In situ structure and priming mechanism of the rhoptry secretion system in Plasmodium revealed by cryo-electron tomography

Apicomplexan parasites secrete the contents of rhoptries into host cells to permit their invasion and establishment of an infectious niche. The rhoptry secretory apparatus (RSA), which is critical for rhoptry secretion, was recently discovered in Toxoplasma and Cryptosporidium. It is positioned at the cell apex and associates with an enigmatic apical vesicle (AV), which docks one or two rhoptries at the site of exocytosis. The interplay among the rhoptries, the AV, and the parasite plasma membrane for secretion remains unclear. Moreover, it is unknown if a similar machinery exists in the deadly malaria parasite Plasmodium falciparum. In this study, we use in situ cryo-electron tomography to investigate the rhoptry secretion system in P. falciparum merozoites. We identify the presence of an RSA at the cell apex and a morphologically distinct AV docking the tips of the two rhoptries to the RSA. We also discover two new organizations: one in which the AV is absent with one of the two rhoptry tips docks directly to the RSA, and a second in which the two rhoptries fuse together and the common tip docks directly to the RSA. Interestingly, rhoptries among the three states show no significant difference in luminal volume and density, suggesting that the exocytosis of rhoptry contents has not yet occurred, and that these different organizations likely represent sequential states leading to secretion. Using subtomogram averaging, we reveal different conformations of the RSA structure corresponding to each state, including the opening of a gate-like density in the rhoptry-fused state. These conformational changes of the RSA uncover structural details of a priming process for major rhoptry secretion, which likely occur after initial interaction with a red blood cell. Our results highlight a previously unknown step in the process of rhoptry secretion and indicate a regulatory role for the conserved apical vesicle in host invasion by apicomplexan parasites.

Adeno-associated Virus Receptor-binding: Flexible Domains and Alternative Conformations through cryo-Electron Tomography of AAV2 and AAV5 complexes

Recombinant forms of adeno-associated virus (rAAV) are vectors of choice in the development of treatments for a number of genetic dispositions. Greater understanding of AAV's molecular virology is needed to underpin needed improvements in efficiency and specificity. Recent advances have included identification of a near universal entry receptor, AAVR, and structures by cryo-electron microscopy (EM) single particle analysis (SPA) that revealed, at high resolution, only the domains of AAVR most tightly bound to AAV. Here, cryogenic electron tomography (cryo-ET) is applied to reveal the neighboring domains of the flexible receptor. For AAV5, where the PKD1 domain is bound strongly, PKD2 is seen in three configurations extending away from the virus. AAV2 binds tightly to the PKD2 domain at a distinct site, and cryo-ET now reveals four configurations of PKD1, all different from that seen in AAV5. The AAV2 receptor complex also shows unmodeled features on the inner surface that appear to be an equilibrium alternate configuration. Other AAV structures start near the 5-fold axis, but now β-strand A is the minor conformer and, for the major conformer, partially ordered N-termini near the 2-fold axis join the canonical capsid jellyroll fold at the βA-βB turn. The addition of cryo-ET is revealing unappreciated complexity that is likely relevant to viral entry and to the development of improved gene therapy vectors. IMPORTANCE: With 150 clinical trials for 30 diseases underway, AAV is a leading gene therapy vector. Immunotoxicity at high doses used to overcome inefficient transduction, has occasionally proven fatal and highlighted gaps in fundamental virology. AAV enters cells, interacting through distinct sites with different domains of the AAVR receptor, according to AAV clade. Single domains are resolved in structures by cryogenic electron microscopy. Here, the adjoining domains are revealed by cryo-electron tomography of AAV2 and AAV5 complexes. They are in flexible configurations interacting minimally with AAV, despite measurable dependence of AAV2 transduction on both domains.

UVC inactivation of pathogenic samples suitable for cryo-EM analysis

Cryo-electron microscopy has become an essential tool to understand structure and function of biological samples. Especially for pathogens, such as disease-causing bacteria and viruses, insights gained by cryo-EM can aid in developing cures. However, due to the biosafety restrictions of pathogens, samples are often treated by chemical fixation to render the pathogen inert, affecting the ultrastructure of the sample. Alternatively, researchers use in vitro or ex vivo models, which are non-pathogenic but lack the complexity of the pathogen of interest. Here we show that ultraviolet-C (UVC) radiation applied at cryogenic temperatures can be used to eliminate or dramatically reduce the infectivity of Vibrio cholerae and the bacterial virus, the ICP1 bacteriophage. We show no discernable structural impact of this treatment of either sample using two cryo-EM methods: cryo-electron tomography followed by sub-tomogram averaging, and single particle analysis (SPA). Additionally, we applied the UVC irradiation to the protein apoferritin (ApoF), which is a widely used test sample for high-resolution SPA studies. The UVC-treated ApoF sample resulted in a 2.1 Å structure indistinguishable from an untreated published map. This research demonstrates that UVC treatment is an effective and inexpensive addition to the cryo-EM sample preparation toolbox.

Correlative cryogenic montage electron tomography for comprehensive in-situ whole-cell structural studies

Imaging large fields of view while preserving high-resolution structural information remains a challenge in low-dose cryo-electron tomography. Here, we present robust tools for montage electron tomography tailored for vitrified specimens. The integration of correlative cryo-fluorescence microscopy, focused-ion beam milling, and micropatterning produces contextual three-dimensional architecture of cells. Montage tilt series may be processed in their entirety or as individual tiles suitable for sub-tomogram averaging, enabling efficient data processing and analysis.

Microtubule plus-end regulation by centriolar cap proteins

Centrioles are microtubule-based organelles required for the formation of centrosomes and cilia. Centriolar microtubules, unlike their cytosolic counterparts, grow very slowly and are very stable. The complex of centriolar proteins CP110 and CEP97 forms a cap that stabilizes the distal centriole end and prevents its over-elongation. Here, we used in vitro reconstitution assays to show that whereas CEP97 does not interact with microtubules directly, CP110 specifically binds microtubule plus ends, potently blocks their growth and induces microtubule pausing. Cryo-electron tomography indicated that CP110 binds to the luminal side of microtubule plus ends and reduces protofilament peeling. Furthermore, CP110 directly interacts with another centriole biogenesis factor, CPAP/SAS-4, which tracks growing microtubule plus ends, slows down their growth and prevents catastrophes. CP110 and CPAP synergize in inhibiting plus-end growth, and this synergy depends on their direct binding. Together, our data reveal a molecular mechanism controlling centriolar microtubule plus-end dynamics and centriole biogenesis.

Amyloid-like aggregating proteins cause lysosomal defects in neurons via gain-of-function toxicity

The autophagy-lysosomal pathway is impaired in many neurodegenerative diseases characterized by protein aggregation, but the link between aggregation and lysosomal dysfunction remains poorly understood. Here, we combine cryo-electron tomography, proteomics, and cell biology studies to investigate the effects of protein aggregates in primary neurons. We use artificial amyloid-like β-sheet proteins (β proteins) to focus on the gain-of-function aspect of aggregation. These proteins form fibrillar aggregates and cause neurotoxicity. We show that late stages of autophagy are impaired by the aggregates, resulting in lysosomal alterations reminiscent of lysosomal storage disorders. Mechanistically, β proteins interact with and sequester AP-3 μ1, a subunit of the AP-3 adaptor complex involved in protein trafficking to lysosomal organelles. This leads to destabilization of the AP-3 complex, missorting of AP-3 cargo, and lysosomal defects. Restoring AP-3μ1 expression ameliorates neurotoxicity caused by β proteins. Altogether, our results highlight the link between protein aggregation, lysosomal impairments, and neurotoxicity.

Visualizing translation dynamics at atomic detail inside a bacterial cell

Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells. Here, we use cryo-electron tomography and sub-tomogram analysis to visualize the dynamics of translation inside the prokaryote Mycoplasma pneumoniae. We first obtain an in-cell atomic model for the M. pneumoniae ribosome that reveals distinct extensions of ribosomal proteins. Classification then resolves thirteen ribosome states that differ in conformation and composition and reflect intermediates during translation. Based on these states, we animate translation elongation and demonstrate how antibiotics reshape the translation landscape inside cells. During translation elongation, ribosomes often arrange in a defined manner to form polysomes. By mapping the intracellular three-dimensional organization of translating ribosomes, we show that their association into polysomes exerts a local coordination mechanism that is mediated by the ribosomal protein L9. Our work demonstrates the feasibility of visualizing molecular processes at atomic detail inside cells.

Nanoscale details of mitochondrial fission revealed by cryo-electron tomography

Mitochondrial fission is required for proper segregation during cell division, quality control, and cellular homeostasis (metabolism and energy production). Despite its importance, models of the process remain speculative. Here we apply cryogenic electron tomography to image the nanoscale architecture of mitochondrial fission in mammalian cells. We find that constriction of the inner and outer membranes is coordinated, suggesting that force on both membranes is applied externally. While we observe ER at constriction sites, it did not encircle constrictions. Instead, we find long bundles of both unbranched actin and septin filaments enriched at constrictions. Actin bundles align with the central region of division bridges and septin bundles with the necks on either side. Septin bundles appear to guide microtubules to constriction sites, suggesting, along with autolysosomes observed in the vicinity, a pathway for mitophagy. Together, our results rule out several existing models for mitochondrial fission and provide empirical parameters to inform the development of realistic coarse-grained models in the future.