Membrane contact sites (MCS) between the endoplasmic reticulum (ER) and the plasma membrane (PM) play fundamental roles in all eukaryotic cells. ER-PM MCS are particularly abundant in Saccharomyces ...cerevisiae, where approximately half of the PM surface is covered by cortical ER (cER). Several proteins, including Ist2, Scs2/22, and Tcb1/2/3 are implicated in cER formation, but the specific roles of these molecules are poorly understood. Here, we use cryo-electron tomography to show that ER-PM tethers are key determinants of cER morphology. Notably, Tcb proteins (tricalbins) form peaks of extreme curvature on the cER membrane facing the PM. Combined modeling and functional assays suggest that Tcb-mediated cER peaks facilitate the transport of lipids between the cER and the PM, which is necessary to maintain PM integrity under heat stress. ER peaks were also present at other MCS, implying that membrane curvature enforcement may be a widespread mechanism to regulate MCS function.
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•Tethers of ER-plasma membrane (PM) contact sites shape cortical ER (cER) morphology•Tricalbins create peaks of extreme curvature on the cER membrane facing the PM•cER peaks are important to maintain PM integrity under heat stress•cER peaks may facilitate ER-to-PM lipid transport
Using cryo-electron tomography, Collado et al. show that tricalbins generate peaks of extreme curvature on the cortical ER (cER) membrane at ER-plasma membrane (PM) contact sites. Functional assays and theoretical modeling indicate that cER peaks are important to maintain PM integrity under heat stress, possibly by facilitating cER-to-PM lipid transport.
The 26S proteasome is at the executive end of the ubiquitin-proteasome pathway for the controlled degradation of intracellular proteins. While the structure of its 20S core particle (CP) has been ...determined by X-ray crystallography, the structure of the 19S regulatory particle (RP), which recruits substrates, unfolds them, and translocates them to the CP for degradation, has remained elusive. Here, we describe the molecular architecture of the 26S holocomplex determined by an integrative approach based on data from cryoelectron microscopy, X-ray crystallography, residue-specific chemical cross-linking, and several proteomics techniques. The "lid" of the RP (consisting of Rpn3/5/6/7/8/9/11/12) is organized in a modular fashion. Rpn3/5/6/7/9/12 form a horseshoe-shaped heterohexamer, which connects to the CP and roofs the AAA-ATPase module, positioning the Rpn8/Rpn11 heterodimer close to its mouth. Rpn2 is rigid, supporting the lid, while Rpn1 is conformationally variable, positioned at the periphery of the ATPase ring. The ubiquitin receptors Rpn10 and Rpn13 are located in the distal part of the RP, indicating that they were recruited to the complex late in its evolution. The modular structure of the 26S proteasome provides insights into the sequence of events prior to the degradation of ubiquitylated substrates.
In a 3D environment, motile cells accommodate their protruding and retracting activities to geometrical cues. Dictyostelium cells migrating on a perforated film explored its holes by forming actin ...rings around their border and extending protrusions through the free space. The response was initiated when an actin wave passed a hole, and the rings persisted only in the PIP3-rich territories surrounded by a wave. To reconstruct actin structures from cryo-electron tomograms, actin rings were identified by cryo-correlative light and electron microscopy, and thin wedges of relevant regions were obtained by cryo-focused ion-beam milling. Retracting stages were distinguished from protruding ones by the accumulation of myosin-II. Early actin rings consisted of filaments pointing upright from the membrane, entangled with a meshwork of filaments close to the membrane. Branches identified at later stages suggested that formin-based nucleation of filaments was followed by Arp2/3-mediated network stabilization, which prevented buckling of the force-generating filaments.
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•Migrating cells sense substrate curvature to assemble force-generating actin networks•Live imaging and cryo-electron tomography reveal network dynamics and architecture•Close-to-life network structures are visualized in the context of whole vitrified cells•Ion-beam milling and automated filament segmentation are key to structural analysis
Cells migrating on structured surfaces respond to geometrical cues by the assembly of force-generating actin networks. Jasnin et al. reveal their architecture by cryo-electron tomography, correlative light and electron microscopy, and focused ion-beam milling.
Cryogenic electron tomography (cryo-ET) is the application of tomographic principles of data acquisition and reconstruction to frozen-hydrated biological specimens. It combines a close-to-life ...preservation of cellular structures with the power of high-resolution three-dimensional imaging, which allows us to study the molecular architecture of cells, or their molecular sociology, in unprecedented detail.
Cryogenic electron tomography (cryo-ET) is the application of tomographic principles of data acquisition and reconstruction to frozen-hydrated biological specimens. It combines a close-to-life preservation of cellular structures with the power of high-resolution three-dimensional imaging, which allows us to study the molecular architecture of cells, or their molecular sociology, in unprecedented detail.
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.
Close-up view of transport train assembly
Cilia and flagella extend from the cell surface of various eukaryotic cells and perform diverse motility and signaling functions. The base of the cilium controls the entry of large intraflagellar transport trains that carry important cargo proteins throughout this specialized organelle. Defects in the ciliary base result in altered ciliary composition and human diseases. van den Hoek
et al
. used a combination of cryo–electron tomography and expansion microscopy techniques to study the molecular architecture of the ciliary base in the green alga
Chlamydomonas reinhardtii
. Their findings elucidate how intraflagellar transport trains assemble before they enter cilia and demonstrate the possibility of visualizing dynamic events with molecular resolution inside native cells. —SMH
Native molecular structure of the
Chlamydomonas
ciliary transition zone and intraflagellar transport chain assembly is described.
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.
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•Cryo-EM reveals how VIPP1 oligomerizes, hydrolyzes nucleotides, and binds lipids•Lipid binding by VIPP1’s H1 helix maintains thylakoid integrity under high-light stress•VIPP1 coats mediate contact between thylakoids and the chloroplast envelope•VIPP1 is the photosynthetic homolog of ESCRT-III membrane-remodeling proteins
Structure-function analysis and in situ visualization of VIPP1 provide insights into how this ESCRT-III homolog manipulates membranes to support thylakoid biogenesis and maintenance in cyanobacteria, algae, and plants.
Autophagosomes are unique organelles that form de novo as double-membrane vesicles engulfing cytosolic material for destruction. Their biogenesis involves membrane transformations of distinctly ...shaped intermediates whose ultrastructure is poorly understood. Here, we combine cell biology, correlative cryo-electron tomography (cryo-ET), and extensive data analysis to reveal the step-by-step structural progression of autophagosome biogenesis at high resolution directly within yeast cells. The analysis uncovers an unexpectedly thin intermembrane distance that is dilated at the phagophore rim. Mapping of individual autophagic structures onto a timeline based on geometric features reveals a dynamical change of membrane shape and curvature in growing phagophores. Moreover, our tomograms show the organelle interactome of growing autophagosomes, highlighting a polar organization of contact sites between the phagophore and organelles, such as the vacuole and the endoplasmic reticulum (ER). Collectively, these findings have important implications for the contribution of different membrane sources during autophagy and for the forces shaping and driving phagophores toward closure without a templating cargo.
Chloroplast function is orchestrated by the organelle's intricate architecture. By combining cryo-focused ion beam milling of vitreous Chlamydomonas cells with cryo-electron tomography, we acquired ...three-dimensional structures of the chloroplast in its native state within the cell. Chloroplast envelope inner membrane invaginations were frequently found in close association with thylakoid tips, and the tips of multiple thylakoid stacks converged at dynamic sites on the chloroplast envelope, implicating lipid transport in thylakoid biogenesis. Subtomogram averaging and nearest neighbor analysis revealed that RuBisCO complexes were hexagonally packed within the pyrenoid, with ~15 nm between their centers. Thylakoid stacks and the pyrenoid were connected by cylindrical pyrenoid tubules, physically bridging the sites of light-dependent photosynthesis and light-independent carbon fixation. Multiple parallel minitubules were bundled within each pyrenoid tubule, possibly serving as conduits for the targeted one-dimensional diffusion of small molecules such as ATP and sugars between the chloroplast stroma and the pyrenoid matrix.
Electron tomography (ET) is uniquely suited to obtain three-dimensional reconstructions of pleomorphic structures, such as cells, organelles or supramolecular assemblies. Although the principles of ...ET have been known for decades, its use has gathered momentum only in recent years, thanks to technological advances and its combination with improved specimen preparation techniques. The rapid freezing/freeze-substitution preparation is applicable to whole cells and tissues, and it is the method of choice for ET investigations of cellular ultrastructure. The frozen-hydrated preparation provides the best possible structural preservation and allows the imaging of molecules, complexes, and supramolecular assemblies in their native state and their natural environment. Devoid of staining and chemical fixation artifacts, cryo-ET provides a faithful representation of both the surface and internal structure of molecules. In combination with advanced computational methods, such as molecular identification based on pattern recognition techniques, cryo-ET is currently the most promising approach to comprehensively map macromolecular architecture inside cellular tomograms.
Celotno besedilo
Dostopno za:
CMK, DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cryoelectron tomography provides unprecedented insights into the macromolecular and supramolecular organization of cells in a close-to-living state. However because of the limited thickness range ...(< 0.5–1 μm) that is accessible with today’s intermediate voltage electron microscopes only small prokaryotic cells or peripheral regions of eukaryotic cells can be examined directly. Key to overcoming this limitation is the ability to prepare sufficiently thin samples. Cryosectioning can be used to prepare thin enough sections but suffers from severe artefacts, such as substantial compression. Here we describe a procedure, based upon focused ion beam (FIB) milling for the preparation of thin (200–500 nm) lamellae from vitrified cells grown on electron microscopy (EM) grids. The self-supporting lamellae are apparently free of distortions or other artefacts and open up large windows into the cell’s interior allowing tomographic studies to be performed on any chosen part of the cell. We illustrate the quality of sample preservation with a structure of the nuclear pore complex obtained from a single tomogram.
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