In situ architecture of the ciliary base reveals the stepwise assembly of intraflagellar transport trains.

The cilium is an antenna-like organelle that performs numerous cellular functions, including motility, sensing, and signaling. The base of the cilium contains a selective barrier that regulates the entry of large intraflagellar transport (IFT) trains, which carry cargo proteins required for ciliary assembly and maintenance. However, the native architecture of the ciliary base and the process of IFT train assembly remain unresolved. In this work, we used in situ cryo-electron tomography to reveal native structures of the transition zone region and assembling IFT trains at the ciliary base in Chlamydomonas. We combined this direct cellular visualization with ultrastructure expansion microscopy to describe the front-to-back stepwise assembly of IFT trains: IFT-B forms the backbone, onto which bind IFT-A, dynein-1b, and finally kinesin-2 before entry into the cilium.

High-Yield Production, Characterization, and Functionalization of Recombinant Magnetosomes in the Synthetic Bacterium Rhodospirillum rubrum "magneticum".

Recently, the photosynthetic Rhodospirillum rubrum has been endowed with the ability of magnetosome biosynthesis by transfer and expression of biosynthetic gene clusters from the magnetotactic bacterium Magnetospirillum gryphiswaldense. However, the growth conditions for efficient magnetite biomineralization in the synthetic R. rubrum "magneticum", as well as the particles themselves (i.e., structure and composition), have so far not been fully characterized. In this study, different cultivation strategies, particularly the influence of temperature and light intensity, are systematically investigated to achieve optimal magnetosome biosynthesis. Reduced temperatures ≤16 °C and gradual increase in light intensities favor magnetite biomineralization at high rates, suggesting that magnetosome formation might utilize cellular processes, cofactors, and/or pathways that are linked to photosynthetic growth. Magnetosome yields of up to 13.6 mg magnetite per liter cell culture are obtained upon photoheterotrophic large-scale cultivation. Furthermore, it is shown that even more complex, i.e., oligomeric, catalytically active functional moieties like enzyme proteins can be efficiently expressed on the magnetosome surface, thereby enabling the in vivo functionalization by genetic engineering. In summary, it is demonstrated that the synthetic R. rubrum "magneticum" is a suitable host for high-yield magnetosome biosynthesis and the sustainable production of genetically engineered, bioconjugated magnetosomes.

Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity.

Vesicle-inducing protein in plastids 1 (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-remodeling functions. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket on one end of the ring. Inside the ring's lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Using cryo-correlative light and electron microscopy (cryo-CLEM), we observe oligomeric VIPP1 coats encapsulating membrane tubules within the Chlamydomonas chloroplast. Our work provides a structural foundation for understanding how VIPP1 directs thylakoid biogenesis and maintenance.

Architecture of the centriole cartwheel-containing region revealed by cryo-electron tomography.

Centrioles are evolutionarily conserved barrels of microtubule triplets that form the core of the centrosome and the base of the cilium. While the crucial role of the proximal region in centriole biogenesis has been well documented, its native architecture and evolutionary conservation remain relatively unexplored. Here, using cryo-electron tomography of centrioles from four evolutionarily distant species, we report on the architectural diversity of the centriole's proximal cartwheel-bearing region. Our work reveals that the cartwheel central hub is constructed from a stack of paired rings with cartwheel inner densities inside. In both Paramecium and Chlamydomonas, the repeating structural unit of the cartwheel has a periodicity of 25 nm and consists of three ring pairs, with 6 radial spokes emanating and merging into a single bundle that connects to the microtubule triplet via the D2-rod and the pinhead. Finally, we identified that the cartwheel is indirectly connected to the A-C linker through the triplet base structure extending from the pinhead. Together, our work provides unprecedented evolutionary insights into the architecture of the centriole proximal region, which underlies centriole biogenesis.

Preparing samples from whole cells using focused-ion-beam milling for cryo-electron tomography.

Recent advances have made cryogenic (cryo) electron microscopy a key technique to achieve near-atomic-resolution structures of biochemically isolated macromolecular complexes. Cryo-electron tomography (cryo-ET) can give unprecedented insight into these complexes in the context of their natural environment. However, the application of cryo-ET is limited to samples that are thinner than most cells, thereby considerably reducing its applicability. Cryo-focused-ion-beam (cryo-FIB) milling has been used to carve (micromachining) out 100-250-nm-thin regions (called lamella) in the intact frozen cells. This procedure opens a window into the cells for high-resolution cryo-ET and structure determination of biomolecules in their native environment. Further combination with fluorescence microscopy allows users to determine cells or regions of interest for the targeted fabrication of lamellae and cryo-ET imaging. Here, we describe how to prepare lamellae using a microscope equipped with both FIB and scanning electron microscopy modalities. Such microscopes (Aquilos Cryo-FIB/Scios/Helios or CrossBeam) are routinely referred to as dual-beam microscopes, and they are equipped with a cryo-stage for all operations in cryogenic conditions. The basic principle of the described methodologies is also applicable for other types of dual-beam microscopes equipped with a cryo-stage. We also briefly describe how to integrate fluorescence microscopy data for targeted milling and critical considerations for cryo-ET data acquisition of the lamellae. Users familiar with cryo-electron microscopy who get basic training in dual-beam microscopy can complete the protocol within 2-3 d, allowing for several pause points during the procedure.

Charting the native architecture of Chlamydomonas thylakoid membranes with single-molecule precision.

Thylakoid membranes scaffold an assortment of large protein complexes that work together to harness the energy of light. It has been a longstanding challenge to visualize how the intricate thylakoid network organizes these protein complexes to finely tune the photosynthetic reactions. Previously, we used in situ cryo-electron tomography to reveal the native architecture of thylakoid membranes (Engel et al., 2015). Here, we leverage technical advances to resolve the individual protein complexes within these membranes. Combined with a new method to visualize membrane surface topology, we map the molecular landscapes of thylakoid membranes inside green algae cells. Our tomograms provide insights into the molecular forces that drive thylakoid stacking and reveal that photosystems I and II are strictly segregated at the borders between appressed and non-appressed membrane domains. This new approach to charting thylakoid topology lays the foundation for dissecting photosynthetic regulation at the level of single protein complexes within the cell.

A helical inner scaffold provides a structural basis for centriole cohesion.

The ninefold radial arrangement of microtubule triplets (MTTs) is the hallmark of the centriole, a conserved organelle crucial for the formation of centrosomes and cilia. Although strong cohesion between MTTs is critical to resist forces applied by ciliary beating and the mitotic spindle, how the centriole maintains its structural integrity is not known. Using cryo-electron tomography and subtomogram averaging of centrioles from four evolutionarily distant species, we found that MTTs are bound together by a helical inner scaffold covering ~70% of the centriole length that maintains MTTs cohesion under compressive forces. Ultrastructure Expansion Microscopy (U-ExM) indicated that POC5, POC1B, FAM161A, and Centrin-2 localize to the scaffold structure along the inner wall of the centriole MTTs. Moreover, we established that these four proteins interact with each other to form a complex that binds microtubules. Together, our results provide a structural and molecular basis for centriole cohesion and geometry.

Direct visualization of degradation microcompartments at the ER membrane.

To promote the biochemical reactions of life, cells can compartmentalize molecular interaction partners together within separated non-membrane-bound regions. It is unknown whether this strategy is used to facilitate protein degradation at specific locations within the cell. Leveraging in situ cryo-electron tomography to image the native molecular landscape of the unicellular alga Chlamydomonas reinhardtii, we discovered that the cytosolic protein degradation machinery is concentrated within ∼200-nm foci that contact specialized patches of endoplasmic reticulum (ER) membrane away from the ER-Golgi interface. These non-membrane-bound microcompartments exclude ribosomes and consist of a core of densely clustered 26S proteasomes surrounded by a loose cloud of Cdc48. Active proteasomes in the microcompartments directly engage with putative substrate at the ER membrane, a function canonically assigned to Cdc48. Live-cell fluorescence microscopy revealed that the proteasome clusters are dynamic, with frequent assembly and fusion events. We propose that the microcompartments perform ER-associated degradation, colocalizing the degradation machinery at specific ER hot spots to enable efficient protein quality control.

In situ Microfluidic Cryofixation for Cryo Focused Ion Beam Milling and Cryo Electron Tomography.

We present a microfluidic platform for studying structure-function relationships at the cellular level by connecting video rate live cell imaging with in situ microfluidic cryofixation and cryo-electron tomography of near natively preserved, unstained specimens. Correlative light and electron microscopy (CLEM) has been limited by the time required to transfer live cells from the light microscope to dedicated cryofixation instruments, such as a plunge freezer or high-pressure freezer. We recently demonstrated a microfluidic based approach that enables sample cryofixation directly in the light microscope with millisecond time resolution, a speed improvement of up to three orders of magnitude. Here we show that this cryofixation method can be combined with cryo-electron tomography (cryo-ET) by using Focused Ion Beam milling at cryogenic temperatures (cryo-FIB) to prepare frozen hydrated electron transparent sections. To make cryo-FIB sectioning of rapidly frozen microfluidic channels achievable, we developed a sacrificial layer technique to fabricate microfluidic devices with a PDMS bottom wall <5 µm thick. We demonstrate the complete workflow by rapidly cryo-freezing Caenorhabditis elegans roundworms L1 larvae during live imaging in the light microscope, followed by cryo-FIB milling and lift out to produce thin, electron transparent sections for cryo-ET imaging. Cryo-ET analysis of initial results show that the structural preservation of the cryofixed C. elegans was suitable for high resolution cryo-ET work. The combination of cryofixation during live imaging enabled by microfluidic cryofixation with the molecular resolution capabilities of cryo-ET offers an exciting avenue to further advance space-time correlative light and electron microscopy (st-CLEM) for investigation of biological processes at high resolution in four dimensions.

The elusive actin cytoskeleton of a green alga expressing both conventional and divergent actins.

The green alga Chlamydomonas reinhardtii is a leading model system to study photosynthesis, cilia, and the generation of biological products. The cytoskeleton plays important roles in all of these cellular processes, but to date, the filamentous actin network within Chlamydomonas has remained elusive. By optimizing labeling conditions, we can now visualize distinct linear actin filaments at the posterior of the nucleus in both live and fixed vegetative cells. Using in situ cryo-electron tomography, we confirmed this localization by directly imaging actin filaments within the native cellular environment. The fluorescently labeled structures are sensitive to the depolymerizing agent latrunculin B (Lat B), demonstrating the specificity of our optimized labeling method. Interestingly, Lat B treatment resulted in the formation of a transient ring-like filamentous actin structure around the nucleus. The assembly of this perinuclear ring is dependent upon a second actin isoform, NAP1, which is strongly up-regulated upon Lat B treatment and is insensitive to Lat B-induced depolymerization. Our study combines orthogonal strategies to provide the first detailed visual characterization of filamentous actins in Chlamydomonas, allowing insights into the coordinated functions of two actin isoforms expressed within the same cell.

A cryo-FIB lift-out technique enables molecular-resolution cryo-ET within native Caenorhabditis elegans tissue.

Cryo-focused ion beam milling of frozen-hydrated cells has recently provided unprecedented insights into the inner space of cells. In combination with cryo-electron tomography, this method allows access to native structures deep inside cells, enabling structural studies of macromolecules in situ. However, this approach has been mainly limited to individual cells that can be completely vitrified by plunge-freezing. Here, we describe a preparation method that is based on the targeted extraction of material from high-pressure-frozen bulk specimens with a cryo-gripper tool. This lift-out technique enables cryo-electron tomography to be performed on multicellular organisms and tissue, extending the range of applications for in situ structural biology. We demonstrate the potential of the lift-out technique with a structural study of cytosolic 80S ribosomes in a Caenorhabditis elegans worm. The preparation quality allowed for subtomogram analysis with sufficient resolution to distinguish individual ribosomal translocation states and revealed significant cell-to-cell variation in ribosome structure.

Biogenic regions of cyanobacterial thylakoids form contact sites with the plasma membrane.

Little is known about how the photosynthetic machinery is arranged in time and space during the biogenesis of thylakoid membranes. Using in situ cryo-electron tomography to image the three-dimensional architecture of the cyanobacterium Synechocystis, we observed that the tips of multiple thylakoids merge to form a substructure called the 'convergence membrane'. This high-curvature membrane comes into close contact with the plasma membrane at discrete sites. We generated subtomogram averages of 70S ribosomes and array-forming phycobilisomes, then mapped these structures onto the native membrane architecture as markers for protein synthesis and photosynthesis, respectively. This molecular localization identified two distinct biogenic regions in the thylakoid network: thylakoids facing the cytosolic interior of the cell that were associated with both marker complexes, and convergence membranes that were decorated by ribosomes but not phycobilisomes. We propose that the convergence membranes perform a specialized biogenic function, coupling the synthesis of thylakoid proteins with the integration of cofactors from the plasma membrane and the periplasmic space.

Structure of the membrane-assembled retromer coat determined by cryo-electron tomography.

Eukaryotic cells traffic proteins and lipids between different compartments using protein-coated vesicles and tubules. The retromer complex is required to generate cargo-selective tubulovesicular carriers from endosomal membranes1-3. Conserved in eukaryotes, retromer controls the cellular localization and homeostasis of hundreds of transmembrane proteins, and its disruption is associated with major neurodegenerative disorders4-7. How retromer is assembled and how it is recruited to form coated tubules is not known. Here we describe the structure of the retromer complex (Vps26-Vps29-Vps35) assembled on membrane tubules with the bin/amphiphysin/rvs-domain-containing sorting nexin protein Vps5, using cryo-electron tomography and subtomogram averaging. This reveals a membrane-associated Vps5 array, from which arches of retromer extend away from the membrane surface. Vps35 forms the 'legs' of these arches, and Vps29 resides at the apex where it is free to interact with regulatory factors. The bases of the arches connect to each other and to Vps5 through Vps26, and the presence of the same arches on coated tubules within cells confirms their functional importance. Vps5 binds to Vps26 at a position analogous to the previously described cargo- and Snx3-binding site, which suggests the existence of distinct retromer-sorting nexin assemblies. The structure provides insight into the architecture of the coat and its mechanism of assembly, and suggests that retromer promotes tubule formation by directing the distribution of sorting nexin proteins on the membrane surface while providing a scaffold for regulatory-protein interactions.

In situ architecture of the algal nuclear pore complex.

Nuclear pore complexes (NPCs) span the nuclear envelope and mediate nucleocytoplasmic exchange. They are a hallmark of eukaryotes and deeply rooted in the evolutionary origin of cellular compartmentalization. NPCs have an elaborate architecture that has been well studied in vertebrates. Whether this architecture is unique or varies significantly in other eukaryotic kingdoms remains unknown, predominantly due to missing in situ structural data. Here, we report the architecture of the algal NPC from the early branching eukaryote Chlamydomonas reinhardtii and compare it to the human NPC. We find that the inner ring of the Chlamydomonas NPC has an unexpectedly large diameter, and the outer rings exhibit an asymmetric oligomeric state that has not been observed or predicted previously. Our study provides evidence that the NPC is subject to substantial structural variation between species. The divergent and conserved features of NPC architecture provide insights into the evolution of the nucleocytoplasmic transport machinery.

Pleomorphic linkers as ubiquitous structural organizers of vesicles in axons.

Many cellular processes depend on a precise structural organization of molecular components. Here, we established that neurons grown in culture provide a suitable system for in situ structural investigations of cellular structures by cryo-electron tomography, a method that allows high resolution, three-dimensional imaging of fully hydrated, vitrified cellular samples. A higher level of detail of cellular components present in our images allowed us to quantitatively characterize presynaptic and cytoskeletal organization, as well as structures involved in axonal transport and endocytosis. In this way we provide a structural framework into which information from other methods need to fit. Importantly, we show that short pleomorphic linkers (tethers and connectors) extensively interconnect different types of spherical vesicles and other lipid membranes in neurons imaged in a close-to-native state. These linkers likely serve to organize and precisely position vesicles involved in endocytosis, axonal transport and synaptic release. Hence, structural interactions via short linkers may serve as ubiquitous vesicle organizers in neuronal cells.

Molecular and structural architecture of polyQ aggregates in yeast.

Huntington's disease is caused by the expansion of a polyglutamine (polyQ) tract in the N-terminal exon of huntingtin (HttEx1), but the cellular mechanisms leading to neurodegeneration remain poorly understood. Here we present in situ structural studies by cryo-electron tomography of an established yeast model system of polyQ toxicity. We find that expression of polyQ-expanded HttEx1 results in the formation of unstructured inclusion bodies and in some cases fibrillar aggregates. This contrasts with recent findings in mammalian cells, where polyQ inclusions were exclusively fibrillar. In yeast, polyQ toxicity correlates with alterations in mitochondrial and lipid droplet morphology, which do not arise from physical interactions with inclusions or fibrils. Quantitative proteomic analysis shows that polyQ aggregates sequester numerous cellular proteins and cause a major change in proteome composition, most significantly in proteins related to energy metabolism. Thus, our data point to a multifaceted toxic gain-of-function of polyQ aggregates, driven by sequestration of endogenous proteins and mitochondrial and lipid droplet dysfunction.

Proteasomes tether to two distinct sites at the nuclear pore complex.

The partitioning of cellular components between the nucleus and cytoplasm is the defining feature of eukaryotic life. The nuclear pore complex (NPC) selectively gates the transport of macromolecules between these compartments, but it is unknown whether surveillance mechanisms exist to reinforce this function. By leveraging in situ cryo-electron tomography to image the native cellular environment of Chlamydomonas reinhardtii, we observed that nuclear 26S proteasomes crowd around NPCs. Through a combination of subtomogram averaging and nanometer-precision localization, we identified two classes of proteasomes tethered via their Rpn9 subunits to two specific NPC locations: binding sites on the NPC basket that reflect its eightfold symmetry and more abundant binding sites at the inner nuclear membrane that encircle the NPC. These basket-tethered and membrane-tethered proteasomes, which have similar substrate-processing state frequencies as proteasomes elsewhere in the cell, are ideally positioned to regulate transcription and perform quality control of both soluble and membrane proteins transiting the NPC.

The structure of the COPI coat determined within the cell.

COPI-coated vesicles mediate trafficking within the Golgi apparatus and from the Golgi to the endoplasmic reticulum. The structures of membrane protein coats, including COPI, have been extensively studied with in vitro reconstitution systems using purified components. Previously we have determined a complete structural model of the in vitro reconstituted COPI coat (Dodonova et al., 2017). Here, we applied cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to determine the native structure of the COPI coat within vitrified Chlamydomonas reinhardtii cells. The native algal structure resembles the in vitro mammalian structure, but additionally reveals cargo bound beneath ß'-COP. We find that all coat components disassemble simultaneously and relatively rapidly after budding. Structural analysis in situ, maintaining Golgi topology, shows that vesicles change their size, membrane thickness, and cargo content as they progress from cis to trans, but the structure of the coat machinery remains constant.

The Eukaryotic CO2-Concentrating Organelle Is Liquid-like and Exhibits Dynamic Reorganization.

Approximately 30%-40% of global CO2 fixation occurs inside a non-membrane-bound organelle called the pyrenoid, which is found within the chloroplasts of most eukaryotic algae. The pyrenoid matrix is densely packed with the CO2-fixing enzyme Rubisco and is thought to be a crystalline or amorphous solid. Here, we show that the pyrenoid matrix of the unicellular alga Chlamydomonas reinhardtii is not crystalline but behaves as a liquid that dissolves and condenses during cell division. Furthermore, we show that new pyrenoids are formed both by fission and de novo assembly. Our modeling predicts the existence of a "magic number" effect associated with special, highly stable heterocomplexes that influences phase separation in liquid-like organelles. This view of the pyrenoid matrix as a phase-separated compartment provides a paradigm for understanding its structure, biogenesis, and regulation. More broadly, our findings expand our understanding of the principles that govern the architecture and inheritance of liquid-like organelles.

Cryo-electron tomography-the cell biology that came in from the cold.

Cryo-electron tomography (cryo-ET) provides high-resolution 3D views into cells pristinely preserved by vitrification. Recent technical advances such as direct electron detectors, the Volta phase plate and cryo-focused ion beam milling have dramatically pushed image quality and expanded the range of cryo-ET applications. Cryo-ET not only allows mapping the positions and interactions of macromolecules within their intact cellular context, but can also reveal their in situ structure at increasing resolution. Here, we review how recent work using cutting-edge cryo-ET technologies is starting to provide fresh views into different aspects of cellular biology at an unprecedented level of detail. We anticipate that these developments will soon make cryo-ET a fundamental technique in cell biology.