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Techniques for Selective Labeling of Molecules and Subcellular Structures for Cryo-Electron Tomography
Abstract
Electron microscopy (EM) is one of the most efficient methods for studying the fine structure of cells with a resolution thousands of times higher than that of visible light microscopy. The most advanced implementation of electron microscopy in biology is EM tomography of samples stabilized by freezing without water crystallization (cryoET). By circumventing the drawbacks of chemical fixation and dehydration, this technique allows investigating cellular structures in three dimensions at the molecular level, down to resolving individual proteins and their subdomains. However, the problem of efficient identification and localization of objects of interest has not yet been solved, thus limiting the range of targets to easily recognizable or abundant subcellular components. Labeling techniques provide the only way for locating the subject of investigation in microscopic images. CryoET imposes conflicting demands on the labeling system, including the need to introduce into a living cell the particles composed of substances foreign to the cellular chemistry that have to bind to the molecule of interest without disrupting its vital functions and physiology of the cell. This review examines both established and prospective methods for selective labeling of proteins and subcellular structures aimed to enable their localization in cryoET images.
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For macromolecular structures determined by cryogenic electron microscopy, no technique currently exists for mapping elements to defined locations, leading to errors in the assignment of metals and other ions, cofactors, substrates, inhibitors and lipids that play essential roles in activity and regulation. Elemental mapping in the electron microscope is well established for dose-tolerant samples but is challenging for biological samples, especially in a cryo-preserved state. Here we combine electron energy-loss spectroscopy with single-particle image processing to allow elemental mapping in cryo-preserved macromolecular complexes. Proof-of-principle data show that our method, reconstructed electron energy-loss (REEL) analysis, allows a three-dimensional reconstruction of electron energy-loss spectroscopy data, such that a high total electron dose is accumulated across many copies of a complex. Working with two test samples, we demonstrate that we can reliably localize abundant elements. We discuss the current limitations of the method and potential future developments.
Cryo-electron tomography (cryoET) provides sub-nanometer protein structure within the dense cellular environment. Existing sample preparation methods are insufficient at accessing the plasma membrane and its associated proteins. Here, we present a correlative cryo-electron tomography pipeline optimally suited to image large ultra-thin areas of isolated basal and apical plasma membranes. The pipeline allows for angstrom-scale structure determination with sub-tomogram averaging and employs a genetically-encodable rapid chemically-induced electron microscopy visible tag for marking specific proteins within the complex cell environment. The pipeline provides fast, efficient, distributable, low-cost sample preparation and enables targeted structural studies of identified proteins at the plasma membrane of cells.
The detection of specific biological macromolecules in cryogenic electron tomography data is frequently approached by applying cross-correlation-based 3D template matching. To reduce computational cost and noise, high binning is used to aggregate voxels before template matching. This remains a prevalent practice in both practical applications and methods development. Here, the relation between template size, shape and angular sampling is systematically evaluated to identify ribosomes in a ground-truth annotated data set. It is shown that at the commonly used binning, a detailed subtomogram average, a sphere and a heart emoji result in near-identical performance. These findings indicate that with current template-matching practices macromolecules can only be detected with high precision if their shape and size are sufficiently different from the background. Using theoretical considerations, the experimental results are rationalized and it is discussed why primarily low-frequency information remains at high binning and that template matching fails to be accurate because similarly shaped and sized macromolecules have similar low-frequency spectra. These challenges are discussed and potential enhancements for future template-matching methodologies are proposed.
Cryo-electron tomography (cryo-ET) allows for label-free high-resolution imaging of macromolecular assemblies in their native cellular context. However, the localization of macromolecules of interest in tomographic volumes can be challenging. Here we present a ligand-inducible labeling strategy for intracellular proteins based on fluorescent, 25-nm-sized, genetically encoded multimeric particles (GEMs). The particles exhibit recognizable structural signatures, enabling their automated detection in cryo-ET data by convolutional neural networks. The coupling of GEMs to green fluorescent protein-tagged macromolecules of interest is triggered by addition of a small-molecule ligand, allowing for time-controlled labeling to minimize disturbance to native protein function. We demonstrate the applicability of GEMs for subcellular-level localization of endogenous and overexpressed proteins across different organelles in human cells using cryo-correlative fluorescence and cryo-ET imaging. We describe means for quantifying labeling specificity and efficiency, and for systematic optimization for rare and abundant protein targets, with emphasis on assessing the potential effects of labeling on protein function.
DNA molecules that store genetic information in living creatures can be repurposed as building blocks to construct artificial architectures, ranging from the nanoscale to the microscale. The precise fabrication of self‐assembled DNA nanomaterials and their various applications have greatly impacted nanoscience and nanotechnology. More specifically, the DNA origami technique has realized the assembly of various nanostructures featuring rationally predesigned geometries, precise addressability, and versatile programmability, as well as remarkable biocompatibility. These features have elevated DNA origami from academic interest to an emerging class of drug delivery platform for a wide range of diseases. In this minireview, the latest advances in the burgeoning field of DNA‐origami‐based innovative platforms for regulating biological functions and delivering versatile drugs are presented. Challenges regarding the novel drug vehicle's safety, stability, targeting strategy, and future clinical translation are also discussed.
While genetically encoded reporters are common for fluorescence microscopy, equivalent multiplexable gene reporters for electron microscopy (EM) are still scarce. Here, by installing a variable number of fixation-stable metal-interacting moieties in the lumen of encapsulin nanocompartments of different sizes, we developed a suite of spherically symmetric and concentric barcodes (EMcapsulins) that are readable by standard EM techniques. Six classes of EMcapsulins could be automatically segmented and differentiated. The coding capacity was further increased by arranging several EMcapsulins into distinct patterns via a set of rigid spacers of variable length. Fluorescent EMcapsulins were expressed to monitor subcellular structures in light and EM. Neuronal expression in Drosophila and mouse brains enabled the automatic identification of genetically defined cells in EM. EMcapsulins are compatible with transmission EM, scanning EM and focused ion beam scanning EM. The expandable palette of genetically controlled EM-readable barcodes can augment anatomical EM images with multiplexed gene expression maps.
Advances in cryo‐electron microscopy (EM) enable imaging of protein assemblies within mammalian cells in a near native state when samples are preserved by cryogenic vitrification. To accompany this progress, specialized EM labelling protocols must be developed. Gold nanoparticles (AuNPs) of 2 nm are synthesized and functionalized to bind selected intracellular targets inside living human cells and to be detected in vitreous sections. As a proof of concept, thioaminobenzoate‐, thionitrobenzoate‐coordinated gold nanoparticles are functionalized on their surface with SV40 Nuclear Localization Signal (NLS)‐containing peptides and 2 kDa polyethyleneglycols (PEG) by thiolate exchange to target the importin‐mediated nuclear machinery and facilitate cytosolic diffusion by shielding the AuNP surface from non‐specific binding to cell components, respectively. After delivery by electroporation into the cytoplasm of living human cells, the PEG‐coated AuNPs diffuse freely in the cytoplasm but do not enter the nucleus. Incorporation of NLS within the PEG coverage promotes a quick nuclear import of the nanoparticles in relation to the density of NLS onto the AuNPs. Cryo‐EM of vitreous cell sections demonstrate the presence of 2 nm AuNPs as single entities in the nucleus. Biofunctionalized AuNPs combined with live‐cell electroporation procedures are thus potent labeling tools for the identification of macromolecules in cellular cryo‐EM.
Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells¹. Here we use advances in cryo-electron tomography and sub-tomogram analysis2,3 to visualize the structural dynamics of translation inside the bacterium Mycoplasma pneumoniae. To interpret the functional states in detail, we first obtain a high-resolution in-cell average map of all translating ribosomes and build an atomic model for the M. pneumoniae ribosome that reveals distinct extensions of ribosomal proteins. Classification then resolves 13 ribosome states that differ in their conformation and composition. These recapitulate major states that were previously resolved in vitro, and reflect intermediates during active translation. On the basis of these states, we animate translation elongation inside native cells and show how antibiotics reshape the cellular translation landscapes. During translation elongation, ribosomes often assemble in defined three-dimensional arrangements to form polysomes⁴. By mapping the intracellular organization of translating ribosomes, we show that their association into polysomes involves a local coordination mechanism that is mediated by the ribosomal protein L9. We propose that an extended conformation of L9 within polysomes mitigates collisions to facilitate translation fidelity. Our work thus demonstrates the feasibility of visualizing molecular processes at atomic detail inside cells.
Rapid advances in cryo-electron tomography (cryo-ET) are driving a revolution in cellular structural biology. However, unambiguous identification of specific biomolecules within cellular tomograms remains challenging. Overcoming this obstacle and reliably identifying targets in the crowded cellular environment is of major importance for the understanding of cellular function and is a pre-requisite for high-resolution structural analysis. The use of highly-specific, readily visualised and adjustable labels would help mitigate this issue, improving both data quality and sample throughput. While progress has been made in cryo-CLEM and in the development of cloneable high-density tags, technical issues persist and a robust 'cryo-GFP' remains elusive. Readily-synthesized gold nanomaterials conjugated to small 'affinity modules' may represent a solution. The synthesis of materials including gold nanoclusters (AuNCs) and gold nanoparticles (AuNPs) is increasingly well understood and is now within the capabilities of non-specialist laboratories. The remarkable chemical and photophysical properties of <3nm diameter nanomaterials and their emergence as tools with widespread biomedical application presents significant opportunities to the cryo-microscopy community. In this review, we will outline developments in the synthesis, functionalisation and labelling uses of both AuNPs and AuNCs in cryo-ET, while discussing their potential as multi-modal probes for cryo-CLEM.
We present a bimodal endocytic tracer, fluorescent BSA-gold (fBSA-Au), as a fiducial marker for 2D and 3D correlative light and electron microscopy (CLEM) applications. fBSA-Au consists of colloidal gold (Au) particles stabilized with fluorescent BSA. The conjugate is efficiently endocytosed and distributed throughout the 3D endolysosomal network of cells and has an excellent visibility in both fluorescence microscopy (FM) and electron microscopy (EM). We demonstrate that fBSA-Au facilitates rapid registration in several 2D and 3D CLEM applications using Tokuyasu cryosections, resin-embedded material, and cryoelectron microscopy (cryo-EM). Endocytosed fBSA-Au benefits from a homogeneous 3D distribution throughout the endosomal system within the cell, does not obscure any cellular ultrastructure, and enables accurate (50–150 nm) correlation of fluorescence to EM data. The broad applicability and visibility in both modalities makes fBSA-Au an excellent endocytic fiducial marker for 2D and 3D (cryo)CLEM applications.
Electron cryotomography (cryoET), an electron cryomicroscopy (cryoEM) modality, has changed our understanding of biological function by revealing the native molecular details of membranes, viruses, and cells. However, identification of individual molecules within tomograms from cryoET is challenging because of sample crowding and low signal-to-noise ratios. Here, we present a tagging strategy for cryoET that precisely identifies individual protein complexes in tomograms without relying on metal clusters. Our method makes use of DNA origami to produce “molecular signposts” that target molecules of interest, here via fluorescent fusion proteins, providing a platform generally applicable to biological surfaces. We demonstrate the specificity of signpost origami tags (SPOTs) in vitro as well as their suitability for cryoET of membrane vesicles, enveloped viruses, and the exterior of intact mammalian cells.
Genetically encoded tags for single-molecule imaging in electron microscopy (EM) are long-awaited. Here, we report an approach for directly synthesizing EM-visible gold nanoparticles (AuNPs) on cysteine-rich tags for single-molecule visualization in cells. We first uncovered an auto-nucleation suppression mechanism that allows specific synthesis of AuNPs on isolated tags. Next, we exploited this mechanism to develop approaches for single-molecule detection of proteins in prokaryotic cells and achieved an unprecedented labeling efficiency. We then expanded it to more complicated eukaryotic cells and successfully detected the proteins targeted to various organelles, including the membranes of endoplasmic reticulum (ER) and nuclear envelope, ER lumen, nuclear pores, spindle pole bodies and mitochondrial matrices. We further implemented cysteine-rich tag–antibody fusion proteins as new immuno-EM probes. Thus, our approaches should allow biologists to address a wide range of biological questions at the single-molecule level in cellular ultrastructural contexts. Genetically encoded cysteine-rich tags enable formation of gold nanoparticles in situ for single-molecule imaging of individual proteins in the context of cellular ultrastructure in bacterial, yeast and mammalian cells.
Iron storage proteins are essential for cellular iron homeostasis and redox balance. Ferritin proteins are the major storage units for bioavailable forms of iron. Some organisms lack ferritins, and it is not known how they store iron. Encapsulins, a class of protein-based organelles, have recently been implicated in microbial iron and redox metabolism. Here, we report the structural and mechanistic characterization of a 42 nm two-component encapsulin-based iron storage compartment from Quasibacillus thermotolerans. Using cryo-electron microscopy and x-ray crystallography, we reveal the assembly principles of a thermostable T = 4 shell topology and its catalytic ferroxidase cargo and show interactions underlying cargo-shell co-assembly. This compartment has an exceptionally large iron storage capacity storing over 23,000 iron atoms. Our results reveal a new approach for survival in diverse habitats with limited or fluctuating iron availability via an iron storage system able to store 10 to 20 times more iron than ferritin.
We describe a method, termed cryoAPEX, which couples chemical fixation and high-pressure freezing of cells with peroxidase tagging (APEX) to allow precise localization of membrane proteins in the context of a well-preserved subcellular membrane architecture. Further, cryoAPEX is compatible with electron tomography. As an example, we apply cryoAPEX to obtain a high-resolution three-dimensional contextual map of the human FIC (filamentation induced by cAMP) protein, HYPE (also known as FICD). HYPE is a single-pass membrane protein that localizes to the endoplasmic reticulum (ER) lumen and regulates the unfolded protein response. Alternate cellular locations for HYPE have been suggested. CryoAPEX analysis shows that, under normal and/or resting conditions, HYPE localizes robustly within the subdomains of the ER and is not detected in the secretory pathway or other organelles. CryoAPEX is broadly applicable for assessing both lumenal and cytosol-facing membrane proteins.
A current challenge is to develop tags to precisely visualize proteins in cells by light and electron microscopy. Here, we introduce FerriTag, a genetically-encoded chemically-inducible tag for correlative light-electron microscopy. FerriTag is a fluorescent recombinant electron-dense ferritin particle that can be attached to a protein-of-interest using rapamycin-induced heterodimerization. We demonstrate the utility of FerriTag for correlative light-electron microscopy by labeling proteins associated with various intracellular structures including mitochondria, plasma membrane, and clathrin-coated pits and vesicles. FerriTagging has a good signal-to-noise ratio and a labeling resolution of approximately 10 nm. We demonstrate how FerriTagging allows nanoscale mapping of protein location relative to a subcellular structure, and use it to detail the distribution and conformation of huntingtin-interacting protein 1 related (HIP1R) in and around clathrin-coated pits.
We genetically controlled compartmentalization in eukaryotic cells by heterologous
expression of bacterial encapsulin shell and cargo proteins to engineer enclosed enzymatic
reactions and size-constrained metal biomineralization. The shell protein (EncA) from Myxococcus
xanthus auto-assembles into nanocompartments inside mammalian cells to which
sets of native (EncB,C,D) and engineered cargo proteins self-target enabling localized
bimolecular fluorescence and enzyme complementation. Encapsulation of the enzyme tyrosinase
leads to the confinement of toxic melanin production for robust detection via multispectral
optoacoustic tomography (MSOT). Co-expression of ferritin-like native cargo
(EncB,C) results in efficient iron sequestration producing substantial contrast by magnetic
resonance imaging (MRI) and allowing for magnetic cell sorting. The monodisperse, spherical,
and iron-loading nanoshells are also excellent genetically encoded reporters for electron
microscopy (EM). In general, eukaryotically expressed encapsulins enable cellular engineering
of spatially confined multicomponent processes with versatile applications in multiscale
molecular imaging, as well as intriguing implications for metabolic engineering and
cellular therapy.
In recent years, DNA origami nanorulers for superresolution (SR) fluorescence microscopy have been developed from fundamental proof-of-principle experiments to commercially available test structures. The self-assembled nanostructures allow placing a defined number of fluorescent dye molecules in defined geometries in the nanometer range. Besides the unprecedented control over matter on the nanoscale, robust DNA origami nanorulers are reproducibly obtained in high yields. The distances between their fluorescent marks can be easily analysed yielding intermark distance histograms from many identical structures. Thus, DNA origami nanorulers have become excellent reference and training structures for superresolution microscopy. In this work, we go one step further and develop a calibration process for the measured distances between the fluorescent marks on DNA origami nanorulers. The superresolution technique DNA-PAINT is used to achieve nanometrological traceability of nanoruler distances following the guide to the expression of uncertainty in measurement (GUM). We further show two examples how these nanorulers are used to evaluate the performance of TIRF microscopes that are capable of single-molecule localization microscopy (SMLM).
Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial toxin which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG’s to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology. When combined with Oxyrase, a cell-friendly photostabilizer, a ~20x improvement in fluorescence photostability is achieved. By adding in glutathione, fluorophores are made to blink, enabling super-resolution fluorescence with 20–30 nm resolution over a long time (~30 min) under continuous illumination. Example applications in conventional and super-resolution imaging of native and transfected cells include p65 signal transduction activation, single molecule tracking of kinesin, and specific labeling of a series of nuclear and cytoplasmic protein complexes.
DOI: http://dx.doi.org/10.7554/eLife.20378.001
Single genomic loci are often related to specific cellular functions, genetic diseases, or pathogenic infections. Visualization of single genomic loci in live human cells is currently of great interest, yet it remains challenging. Here, we describe a strategy for live cell imaging of single genomic loci by combining transcription activator-like effectors (TALEs) with a quantum dot labelling technique. We design and select a pair of TALEs that specifically target HIV-1 proviral DNA sequences, and use bioorthogonal ligation reactions to label them with different colour quantum dots (QDs). These QD-labelled TALEs are able to enter the cell nucleus to provide fluorescent signals to identify single gene loci. Based on the co-localization of the pair of different coloured QD-labelled TALEs, we determine and map single-copy HIV-1 provirus loci in human chromosomes in live host cells.
Bioinspired design of ligands for nanoparticle coating with remarkable precision in controlling anisotropic connectivity and with universal binding efficiency to the membrane have made a great impact on nanoparticle self-assembly. We utilize HIV-1 derived trans-activator of transcription peptide (TAT), a member of the cell-penetrating peptides, as a soft shell coating on gold nanoparticles (GNPs), and characterize TAT pepide-mediated binding behaviors of GNPs on the lipid membrane. Whereas the peptides enable GNPs to firmly attach to the membrane, the binding structures are driven by two electrostatic forces: the interparticle peptide-repulsion and the peptide-membrane attraction. Although transmission electron microscopic images showed that the densities of membrane-embedded GNPs were almost equal, X-ray reflectivity revealed significant difference in binding structures of GNPs along the surface normal upon the increase of charged densities (Φ) of the membrane. Especially, GNPs densely suspended at Φ = 70%, while they adopted an additional well-defined layer underneath the membrane at Φ = 100%, in addition to a translocation of the initially-bound particles into the membrane. The observed behaviors of GNPs manifest a 3D to 2D transformation of the self-assembled structures from the diffused state to the closely packed state upon the increase of the charged density of the membrane. The present study also provides insights on the binding mechanisms of the cell-penetrating peptide-coated nanoparticles to the lipid membranes, which is a common theme of delivery systems in pharmaceutical research.
‘Bottom-up fabrication’, which exploits the intrinsic properties of atoms and molecules to direct their self-organization, is widely used to make relatively simple nanostructures. A key goal for this approach is to create nanostructures of high complexity, matching that routinely achieved by ‘top-down’ methods. The self-assembly of DNA molecules provides an attractive route towards this goal. Here I describe a simple method for folding long, single-stranded DNA molecules into arbitrary two-dimensional shapes. The design for a desired shape is made by raster-filling the shape with a 7-kilobase single-stranded scaffold and by choosing over 200 short oligonucleotide ‘staple strands’ to hold the scaffold in place. Once synthesized and mixed, the staple and scaffold strands self-assemble in a single step. The resulting DNA structures are roughly 100 nm in diameter and approximate desired shapes such as squares, disks and five-pointed stars with a spatial resolution of 6 nm. Because each oligonucleotide can serve as a 6-nm pixel, the structures can be programmed to bear complex patterns such as words and images on their surfaces. Finally, individual DNA structures can be programmed to form larger assemblies, including extended periodic lattices and a hexamer of triangles (which constitutes a 30-megadalton molecular complex).
Pseudomonas moraviensis stanleyae was recently isolated from the roots of the selenium (Se) hyperaccumulator plant Stanleya pinnata. This bacterium tolerates normally lethal concentrations of SeO3(2-) in liquid culture, where it also produces Se nanoparticles. Structure and cellular ultrastructure of the Se nanoparticles as determined by cellular electron tomography shows the nanoparticles as intracellular, of narrow dispersity, symmetrically irregular and without any observable membrane or structured protein shell. Protein mass spectrometry of a fractionated soluble cytosolic material with selenite reducing capability identified nitrite reductase and glutathione reductase homologues as NADPH dependent candidate enzymes for the reduction of selenite to zerovalent Se nanoparticles. In vitro experiments with commercially sourced glutathione reductase revealed that the enzyme can reduce SeO3(2-) (selenite) to Se nanoparticles in an NADPH-dependent process. The disappearance of the enzyme as determined by protein assay during nanoparticle formation suggests that glutathione reductase is associated with or possibly entombed in the nanoparticles whose formation it catalyzes. Chemically dissolving the nanoparticles releases the enzyme. The size of the nanoparticles varies with SeO3(2-) concentration, varying in size form 5 nm diameter when formed at 1.0 μM [SeO3(2-)] to 50 nm maximum diameter when formed at 100 μM [SeO3(2-)]. In aggregate, we suggest that glutathione reductase possesses the key attributes of a clonable nanoparticle system: ion reduction, nanoparticle retention and size control of the nanoparticle at the enzyme site.
Numerous methods have been developed for immunogold labeling of thick, cryo-preserved biological specimens. However, most of the methods are permutations of chemical fixation and sample sectioning, which select and isolate the immunolabeled region of interest. We describe a method for combining immunogold labeling with cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) of the surface proteins of intact mammalian cells or the surface glycoproteins of assembling and budding viruses in the context of virus-infected mammalian cells cultured on EM grids. In this method, the cells were maintained in culture media at physiologically relevant temperatures while sequentially incubated with the primary and secondary antibodies. Subsequently, the immunogold-labeled specimens were vitrified and observed under cryo-conditions in the transmission electron microscope. Cryo-EM and cryo-ET examination of the immunogold-labeled cells revealed the association of immunogold particles with the target antigens. Additionally, the cellular structure was unaltered by pre-immunolabeling chemical fixation and retained well-preserved plasma membranes, cytoskeletal elements, and macromolecular complexes. We think this technique will be of interest to cell biologists for cryo-EM and conventional studies of native cells and pathogen-infected cells.
Labeling nuclear proteins with electron dense probes in living cells has been a major challenge due to their inability to penetrate into nuclei. We developed a lipid-based approach for delivering antibodies coupled to 0.8 nm ultrasmall gold particles into the nucleus to label RNA polymerase II. Focussed Ion Beam slicing coupled to Scanning Electron Microscopy (FIB/SEM) enabled visualization of entire cells with probe localization accuracy in the 10 nm range.
Cryo-electron tomography (cryoET) is an important imaging technique that can provide 3D datasets of organelles and proteins at nanometer and sub-nanometer resolution. Recently, combining cryoET with subtomogram averaging has pushed the resolution to 3-4 A. However, one main challenge for cryoET is locating target proteins in live cells. Conventional methods such as fluorescent protein tagging and immunogold labeling are not entirely suitable to label small structures in live cells with molecular resolution in vitrified samples. If large proteins, which can be visually identified in cryoET, are directly linked to the target protein, the large tag may alter the target protein structure, localization and function. To address this challenge, we used the rapamycin-induced oligomer formation system, which involves two tags (FKBP and FRB) that can bind together within rapamycin. In our system, the FKBP tag is linked to target protein and the FRB tag is linked to a large protein to create a marker. We chose ferritin as the marker protein because it is a large complex (10-12 nm) and can bind iron to create strong contrast in cryoET. After adding rapamycin to the cell medium, the iron-loaded ferritin accurately indicates the location of the target protein. Recently, in-situ cryoET with subtomogram averaging has been rapidly developing. However, it is still challenging to locate target proteins in live cells, and this method provides a much-needed solution.
Cryo-electron tomography (cryo-ET) combined with sub-tomogram averaging (STA) allows the determination of protein structures imaged within the native context of the cell at near-atomic resolution. Particle picking is an essential step in the cryo-ET/STA image analysis pipeline that consists in locating the position of proteins within crowded cellular tomograms so that they can be aligned and averaged in 3D to improve resolution. While extensive work in 2D particle picking has been done in the context of single-particle cryo-EM, comparatively fewer strategies have been proposed to pick particles from 3D tomograms, in part due to the challenges associated with working with noisy 3D volumes affected by the missing wedge. While strategies based on 3D template-matching and deep learning are commonly used, these methods are computationally expensive and require either an external template or manual labelling which can bias the results and limit their applicability. Here, we propose a size-based method to pick particles from tomograms that is fast, accurate, and does not require external templates or user provided labels. We compare the performance of our approach against a commonly used algorithm based on deep learning, crYOLO, and show that our method: i) has higher detection accuracy, ii) does not require user input for labeling or time-consuming training, and iii) runs efficiently on non-specialized CPU hardware. We demonstrate the effectiveness of our approach by automatically detecting particles from tomograms representing different types of samples and using these particles to determine the high-resolution structures of ribosomes imaged in vitro and in situ.
Imagine you need to look inside a living organism. You would have only two options: using invasive techniques or applying tomography. In this non-invasive imaging method, two-dimensional (2D) images of slices, or sections, within an organism are analyzed to determine the three-dimensional (3D) localization of internal objects. These sections can be so thin that the target objects are unobscured by the superposition of over- or underlying structures. The challenge of seeing internal objects that are free of superposition originated in medicine, where such 3D localization is necessary for accurate diagnosis and treatment.
One of the main benefits of cryo-electron tomography (ET) is direct visualisation of the sample itself, without the need for stains or fixation. A consequence of this direct imaging is that every molecule in the sample contributes to the resulting tomogram, and for complex specimens such as cells or viruses, it can be challenging to identify a specific feature of interest. This chapter discusses two approaches to help guide collection and interpretation of data from cryo-ET: correlative light and electron microscopy (CLEM) and molecular tagging. These methods can be used independently or together and help cryo-ET practitioners identify areas for data collection and localise or identify specific molecules in their reconstructed tomograms.
This chapter takes a close look at the hardware side of transmission electron microscopy and, by extension, its applications in cryo-electron tomography. Before we throw ourselves into the fray, we are briefly soaking in some information on notable development milestones of the past. I firmly believe there is value in appreciating the historical context of electron microscopy to gain a better understanding of the many advances that were necessary to get us to where we are today. In the chapter’s main part, we will then learn how to “make” electrons and how to make use of them in imaging techniques. We will deal with electron lenses and how an electron microscope is designed to create a space for the electron beam to shape and interact with our sample. We will discover the various ways in which these interactions take place and how, through the principle of phase contrast, a “micrograph” is eventually recorded in the image plane—by ever more sophisticated detectors. As we acknowledge that our sample fries all too quickly, we l also discuss low dose techniques and phase plates. Finally, we provide a short outlook towards exciting developments that may well push cryo-electron tomography to the next level.
Immunogold labeling in transmission electron microscopy (TEM) utilizes the high electron density of gold nanoparticles conjugated to proteins to identify specific antigens in biological samples. In this work we applied the concept of immunogold labeling for the labeling of negatively charged phospholipids, namely phosphatidylserine, by a simple protocol, performed entirely in the liquid-phase, from which cryo-TEM specimens can be directly prepared. Labeling included a two-step process using biotinylated annexin-V and gold-conjugated streptavidin. We initially applied it on liposomal systems demonstrating its specificity and selectivity, differentiating between 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) membranes. We also observed specific labeling on extracellular vesicle samples isolated from THP1 cells and from MDA-468 cells that underwent stimulations. Finally, we compared the levels of annexin-V labeling on the cells vs. on their isolated EVs by flow cytometry and found a good correlation with cryo-TEM results. This simple, yet effective labeling technique makes it possible to differentiate between negatively charged to non-negatively charged membranes, thus shedding light on their possible EV shedding mechanism.
The application of cryo-correlative light and cryo-electron microscopy (cryo-CLEM) gives us a way to locate structures of interest in the electron microscope. In brief, the structures of interest are fluorescently tagged, and images from the cryo-fluorescent microscope (cryo-FM) maps are superimposed on those from the cryo-electron microscope (cryo-EM). By enhancing cryo-FM to include single-molecule localization microscopy (SMLM), we can achieve much better localization. The introduction of cryo-SMLM increased the yield of photons from fluorophores, which can benefit localization efforts. Dahlberg and Moerner (2021, Annual Review of Physical Chemistry, 72 , 253–278) have a recent broad and elegant review of super-resolution cryo-CLEM. This paper focuses on cryo(F)PALM/STORM for the cryo-electron tomography community. I explore the current challenges to increase the accuracy of localization by SMLM and the mapping of those positions onto cryo-EM images and maps. There is much to consider: we need to know if the excitation of fluorophores damages the structures we seek to visualize. We need to determine if higher numerical aperture (NA) objectives, which add complexity to image analysis but increase resolution and the efficiency of photon collection, are better than lower NA objectives, which pose fewer problems. We need to figure out the best way to determine the axial position of fluorophores. We need to have better ways of aligning maps determined by FM with those determined by EM. We need to improve the instrumentation to be easier to use, more accurate, and ice-contamination free. The bottom line is that we have more work to do.
A long-standing challenge in cell biology is elucidating the spatial distribution of individual membrane-bound proteins, protein complexes and their interactions in their native environment. Here, we describe a workflow that combines on-grid immunogold labeling, followed by cryo-electron tomography (cryoET) imaging and structural analyses to identify and characterize the structure of photosystem II (PSII) complexes. Using an antibody specific to a core subunit of PSII, the D1 protein (uniquely found in the water splitting complex in all oxygenic photoautotrophs), we identified PSII complexes in biophysically active thylakoid membranes isolated from a model marine diatom Phaeodactylum tricornutum. Subsequent cryoET analyses of these protein complexes resolved two PSII structures: supercomplexes and dimeric cores. Our integrative approach establishes the structural signature of multimeric membrane protein complexes in their native environment and provides a pathway to elucidate their high-resolution structures.
Quantum dots (QDs), as one of the emerging nanomaterials, have been widely studied by scientists due to their advantages and potential in bioimaging, especially cell imaging. Cadmium (Cd)-based QDs, which have the best photoluminescence property, have received widespread attention. However, due to the obvious toxicity problem of these QDs, their cell imaging application is hindered. Recently, the emergence of Cd-free-metal and metal-free QDs with lower toxicity makes people consider that Cd-based QDs may be substituted by these QDs. However, problems of these QDs in cytotoxicity also cannot be ignored. Some reports claimed that their prepared emerging QDs for cell imaging were low toxicity, but there still exist up-regulation of oxidative stress, inflammation, and apoptosis from other reports. And, up to now, few reports have separately summarized and discussed the issues of cell imaging and cytotoxicity of different types of QDs. Therefore, in this review, we classify QDs as following three types, Cd-based, Cd-free-metal and metal-free QDs, focus on the cell, the essential unit of life, discuss cell imaging application and cytotoxicity of QDs, and finally elucidate main mechanisms of cytotoxicity respectively. This review provides reference for the toxicity evaluation of QDs and highlights the potential cytotoxicity of Cd-free QDs.
The nanoprobes for identification of cancer metastases in the mononuclear phagocyte system (MPS) organs are of significant importance, but are limited due to the long-standing challenge of low tumor-targeting specificity with inadequate targeting efficiency and high nonspecific accumulation. Here, we report a surface regulation strategy that integrates the tumor-acidity-activated charge-reversal behavior and precise control in both hydrodynamic diameter (HD) and surface charge on ultrasmall luminescent gold nanoparticles (AuNPs) to achieve significantly high tumor-targeting specificity. The precise regulation of AuNPs to a rational HD and surface charge could rapidly and selectively recognize the small metastatic tumors (~1 mm) in liver and lung with high signal-to-noise ratios of 4.6 and 4.5, respectively. These results help further understand the in vivo transport of nanoprobes, and provide guidance for design of translatable nanosized nanomedicines in cancer metastasis theranostics.
Tracking intracellular proteins in live cells has many challenges. The most widely-used method, fluorescent protein fusions, can track proteins in their native cellular environment and has led to significant discoveries in cell biology. Fusion proteins add steric bulk to the target protein and can negatively affect native protein function. The use of exogenous probes such as antibodies or protein labels is problematic since these cannot cross the plasma membrane on their own and thus cannot label intracellular targets in cells. We developed a labeling platform, VIPERnano, for live cell imaging of intracellular proteins using a peptide fusion tag (CoilE) to the protein of interest and delivery of a fluorescently labeled probe peptide (CoilR). CoilR and CoilE form an α-helical heterodimer with the protein of interest, rendering a labeled protein. Delivery of CoilR into the cell uses hollow gold nanoshells (HGN) as the primary delivery vehicle. The technology relies on the conjugation and light activated release of the CoilR peptide on the surface of the HGNs. We demonstrate light-activated VIPERnano delivery and labeling with two intracellular proteins, localized either in the mitochondria or the nucleus. This technology has the ability to study intracellular protein dynamics and spatial tracking while lessening the steric bulk of tags associated with the protein of interest.
RNA interference is one of the prosperous approaches for cancer treatment. However, small interfering RNA (siRNA) delivery to cancer cells has been faced with various challenges restricting their clinical application over the decades. Since ROR1 is an onco‐embryonic gene overexpressed in many malignancies, suppression of ROR1 by siRNA can potentially fight cancer. Herein, a delivery system for ROR1 siRNA based on HIV‐1 TAT peptide‐capped gold nanoparticles (GNPs) was developed to treat breast cancer. Besides, we introduced a new feasible method for conjugating the peptide to the nanoparticles. Since the GNPs have high affinity to the sulfur, the findings demonstrated the peptide successfully conjugated to the nanoparticles via Au–S bonds. As positively charged nanoparticles showed high cellular uptake, we could use a low concentration of nanoparticles led to high efficient gene transfection with negligible cytotoxicity that was confirmed by flow cytometry, confocal microscopy, gel retardation, and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. Following transfection, downregulation of ROR1 and its targeted gene, CCND1, induced apoptosis in cancer cells. In conclusion, the reported capped GNPs could be potentially utilized for delivering negatively charged therapeutic agents in particular genes. HIV‐1 TAT cell penetrating peptide is conjugated to gold nanoparticles by replacing with cetyltriethyl ammonium bromide molecules. The positively charged nanoparticles are able to interact with ROR1 small interfering RNA electrostatically. HIV‐1 TAT peptides enhance cellular uptake of the prepared complexes.
The trans-acting activator of transcription (TAT) peptide, which derived from human immunodeficiency virus-1 (HIV-1), has been widely used as an effective nanocarrier to transport extracellular substances into cells. However, the underlying translocation mechanism of TAT peptide across cell membranes still remains controversial. Besides, the molecular process of TAT peptide facilitating the transport of extracellular substances into cells is largely unknown. In this study, we explore the interactions between TAT peptides and its conjugated gold nanoparticles with lipid membranes by coarse-grained molecular dynamics simulations. It is found that the TAT peptides can hardly penetrate through the membrane at low peptide concentrations; after the concentration increases to a threshold value, they can cross the membrane through an induced nanopore due to the transmembrane electrostatic potential difference. The translocation of TAT peptides is mainly caused by the overall structural changes of membranes. Furthermore, we demonstrate that the translocation of gold nanoparticles (AuNPs) across the membrane is significantly affected by the number of grafted TAT peptides on the particle surface. The transmembrane efficiency of AuNPs may even be reduced when little peptides are modified; whereas the number of grafted peptides increases to a certain value, the TAT-AuNPs complex can translocate across the membrane in a pore-mediated way. Based on our findings, an effective strategy has been proposed to enhance the delivery efficiency of AuNPs. The present study can improve our understanding on the interactions between TAT peptides and cell membranes; it may also give some insightful suggestions on the design and development of nanocarriers with high efficiency for the delivery of nanoparticles and drugs.
MTs are small cysteine-rich proteins that, chelate metal ions such as Cu⁺ and Zn²⁺, and are widely distributed in several life domains in particular the eukaryotic one. They are present in the following phyla: Opisthokonta (mainly Fungi and Metazoa), Chloroplastida, Alveolata (Ciliates) and Excavata (Trichomonas) for Eukaryota and Cyanobacteria, Actinobacteria, Proteobacteria and Firmicutes for Bacteria. However, their absence in some phyla underlines that MTs are far from being fully known. The MT amino acid sequences show a great diversity of sizes and structures both in terms of cysteine motifs and organization of these motifs. This review also highlights the different oxidized, apoprotein and metalated forms of MTs, the diversity of interactions they can establish with different molecules and their central and multifunctional cellular role. We present MTs as a protein system that could be a hub in molecular interaction networks. Studying MTs as a hub in cellular interaction networks should provide new insights for a better understanding of MT functioning and cellular processes.
Nucleic acid aptamers are small three-dimensional structures of oligonucleotides selected to bind to a target of interest with high affinity and specificity. In vitro, aptamers already compete with antibodies to serve as imaging probes, e.g. for microscopy or flow cytometry. However, they are also increasingly used for in vivo molecular imaging. Accordingly, aptamers have been evaluated over the last twenty years in almost every imaging modality, including single photon emission computed tomography, positron emission tomography, magnetic resonance imaging, fluorescence imaging, echography, and x-ray computed tomography. This review focuses on the studies that were conducted in vivo with aptamer-based imaging probes. It also presents how aptamers have been recently used to develop new types of probes for multimodal imaging and theranostic applications.
Gold nanoparticles (AuNPs) and their conjugation to biological samples have numerous potential applications. When combined with cryo-electron microscopy and tomography analysis, AuNPs may provide a versatile and powerful tool to identify and precisely localize proteins even when attached to cellular components. Here, we describe a general and facile approach for the synthesis of homogeneous and stable AuNPs, which can readily be conjugated to a molecule of interest and imaged by cryo-electron tomography (cryo-ET). We demonstrate the synthesis of 2.2 ± 0.45-nm tiopronin-protected AuNPs, followed by their conjugation with recombinant proteins and peptides. Visualization of the ∼2.2-nm gold-tagged peptides by cryo-ET reveals the potential use of this strategy to label and localize accessible proteins in a cellular environment with nanometric resolution.
Pressure-freezing has often been regarded as a method for highly technical specialists. At the beginning of its development, this may have been true: it was introduced in 1968 by Moor and Riehle at the European conference on electron microscopy in Rome. The interest of the audience was not overwhelming, because everybody thought that this approach is oversophisticated and in principle unnecessary. In the following decade, many technically less pretentious freezing methods have been developed, which work in the absence of pressure. All of them became standardized and their methodology has been described in numerous reviews and textbooks (e.g. Rash 1983; Gilkey and Staehlin 1986; see also Sitte et al., Chap. 4, this Vol.). The compiled experience shows the manifold profits of applying impact-, plunge-, jet- and spray-freezing. In one aspect, however, all of these techniques are inadequate: namely they only enable satisfactory cryofixation of objects or superficial layers, which are not thicker than 10–20 μm. This limitation is caused by the physical properties of aqueous systems and it indicates that thicker specimens can be well cryofixed only if these properties are altered.
DNA provides an ideal substrate for the engineering of versatile nanostructures due to its reliable Watson-Crick base pairing and well-characterized conformation. One of the most promising applications of DNA nanostructures arises from the site-directed spatial arrangement with nanometer precision of guest components such as proteins, metal nanoparticles, and small molecules. Two-dimensional DNA origami architectures in particular offer a simple design, high yield of assembly, and large surface area for use as nano-platform. However, such single-layer DNA origami were recently found to be structurally polymorphous due to their high flexibility, leading to the development of conformationally restrained multi-layered origami that lack some of the advantages of the single-layer designs. Here we monitored single-layer DNA origami by transmission electron microscopy (EM) and discovered that their conformational heterogeneity is dramatically reduced in the presence of a low concentration of dimethyl sulfoxide (DMSO), allowing for an efficient flattening onto the carbon support of an EM grid. We further demonstrated that streptavidin and a biotinylated target protein (cocaine esterase - CocE) can be captured at pre-designated sites on these flattened origami while maintaining their functional integrity. Our demonstration that protein assemblies can be constructed with high spatial precision (within ~2 nm of their predicted position on the platforms) by using strategically flattened single-layer origami paves the way for exploiting well-defined guest molecule assemblies for biochemistry and nanotechnology applications.
Microscopy has gone hand in hand with the study of living systems since van Leeuwenhoek observed living microorganisms and cells in 1674 using his light microscope. A spectrum of dyes and probes now enable the localization of molecules of interest within living cells by fluorescence microscopy. With electron microscopy (EM), cellular ultrastructure has been revealed. Bridging these two modalities, correlated light microscopy and EM (CLEM) opens new avenues. Studies of protein dynamics with fluorescent proteins (FPs), which leave the investigator 'in the dark' concerning cellular context, can be followed by EM examination. Rare events can be preselected at the light microscopy level before EM analysis. Ongoing development-including of dedicated probes, integrated microscopes, large-scale and three-dimensional EM and super-resolution fluorescence microscopy-now paves the way for broad CLEM implementation in biology.
Protein structures such as ferritin in combination with synthetic as well as genetic alterations has proven to be highly interesting for the production of new materials. Ferritin describes a family of iron storage proteins with ubiquitous distribution among all life forms, with the notable exception of yeast and they are the most abundant members of the ferritin-like superfamily and may have developed from a rubrerythrin-like ancestor protein with two homologous pairs of antiparallel helices as main structural feature. The protein shell of mammalian ferritin is usually heterogeneous and consists of a mixture of two subunits of about 21 kDa, termed H for heavy (predominant in heart) and of about 19 kDa, termed L for light chain (predominant in liver), with around 55% amino acid homology for human H- and Lferritin. Apoferritin can be readily disassembled and reassembled by reducing the pH to a value as low as pH 2 and increasing it above pH 7, respectively. Holo-ferritin exhibits a remarkable affinity for anions and some nonferrous metal ions. Direct demineralization of the iron core in ferritins can be induced with strong Fe(III) chelators.