Cryo-electron tomography of cells: connecting structure and function
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Cryo-electron tomography (cryo-ET) allows the visualization of cellular structures under close-to-life conditions and at molecular resolution. While it is inherently a static approach, yielding structural information about supramolecular organization at a certain time point, it can nevertheless provide insights into function of the structures imaged, in particular, when supplemented by other approaches. Here, we review the use of experimental methods that supplement cryo-ET imaging of whole cells. These include genetic and pharmacological manipulations, as well as correlative light microscopy and cryo-ET. While these methods have mostly been used to detect and identify structures visualized in cryo-ET or to assist the search for a feature of interest, we expect that in the future they will play a more important role in the functional interpretation of cryo-tomograms.
KeywordsCryo-electron tomography Correlative light microscopy Electron microscopy
Among techniques used in cell biology, cryo-electron tomography (cryo-ET) is a relatively recent one. It combines the advantages of 3D imaging with a close-to-life preservation and it allows studying biological material at molecular resolution (rev. in Lucic et al. 2005; Nicastro et al. 2005). Rapid freezing followed by the investigation of the frozen-hydrated samples avoids artifacts notorious to chemical fixation and dehydration procedures (Dubochet et al. 1988). Furthermore, the biological material is observed directly, without heavy metal staining, avoiding problems in interpretation caused by an unpredictable accumulation of staining material. Consequently, cryo-ET of whole cells has the advantage that the supramolecular architecture can be studied in unperturbed cellular environments (Baumeister 2002).
We can distinguish two types of cryo-preparation. Thin samples (typically below 0.5 μm in thickness) can be rapidly frozen by plunging into liquid ethane (Dubochet et al. 1988) and imaged in an electron microscope without further processing. While plunge-freezing is limited to small cells and thin regions of cells, thicker samples are frozen by high-pressure freezing (Moor 1987) and then cryo-sectioned, that is sectioned to typically 50–200 nm thick sections at cryogenic temperatures (Al-Amoudi et al. 2004). In both cases the high freezing rates ensure that a sample is vitrified, i.e. formation of ice crystals is avoided. Also, samples are kept and imaged in EM at cryogenic temperatures to avoid devitrification.
Electron microscopy (EM) in general, and cryo-ET being no exception, delivers static images of a biological system, as does X-ray crystallography. In spite of this apparent limitation, both methods can provide a structural framework for a mechanistic understanding of molecular and cellular functions. Cryo-ET has already pushed the resolution limits and increased the fidelity of the images, and we can expect this trend to continue. In this review, we focus on the experimental methods that supplement cryo-ET imaging of whole cells. Various approaches were used to assist the detection or identification of imaged structures and also to allow cryo-ET imaging of intracellular structures arrested in a particular functional state. This is a very young field, and consequently the current literature is not extensive. We paid particular attention to the methods used and to the development of methods that might be relevant for the future. The methods were separated in two main categories. On one side are genetic and pharmacological manipulations that are used along the lines of the well-proven basic paradigm that involves investigations of untreated, treated and (when applicable) control samples. When applied to cryo-ET, a number of tomograms are recorded for samples in each group and a value is associated with each tomogram in order to assess the effect of a treatment. Correlative light microscopy (LM) and EM methods fall in the second category. The advantage of this approach is that LM provides information about the larger environment and the functional states of structures visualized in cryo-ET, or alternatively, cryo-ET effectively extends the resolution achievable in LM.
Genetic, pharmacological and related treatments integrated with cryo-ET
Finding and identifying a structure of interest in tomograms recorded under low-dose conditions having a poor signal-to-noise ratio is one of the basic problems in cryo-ET. It is also a prerequisite for providing insights to its function. Although a large number of observations obtained from conventional EM are certainly very useful in this respect, more sophisticated detection methods are needed if cryo-ET is expected to reach beyond ultrastructure to the molecular architecture level. In straightforward cases, when features of interest possess strong intrinsic contrast, they can be easily identified. For example, the location and spatial arrangement of magnetosomes, small organelles containing magnetite crystals present in magnetotactic bacteria, were compared in wild-type and mutant cells lacking proteins that were expected to be relevant for magnetosome organization and function (Komeili et al. 2006; Scheffel et al. 2006). In both studies, the wild-type organization of magnetosomes was restored after overexpression of the deleted proteins. Cryo-preparation was necessary for the preservation of cytoskeletal filaments of Spiroplasma melliferum (Kurner et al. 2005) and of cell surface filaments of Flavobacterium johnsoniae (Liu et al. 2007) and allowed a relatively straightforward detection. This in turn made an integrative approach possible, whereby the genetic deletion of GldF, one of the envelope-associated gliding proteins, led to a complete loss of filaments (Liu et al. 2007). The reintroduction of the deleted protein rescued the filaments, thus, providing new evidence for the involvement of GldF in filament formation. Another challenge inherent to cryo-ET is to obtain enough cryo-structures so that they can be statistically analyzed at a reasonable confidence level. Difficulties in sample preparation, as well as the time needed to record, reconstruct and analyze a cryo-tomogram limit throughput. For example, the investigation of actin networks in filopodia of slime mold Dictyostelium cells was facilitated by using genetically modified cells that overexpress small GTPase Rac1A and show an abundance of filopodia (Medalia et al. 2007). Tomograms of wild-type filopodia were used as a control, to make sure that the overexpression of Rac1A did not alter actin networks.
Cytoskeletal filament bundles observed in cryo-tomograms of wild-type Caulobacter crescentus were classified in four types based on their dimensions and cellular localization (Briegel et al. 2006). In an attempt to identify at least some of the filaments, the authors also imaged cells lacking crescentin, one of the previously identified cytoskeletal proteins. Additionally, they imaged both wild-type and crescentin knockout cells that were treated with a drug that is known to depolymerize filaments composed of another cytoskeletal protein, MreB. While crescentin and MreB could be ruled out as constituents of some filament types, the interpretation of the results was complicated by ambiguous results. For example, the lack of one type of filament in the MreB depolymerizing-drug-treated crescentin knockout cells, but not in the non-treated knockout and treated wild-type cells, points to a particular interaction between crescentin, MreB and an as yet unidentified molecule forming these filaments. Also, although the authors analyzed a fair number of tomograms, another type of filament could not be identified because its abundance in wild-type cells was too low. Consequently, this example shows how even a moderately complicated experimental system (four conditions, four filament types) can go beyond current limits of the interpretability of cellular cryo-ET. An even more elaborate version of the same wild-type—mutant—rescue scheme was applied in the investigation of Escherichia coli chemotaxis receptor arrays (Zhang et al. 2004, 2007). It involved genetic deletion of chemoreceptors and/or another two proteins of the chemosensory system, CheA and CheW, and their subsequent expression at different relative levels. As a result, it was confirmed that the periodic structures located at cell poles represent chemoreceptors, illustrating how a genetic approach can assist the identification of structures observed in cryo-tomograms.
In all examples presented so far, various experimental assays were used to assist detection and identification of structures of interest in cryo-tomograms, but not to investigate cells in a well-defined functional or developmental stage. The latter approach was implemented by partially synchronizing the growth phase of the unicellular eukaryote Ostreococcus tauri by light–dark cycles and revealed the detailed ultrastructure of whole cells, tightly packed with organelles, even though the resolution was effectively limited by the thickness of this sample (Henderson et al. 2007).
Rapid freezing techniques currently allow EM imaging of the same process at well-defined time points. This method, called time-resolved cryo-EM, was used to observe growth and shrinkage in reconstituted microtubules (Mandelkow et al. 1991) and liposome formation under different conditions (Frederik and Sommerdijk 2005). A very precise control of reaction time can be achieved by spraying an interacting component on a sample just milliseconds before vitrification (Berriman and Unwin 1994), or by flash-photolysis (Subramaniam et al. 1993). While it may not be straightforward to achieve such precise timings for cryo-ET of whole cells, its application for investigations of cellular processes in vitro may complement cellular studies.
Correlative light microscopy and cryo-ET
In cell biology journals, green pictures abound. The vast array of currently available fluorescent probes as well as the pervasive use of fluorescence LM contributed to our knowledge about the identity and the spatial distribution of molecular players involved in many cellular processes, but left many open questions regarding the molecular and supramolecular architecture of these players. Resolving these questions may help us understand the principles underlying a large number of cellular processes. Correlative LM and EM methods allow imaging of the same feature over multiple length scales and can combine the advantages of both methods, namely live cell imaging and the ability to obtain high-resolution information. Approaches that combine LM with other EM preservation methods are already showing their importance for the integration of structure and function and are beginning to become important tools in cell biology (rev. in Koster and Klumperman 2003). We first review the current state of this topic and then outline some future directions.
Lessons from correlative approaches involving other EM preparations
Conventional sample preparation in EM involves a sequence of steps: a chemical fixation, dehydration, embedding, staining and sectioning. Better preservation can be obtained if instead of a chemical fixation and dehydration a sample is rapidly frozen using a high-pressure freezer and then freeze substituted. In this type of EM, labeling by electron-dense markers is typically achieved using the immunolabeling methods and it is a trade-off between obtaining good structural preservation and good labeling. In general, pre-embedding labeling methods (typically a sample is mildly fixed, permeabilized and labeled by primary reagents before it is embedded and sectioned) can achieve more efficient labeling; post-embedding methods provide better preservation, while the Tokuyasu method often provides the best compromise (Griffiths 1993).
For correlative EM–LM, one way of introducing fluorescent markers is by immunolabeling, where one proceeds in a similar fashion to immunolabeling with electron-dense labels. However, postembedding labeling is generally more difficult to achieve and it is incompatible with the use of osmium tetroxide, a common fixative and staining agent (Giepmans et al. 2005; van Driel et al. 2008). The use of genetic manipulations allows non-invasive introduction of fluorescence into living cells and facilitates the monitoring of various cellular processes by time-lapse or laser scan fluorescence imaging. This in turn allows the identification of a time point where a process that is followed reaches a particular stage, so that the process can be arrested in that state and examined in EM. For example, individual tubulovesicular transport compartments in axons of cultured neurons were labeled by the overexpression of GFP-fusion proteins that are known to associate with these organelles and their movement was observed by recovery after photobleaching of small areas of axons (Nakata et al. 1998).
The movement was arrested by chemical fixation and the area where the fluorescence was observed was imaged by EM. Similarly, the simultaneous labeling by a GFP-fusion protein and a membrane dye allowed the authors to detect the exact time point when individual synapses were formed in neuronal cultures and to arrest further development by chemical fixation in order to image the nascent synapses in conventional EM (Ahmari et al. 2000). Thanks to the clearly identifiable features of neurons in culture and with the help of external orientation marks in both studies the same areas were imaged in both LM and EM thus revealing the cellular environment at the ultrastructural level of the fluorescently labeled tubulovesicular structures involved in the transport of presynaptic components and the synaptogenesis.
The ability of horseradish peroxidase to catalyze the oxidation of diaminobenzidine (DAB) and induce formation of diffusible osmiophilic precipitate has been extensively exploited for immunolabeling in conventional EM. DAB is introduced to an osmium treated and immunolabeled sample, where horseradish peroxidase is typically conjugated to the secondary antibodies, to form an electron-dense reaction product visible in EM. This approach was used to investigate the ultrastructure and identify components of the secretory pathway involved in the transport of cargo from the Golgi apparatus towards the plasma membrane (Polishchuk et al. 2000), within Golgi apparatus (Mironov et al. 2001), and between endoplasmic reticulum and Golgi (Mironov et al. 2003). Pre-embedding fluorescence labeling of structures involved in the transport allowed the authors to follow the transport by time-lapse fluorescence microscopy and to stop it at a desired moment. Furthermore, the immunolabeling was directed against the lumenal part of the transport structures, and so the precipitate formed by the subsequent oxidation of DAB delineated these structures and allowed their detection in EM.
The photooxidation of DAB obtained upon strong illumination of a fluorescent molecule was also used for labeling in conventional EM, and was first shown for Lucifer yellow (Maranto 1982). It brings the additional benefit of observing first a fluorescent label directly in LM and then the product of the photoconversion in EM. FM dyes, widely used labels for studies of synaptic vesicle endocytosis and exocytosis, can be applied to live cells and, under suitable experimental conditions, provide electron-dense labeling of internalized synaptic vesicles, thus functionally distinguishing synaptic vesicle pools (Henkel et al. 1996). Also, fluorescence from a membrane-permeable biarsenical dye ReAsH, induced by its binding to any of the specially designed short genetically encoded tetra-cysteine motives as well as from GFP can induce photooxidation of DAB; these compounds were used for correlative LM and EM (Gaietta et al. 2002; Grabenbauer et al. 2005).
Rapid freezing arrests intracellular processes within milliseconds, and therefore avoids structural changes associated with chemical fixation. For example, the development of Caenorhabditis elegans embryos was followed in LM and stopped at specific stages of early mitosis using high-pressure freezing (Muller-Reichert et al. 2007). In some of the previously mentioned publications on the secretory pathway, tomograms of high pressure frozen, freeze-substituted samples were obtained (Mironov et al. 2001, 2003). However, because pre-embedding immunolabeling methods require chemical fixation and permeabilization and consequently are not compatible with rapid freezing, it was not possible to obtain direct correlation between fluorescent labeling and structures observed in tomograms. Another possible approach for fluorescent labeling of high-pressure frozen samples is to add a fluorescent dye during freeze-substitution (Biel et al. 2003). This type of fluorescence labeling allows imaging of the sample in LM before sectioning, but unlike the pre-embedding methods does not interfere with the sample structure.
Examples of correlation between LM and cryo-ET
Strategies for correlative LM and cryo-ET
Many genetically encoded or non-toxic fluorescent indicators can be introduced into living cells. These include different membrane dyes, probes for various organelles, and metal-ion or pH indicators that can be used for correlations to detect and identify a cellular compartment of interest. Promising candidates for this approach might be smaller membrane-enclosed compartments, the identity and function of which cannot be readily inferred from their pleomorphic structures. This approach can also be useful in cases where the cellular localization or a larger environment of a structure of interest (molecule, membranous structure or an organelle) needs to be determined, or to capture the functional state of a structure of interest and its environment in the case where the fluorescent probe indeed provides such information. In addition, fluorescent fusion proteins the overexpression of which causes functional changes in a cell offer numerous possibilities to identify structures involved in these functional changes.
Considering the observation that imaging in EM rapidly quenches fluorescence (Lucic et al. 2007; van Driel et al. 2008), it follows that fluorescence imaging needs to precede acquisition of a cryotomogram, but the decision between live and cryo fluorescent imaging, or both, often depends on other considerations. We are particularly interested in the approach where correlation is used to navigate the search in cryo-EM to the position of a given fluorescent spot, essentially replacing the search for a feature of interest in EM. However, sample vitrification, especially in cellular tomography, tends to be an unreliable process, yielding ice of heterogenous thickness and not always providing the best preservation, so it is advisable to image a large portion of, if not a whole grid, in order to increase chances that a region where a tomogram can be recorded was imaged in LM. Obviously, the higher the resolution required, the more LM images are needed. Considering that grids are usually slightly bent, z-stacks have to be recorded at each position in order to obtain in-focus images, which greatly increases the time needed for the imaging and handling of the images. On the other hand, imaging with a cryo-stage for LM ensures that the location of imaged structures remains the same in both LM and cryo-EM. Cryo-phase contrast images have poor resolution, but they can be used to estimate ice thickness (Sartori et al. 2007) and to direct (higher resolution) fluorescence imaging only to regions where ice thickness is appropriate for cryo-ET, thus greatly reducing the number of images taken.
As for the correlative approaches involving cryo-sections, additional complications arising from the 3D nature of the frozen samples argue for cryo-fluorescence imaging. Development of special devices and protocols that would allow easier manipulation and confocal imaging of frozen samples prior to cryo-sectioning, perhaps along the lines as for the conventionally prepared or freeze-substituted material (Pfeiffer et al. 2003; Biel et al. 2003), may be quite useful to assist cryo-sectioning. A clonable label for EM would represent one way of localizing supramolecular structures within the cellular context. At first glance, such an approach would be analogous to the use of green fluorescent protein in live cell imaging. Small metal-binding proteins such as metallothionein can be fused to cellular proteins, thus acting as nucleation sites for (electron-dense) gold atoms taken up from the surrounding medium (Mercogliano and DeRosier 2007). This approach is not completely straightforward, however, since it is difficult to distinguish between non-specific uptake/precipitation and bona fide labeling. Metallothioneins occur naturally in many organisms, thus relegating the use of this and similar strategies to organisms where endogenous expression has been suppressed. Furthermore, the need for relatively high concentrations of gold salts in the growth medium and their usefulness as chemotherapeutic agents raises the issue of cytotoxicity or other physiological effects. One must also bear in mind that strategies aimed at pinpointing certain proteins indirectly can only hope to visualize a very small subset of the proteome simultaneously, as opposed to a pattern recognition-based approach with a theoretically comprehensive/complete database of unique templates. On the other hand, information on the 3D subcellular distribution of one particular protein may be a valuable piece of information if morphology is well preserved. Classical immunolabeling of sectioned material grossly underestimates numbers of a particular protein in a 3D volume because high-affinity binding with gold-conjugated antibodies relies on accessibility of epitopes. Hence, labeling of sectioned cells is essentially a 2D (surface) phenomenon. The insertion of a clonable label provides possibilities for 3D labeling but relies on high-affinity binding, or simultaneous nucleation of gold at all target sites, which clearly conflicts with the dynamic nature of protein turnover. Therefore, neither of these approaches can be regarded as quantitative.
Summary and outlook
Furthermore, we expect that genetic or pharmacological manipulations that can bring a cell or a cellular process into a well-defined state will be more prominent in future cryo-ET work. These investigations might benefit from hybrid approaches involving complementary techniques that can provide information about the molecular composition of a complete cell or some of its organelles, interaction networks, formation of transient molecular complexes, or spatial proximity of different molecules. However, these are mostly “bulk” measurements that do not account for the underlying variability that is evident at the level of single molecules. In order to reconcile this with the “individualistic” nature of cryo-ET, where structures are accessed individually and only a limited number of cryo-tomograms can be analyzed, great care should be taken to assure that the manipulations used are reliable and show high efficiency.
It is somewhat of a paradox that increasing magnification and the concomitant increase in information at a certain structural level also carries the risk of losing the overall picture. In other words, so-called ‘representative images’ may indeed represent the exception rather than the rule. This issue is very much related to specimen quality, since aesthetically acceptable regions of a specimen (suitable ice thickness in a plunge-frozen specimen, for example) will naturally bias a microscopist’s data collection strategy. Thinner ice may, however, simply be the result of smaller cells that in turn may not be representative of a population of cells. Pre-selection of live cells for a particular phenotype by flow cytometry may increase the chances or even ensure that any imaged cell is representative of that phenotype. Also, selection of small cells and their subsequent vitrification, for example, can lead to more efficient data collection, provided that this phenotype is not an artifact, such as a starved or dormant cell phenotype.
The correlative LM and cryo-ET approaches, where the same feature is imaged in both LM (particularly in fluorescence) and cryo-ET, fall in the second category of experimental methods that supplement cryo-ET with information about the dynamics or a functional state of a cell under investigation. This approach was used to help navigate in cryo-EM to a structure of interest, provide information about the wider environment, and to associate dynamic information with structures visualized in cryo-tomograms.
Although correlating LM and cryo-ET is still time-consuming, there are several improvements that are expected to make the correlation approach more productive. Straightforward improvements focusing on flexibility are needed to streamline the software aspect of correlative work. Typically, different computers and operating systems are used, while different samples and microscopes require different correlative approaches. It would be useful to develop quick and flexible computer routines that cover several correlation scenarios. It is crucial to choose open file formats to allow easy exchange of data (such as coordinates of correlated spots) between a program that controls EM and other correlation-related programs, allowing users to develop their own correlation scripts that best fit their needs. If a correlation is precise, reliable procedures for automated recording of multiple tomographic series and procedures for automated tomographic reconstruction may then allow us to obtain several tomograms correlated to features of interest in LM, with minimal user interference.
One can envisage correlative approaches based on advanced LM techniques, such as time-lapse microscopy, or FRET-based methods that allow monitoring of different functional states of molecules in living cells. Although these methods can provide precise information about complex formation and translocation, they do not reveal structural components. Consequently, their correlation with cryo-ET has great potential in identifying structural correlates of many functional states. Furthermore, recent fluorescence imaging techniques that can reach resolutions above the diffraction limit, such as stimulated emission depletion (Hell 2003), photo-activated localization microscopy (Betzig et al. 2006), stochastic optical reconstruction microscopy (Rust et al. 2006), or nonlinear structured illumination (Gustafsson 2005), may in principle allow detection of individual fluorescently labeled molecules in correlated cryo-tomograms. While in principle any information from LM can be used for a correlation, difficulties associated with cellular cryo-ET might seriously limit such an approach. The diffraction limit breaking methods typically require high illumination intensities, which may limit their applicability to intact cells and make the correlative approach even more involved. In any case, one would need to ensure that a fair number of fluorescently labeled structures exist on a given sample, so that the chances of recording a cryo-tomogram correlated to one of these structures are reasonably high. Cryo-X-ray tomography provides the enticing possibility to visualize entire cells in 3D at a resolution intermediate between that of LM and EM, and to directly correlate tomograms with 3D LM volumes of the same cell (Schneider 1998). In fact, a light microscope can be integrated into the system to directly screen for features of interest prior to tomography. Interpretation of X-ray microscopy data is straightforward because image formation is based on absorption contrast (note that phase contrast imaging using a Zernicke-type phase stop is also possible). A further attractive feature of this technique is that specimens can be prepared in a manner identical to that used for cryo-ET. Vitreous ice is essentially transparent to X-rays between the K-absorption edges of carbon and oxygen to a depth of 10 µm, thus allowing imaging of relatively large structures at these energies. Ice embedded specimens with suitable size/geometry can be tilted over the full angular range, giving rise to tomograms with isotropic resolution. Resolution in X-ray tomography is largely a function of the outermost ring of the zone plate used to focus the X-rays with the caveat that the use of high-resolution zone plates reduces the amount of the specimen kept in focus: for example, a 40 nm outer ring corresponds to a 1.3 μm depth of focus, and 25 nm zone plates are now being introduced. Fortunately, cryogenically prepared cells can tolerate large doses of X-ray radiation before the onset of visible damage (>1010 Gray), which makes the acquisition of a focus series for each tilt angle a viable proposition.
Arguably, the careful and often tedious optimization of sample preparation remains among the most important objectives and holds the key for the further integration of cryo-ET with other experimental methods, be it for sophisticated treatment schemes or correlations using advanced LM methods. We expect that these, as well as computational and technical developments will lead to attributing further functional aspects to cellular structures visualized in cryo-ET.
We thank Gabriela J. Greif for critical reading of the manuscript. This work was supported by DFG SPP 1128 grant and by the EU within the Network of Excellence NoE-3DEM.
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