The demand for high-throughput data collection in electron microscopy is increasing for applications in structural and cellular biology. Here we present a combination of software tools that enable ...automated acquisition guided by image analysis for a variety of transmission electron microscopy acquisition schemes. SerialEM controls microscopes and detectors and can trigger automated tasks at multiple positions with high flexibility. Py-EM interfaces with SerialEM to enact specimen-specific image-analysis pipelines that enable feedback microscopy. As example applications, we demonstrate dose reduction in cryo-electron microscopy experiments, fully automated acquisition of every cell in a plastic section and automated targeting on serial sections for 3D volume imaging across multiple grids.
A global concern has emerged with the pandemic spread of Zika virus (ZIKV) infections that can cause severe neurological symptoms in adults and newborns. ZIKV is a positive-strand RNA virus ...replicating in virus-induced membranous replication factories (RFs). Here we used various imaging techniques to investigate the ultrastructural details of ZIKV RFs and their relationship with host cell organelles. Analyses of human hepatic cells and neural progenitor cells infected with ZIKV revealed endoplasmic reticulum (ER) membrane invaginations containing pore-like openings toward the cytosol, reminiscent to RFs in Dengue virus-infected cells. Both the MR766 African strain and the H/PF/2013 Asian strain, the latter linked to neurological diseases, induce RFs of similar architecture. Importantly, ZIKV infection causes a drastic reorganization of microtubules and intermediate filaments forming cage-like structures surrounding the viral RF. Consistently, ZIKV replication is suppressed by cytoskeleton-targeting drugs. Thus, ZIKV RFs are tightly linked to rearrangements of the host cell cytoskeleton.
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•ZIKV induces ER membrane invaginations similar to Dengue virus•ZIKV induces profound alterations of the cytoskeleton•Microtubules and intermediate filaments surround the ZIKV replication factory•ZIKV replication is sensitive to cytoskeleton-targeting drugs
Cortese et al. show that ZIKV infection in both human hepatoma and neuronal progenitor cells induces drastic structural modification of the cellular architecture. Microtubules and intermediate filaments surround the viral replication factory composed of vesicles corresponding to ER membrane invagination toward the ER lumen. Importantly, alteration of microtubule flexibility impairs ZIKV replication.
The nuclear envelope has to be reformed after mitosis to create viable daughter cells with closed nuclei. How membrane sealing of DNA and assembly of nuclear pore complexes (NPCs) are achieved and ...coordinated is poorly understood. Here, we reconstructed nuclear membrane topology and the structures of assembling NPCs in a correlative 3D EM time course of dividing human cells. Our quantitative ultrastructural analysis shows that nuclear membranes form from highly fenestrated ER sheets whose holes progressively shrink. NPC precursors are found in small membrane holes and dilate radially during assembly of the inner ring complex, forming thousands of transport channels within minutes. This mechanism is fundamentally different from that of interphase NPC assembly and explains how mitotic cells can rapidly establish a closed nuclear compartment while making it transport competent.
During clathrin-mediated endocytosis (CME), plasma membrane regions are internalized to retrieve extracellular molecules and cell surface components. Whether endocytosis occurs by direct clathrin ...assembly into curved lattices on the budding vesicle or by initial recruitment to flat membranes and subsequent reshaping has been controversial. To distinguish between these models, we combined fluorescence microscopy and electron tomography to locate endocytic sites and to determine their coat and membrane shapes during invagination. The curvature of the clathrin coat increased, whereas the coated surface area remained nearly constant. Furthermore, clathrin rapidly exchanged at all stages of CME. Thus, coated vesicle budding appears to involve bending of a dynamic preassembled clathrin coat.
Extracellular vesicles (EVs) are produced by all known organisms and are important for cell communication and physiology. Great morphological diversity has been described regarding EVs found in body ...fluids such as blood plasma, breast milk, and ejaculate. However, a detailed morphological analysis has never been performed on exosomes when purified from a single cell type.
In this study we analysed and quantified, via multiple electron microscopy techniques, the morphology of exosomes purified from the human mast cell line HMC-1. The results revealed a wide diversity in exosome morphology, suggesting that subpopulations of exosomes with different and specific functions may exist. Our findings imply that a new, more efficient way of defining exosome subpopulations is necessary. A system was proposed where exosomes were classified into nine different categories according to their size and shape. Three additional morphological features were also found in exosomes regardless of their morphological classification.
These findings show that exosomes purified from a single cell line are also morphologically diverse, similar to previous observations for EVs in body fluids. This knowledge can help to improve the interpretation of experimental results and widen our general understanding of the biological functions of exosomes.
The nuclear pore complex (NPC) mediates nucleocytoplasmic transport through the nuclear envelope. How the NPC assembles into this double membrane boundary has remained enigmatic. Here, we captured ...temporally staged assembly intermediates by correlating live cell imaging with high-resolution electron tomography and super-resolution microscopy. Intermediates were dome-shaped evaginations of the inner nuclear membrane (INM), that grew in diameter and depth until they fused with the flat outer nuclear membrane. Live and super-resolved fluorescence microscopy revealed the molecular maturation of the intermediates, which initially contained the nuclear and cytoplasmic ring component Nup107, and only later the cytoplasmic filament component Nup358. EM particle averaging showed that the evagination base was surrounded by an 8-fold rotationally symmetric ring structure from the beginning and that a growing mushroom-shaped density was continuously associated with the deforming membrane. Quantitative structural analysis revealed that interphase NPC assembly proceeds by an asymmetric inside-out extrusion of the INM.
Correlative light and electron microscopy allows features of interest defined by fluorescence signals to be located in an electron micrograph of the same sample. Rare dynamic events or specific ...objects can be identified, targeted and imaged by electron microscopy or tomography. To combine it with structural studies using cryo-electron microscopy or tomography, fluorescence microscopy must be performed while maintaining the specimen vitrified at liquid-nitrogen temperatures and in a dry environment during imaging and transfer. Here we present instrumentation, software and an experimental workflow that improves the ease of use, throughput and performance of correlated cryo-fluorescence and cryo-electron microscopy. The new cryo-stage incorporates a specially modified high-numerical aperture objective lens and provides a stable and clean imaging environment. It is combined with a transfer shuttle for contamination-free loading of the specimen. Optimized microscope control software allows automated acquisition of the entire specimen area by cryo-fluorescence microscopy. The software also facilitates direct transfer of the fluorescence image and associated coordinates to the cryo-electron microscope for subsequent fluorescence-guided automated imaging. Here we describe these technological developments and present a detailed workflow, which we applied for automated cryo-electron microscopy and tomography of various specimens.
The assembly of HIV-1 is mediated by oligomerization of the major structural polyprotein, Gag, into a hexameric protein lattice at the plasma membrane of the infected cell. This leads to budding and ...release of progeny immature virus particles. Subsequent proteolytic cleavage of Gag triggers rearrangement of the particles to form mature infectious virions. Obtaining a structural model of the assembled lattice of Gag within immature virus particles is necessary to understand the interactions that mediate assembly of HIV-1 particles in the infected cell, and to describe the substrate that is subsequently cleaved by the viral protease. An 8-Å resolution structure of an immature virus-like tubular array assembled from a Gag-derived protein of the related retrovirus Mason–Pfizer monkey virus (M-PMV) has previously been reported, and a model for the arrangement of the HIV-1 capsid (CA) domains has been generated based on homology to this structure. Here we have assembled tubular arrays of a HIV-1 Gag-derived protein with an immature-like arrangement of the C-terminal CA domains and have solved their structure by using hybrid cryo-EM and tomography analysis. The structure reveals the arrangement of the C-terminal domain of CA within an immature-like HIV-1 Gag lattice, and provides, to our knowledge, the first high-resolution view of the region immediately downstream of CA, which is essential for assembly, and is significantly different from the respective region in M-PMV. Our results reveal a hollow column of density for this region in HIV-1 that is compatible with the presence of a six-helix bundle at this position.
Electron microscopy is the gold standard to characterize cellular ultrastructure. However, production of significant morphometrical data is highly limited by acquisition time. Here, we describe a ...semi-automated high-throughput strategy using single-axis serial section electron tomography to investigate and analyze centriole ultrastructure in bone-marrow-derived, primary human CD138pos plasma cells. The protocol comprises steps for electron microscopy sample preparation, semi-automated transmission electron microscopy screening, and screening evaluation for cells of interest. Thereafter, we detail tomography acquisition, data reconstruction, and joining.
For complete details on the use and execution of this protocol, please refer to Dittrich et al.1
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•High-throughput production of volumetric EM data•From sample acquisition to tomography data in one week•Protocol can be used in both cell lines and primary patient material•Protocol is adaptable to various organelles and subcellular features
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Electron microscopy is the gold standard to characterize cellular ultrastructure. However, production of significant morphometrical data is highly limited by acquisition time. Here, we describe a semi-automated high-throughput strategy using single-axis serial section electron tomography to investigate and analyze centriole ultrastructure in bone-marrow-derived, primary human CD138pos plasma cells. The protocol comprises steps for EM sample preparation, semi-automated transmission electron microscopy screening, and screening evaluation for cells of interest. Thereafter, we detail tomography acquisition, data reconstruction, and joining.
Microtubules are key to multiple neuronal functions involving the transport of organelles, however, their relationship to neurotransmitter release is still unresolved. Here, we show that microtubules ...present in the presynaptic compartment of cholinergic autaptic synapses are dynamic. To investigate how the balance between microtubule growth and shrinkage affects neurotransmission we induced synchronous microtubule depolymerization by photoactivation of the chemical inhibitor SBTub3. The consequence was an increase in spontaneous neurotransmitter release. An analogous effect was obtained by dialyzing the cytosol with Kif18A, a plus-end-directed kinesin with microtubule depolymerizing activity. Kif18A also inhibited the refilling of the readily releasable pool of synaptic vesicles during high frequency stimulation. The action of Kif18A was associated to one order of magnitude increases in the numbers of exo-endocytic pits and endosomes present in the presynaptic terminal. An enhancement of spontaneous neurotransmitter release was also observed when neurons were dialyzed with stathmin-1, a protein with a widespread presence in the nervous system that induces microtubule depolymerization. Taken together, these results support that microtubules restrict spontaneous neurotransmitter release as well as promote the replenishment of the readily releasable pool of synaptic vesicles.
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