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.

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.

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.

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.

Charting the native architecture of 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.

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.

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 , 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.

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.

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

The green alga 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 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.

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.

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.

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 membranes. Conserved in eukaryotes, retromer controls the cellular localization and homeostasis of hundreds of transmembrane proteins, and its disruption is associated with major neurodegenerative disorders.

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.

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.

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.

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 , we observed that nuclear 26S proteasomes crowd around NPCs.

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 reconstitution systems using purified components. Previously we have determined a complete structural model of the reconstituted COPI coat (Dodonova et al.

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

Approximately 30%-40% of global CO 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 CO-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.

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.

Determining the bacterial cell biology of Planctomycetes.

Bacteria of the phylum Planctomycetes have been previously reported to possess several features that are typical of eukaryotes, such as cytosolic compartmentalization and endocytosis-like macromolecule uptake. However, recent evidence points towards a Gram-negative cell plan for Planctomycetes, although in-depth experimental analysis has been hampered by insufficient genetic tools. Here we develop methods for expression of fluorescent proteins and for gene deletion in a model planctomycete, Planctopirus limnophila, to analyse its cell organization in detail.

Dissecting the molecular organization of the translocon-associated protein complex.

In eukaryotic cells, one-third of all proteins must be transported across or inserted into the endoplasmic reticulum (ER) membrane by the ER protein translocon. The translocon-associated protein (TRAP) complex is an integral component of the translocon, assisting the Sec61 protein-conducting channel by regulating signal sequence and transmembrane helix insertion in a substrate-dependent manner. Here we use cryo-electron tomography (CET) to study the structure of the native translocon in evolutionarily divergent organisms and disease-linked TRAP mutant fibroblasts from human patients.

Cryo-electron microscopy of an extremely halophilic microbe: technical aspects.

Most halophilic Archaea of the class Halobacteriaceae depend on the presence of several molar sodium chloride for growth and cell integrity. This poses problems for structural studies, particularly for electron microscopy, where the high salt concentration results in diminished contrast. Since cryo-electron microscopy of intact cells provides new insights into the cellular and molecular organization under close-to-live conditions, we evaluated strategies and conditions to make halophilic microbes available for investigations in situ.

Optimized cryo-focused ion beam sample preparation aimed at in situ structural studies of membrane proteins.

While cryo-electron tomography (cryo-ET) can reveal biological structures in their native state within the cellular environment, it requires the production of high-quality frozen-hydrated sections that are thinner than 300nm. Sample requirements are even more stringent for the visualization of membrane-bound protein complexes within dense cellular regions. Focused ion beam (FIB) sample preparation for transmission electron microscopy (TEM) is a well-established technique in material science, but there are only few examples of biological samples exhibiting sufficient quality for high-resolution in situ investigation by cryo-ET.

Cryo-focused Ion Beam Sample Preparation for Imaging Vitreous Cells by Cryo-electron Tomography.

Cryo-electron tomography (CET) is a well-established technique for imaging cellular and molecular structures at sub-nanometer resolution. As the method is limited to samples that are thinner than 500 nm, suitable sample preparation is required to attain CET data from larger cell volumes. Recently, cryo-focused ion beam (cryo-FIB) milling of plunge-frozen biological material has been shown to reproducibly yield large, homogeneously thin, distortion-free vitreous cross-sections for state-of-the-art CET.