-LM stage is that photobleaching is slower at cryogenic temperatures (−140°C) than at room temperature.

Many organisms divide chromosomes within the confines of the nuclear envelope (NE) in a process known as closed mitosis. Thus, they must ensure coordination between segregation of the genetic material and division of the NE itself. Although many years of work have led to a reasonably clear understanding of mitotic spindle function in chromosome segregation, the NE division mechanism remains obscure. Here, we show that fission yeast cells overexpressing the transforming acid coiled coil (TACC)-related protein, Mia1p/Alp7p, failed to separate the spindle pole bodies (SPBs) at the onset of mitosis, but could assemble acentrosomal bipolar and antiparallel spindle structures. Most of these cells arrested in anaphase with fully extended spindles and nonsegregated chromosomes. Spindle poles that lacked the SPBs did not lead the division of the NE during spindle elongation, but deformed it, trapping the chromosomes within. When the SPBs were severed by laser microsurgery in wild-type cells, we observed analogous deformations of the NE by elongating spindle remnants, resulting in NE division failure. Analysis of dis1Δ cells that elongate spindles despite unattached kinetochores indicated that the SPBs were required for maintaining nuclear shape at anaphase onset. Strikingly, when the NE was disassembled by utilizing a temperature-sensitive allele of the Ran GEF, Pim1p, the abnormal spindles induced by Mia1p overexpression were capable of segregating sister chromatids to daughter cells, suggesting that the failure to divide the NE prevents chromosome partitioning. Our results imply that the SPBs preclude deformation of the NE during spindle elongation and thus serve as specialized structures enabling nuclear division during closed mitosis in fission yeast., Author Summary Chromosomes replicate in the nucleus, which is surrounded by the double-layered nuclear envelope (NE). During so-called open mitosis, the NE breaks down to allow spindle microtubules (which do the work of pulling the replicated chromosomes apart) access to the nuclear interior. Alternatively, in closed mitosis, many species assemble spindles inside the nucleus and segregate daughter genomes within the NE. To see how cells coordinate chromosome segregation with NE division in closed mitosis, we looked at the fission yeast Schizosaccharomyces pombe in which spindle poles are anchored at the spindle pole bodies (SPBs), specialized organelles inserted into the NE. We used two experimental manipulations to cause cells to assemble unusual spindles without the proper pole-located SPBs. These spindles deformed the NE and failed to segregate chromosomes. However, spindles lacking the SPBs were capable of segregating chromosomes if the NE was disassembled. We hypothesize that the SPBs have evolved as specialized structures preventing NE deformation during spindle elongation during closed mitosis., Spindle pole bodies allow nuclear division during mitosis, when there is no nuclear envelope breakdown in fission yeast.

Eukaryotic flagella and cilia are built on a 9 + 2 array of microtubules plus >250 accessory proteins, forming a biological machine called the axoneme. Here we describe the three-dimensional structure of rapidly frozen axonemes from Chlamydomonas and sea urchin sperm, using cryoelectron tomography and image processing to focus on the motor enzyme dynein. Our images suggest a model for the way dynein generates force to slide microtubules. They also reveal two dynein linkers that may provide “hard-wiring” to coordinate motor enzyme action, both circumferentially and along the axoneme. Periodic densities were also observed inside doublet microtubules; these may contribute to doublet stability.

Microtubule-organizing centers (MTOCs) concentrate microtubule nucleation, attachment and bundling factors and thus restrict formation of microtubule arrays in spatial and temporal manner. How MTOCs occur remains an exciting question in cell biology. Here, we show that the transforming acidic coiled coil–related protein Mia1p/Alp7p functions in emergence of large MTOCs in interphase fission yeast cells. We found that Mia1p was a microtubule-binding protein that preferentially localized to the minus ends of microtubules and was associated with the sites of microtubule attachment to the nuclear envelope. Cells lacking Mia1p exhibited less microtubule bundles. Microtubules could be nucleated and bundled but were frequently released from the nucleation sites in mia1Δ cells. Mia1p was required for stability of microtubule bundles and persistent use of nucleation sites both in interphase and postanaphase array dynamics. The γ-tubulin–rich material was not organized in large perinuclear or microtubule-associated structures in mia1Δ cells. Interestingly, absence of microtubules in dividing wild-type cells prevented appearance of large γ-tubulin–rich MTOC structures in daughters. When microtubule polymerization was allowed, MTOCs were efficiently assembled de novo. We propose a model where MTOC emergence is a self-organizing process requiring the continuous association of microtubules with nucleation sites.

Spore formation in Saccharomyces cerevisiae requires the de novo formation of prospore membranes. The coalescence of secretory vesicles into a membrane sheet occurs on the cytoplasmic surface of the spindle pole body. Spo14p, the major yeast phospholipase D, is necessary for prospore membrane formation; however, the specific function of Spo14p in this process has not been elucidated. We report that loss of Spo14p blocks vesicle fusion, leading to the accumulation of prospore membrane precursor vesicles docked on the spindle pole body. A similar phenotype was seen when the t-SNARE Sso1p, or the partially redundant t-SNAREs Sec9p and Spo20p were mutated. Although phosphatidic acid, the product of phospholipase D action, was necessary to recruit Spo20p to the precursor vesicles, independent targeting of Spo20p to the membrane was not sufficient to promote fusion in the absence of SPO14. These results demonstrate a role for phospholipase D in vesicle fusion and suggest that phospholipase D-generated phosphatidic acid plays multiple roles in the fusion process.

The fission yeast spindle pole body (SPB) is a nucleus-associated organelle that duplicates once each cell cycle during interphase. Duplicated SPBs serve as the poles of an intranuclear mitotic spindle after their insertion into the nuclear envelope in mitosis (Ding et al., Mol. Biol. Cell 8, 1461–1479). Here, we report the identification and characterization of Schizosaccharomyces pombe cdc31p, a member of the conserved calcium-binding centrin/CDC31 family. Immunofluorescence and immunoelectron microscopy show that cdc31p is a SPB component localized at the half-bridge structure of the SPB. cdc31 is an essential gene and Δcdc31 cells and cdc31 conditional mutant cells arrest in mitosis with a monopolar mitotic spindle organized from a single SPB. EM analysis demonstrates that mutant cdc31 cells fail to duplicate the SPB. In addition, cdc31p exhibits genetic interactions with the SPB component sad1p and is required for sad1p localization. Finally, cdc31 mutant can undergo single or multiple rounds of septation before the exit from mitosis, suggesting that cdc31p activity or SPB duplication may be required for the proper coordination between the exit from mitosis and the initiation of septation.

Spore formation in Saccharomyces cerevisiae occurs via the de novo synthesis of the prospore membrane during the second meiotic division. Prospore membrane formation is triggered by assembly of a membrane-organizing center, the meiotic outer plaque (MOP), on the cytoplasmic face of the spindle pole body (SPB) during meiosis. We report here the identification of two new components of the MOP, Ady4p and Spo74p. Ady4p and Spo74p interact with known proteins of the MOP and are localized to the outer plaque of the SPB during meiosis II. MOP assembly and prospore membrane formation are abolished in spo74 Δ/ spo74 Δ cells and occur aberrantly in ady4 Δ/ ady4 Δ cells. Spo74p and the MOP component Mpc70p are mutually dependent for recruitment to SPBs during meiosis. In contrast, both Ady4p and Spo74p are present at SPBs, albeit at reduced levels, in cells that lack the MOP component Mpc54p. Our findings suggest a model for the assembled MOP in which Mpc54p, Mpc70p, and Spo74p make up a core structural unit of the scaffold that initiates synthesis of the prospore membrane, and Ady4p is an auxiliary component that stabilizes the plaque.

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008