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Cell Biology International (2006) 30, 747–754 (Printed in Great Britain)
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Studying cellular architecture in three dimensions with improved resolution: Ta replicas revisited
Pilar Cabezas and Cristina Risco*
Department of Structure of Macromolecules, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma, Darwin 3, Cantoblanco, 28049 Madrid, Spain

Abstract

Metal replicas have been used for surface analysis of biological structures with a variety of spatial resolutions. Platinum (Pt) has been the metal of choice because it provides very stable replicas and images of high contrast. Some other metals, such as tantalum (Ta) have been reported to provide better resolution on isolated macromolecular complexes and cellular structures. Our goal is to study the gain in detail with Ta and to evaluate if it provides enough detail and resolution to assist in the study of complex volumes of intact cellular structures obtained by methods that reach molecular resolution. To this purpose Pt and Ta replicas of cellular structures and viruses have been studied by transmission electron microscopy (TEM). Replicas of Ta show new details on the surface of two types of isolated viral particles such as 100nm bunyaviruses and large, >300nm, vaccinia virus (VV). Inside cells, the structural pieces that build VV immature particles are visualized only in Ta replicas. Looking for smaller intracellular complexes, new details are also seen in nuclear pores from Ta replicas. Additional masses, most likely representing the cargo during transport, are distinguished in some of the pores. Visualization of proteins in plasma membranes strongly suggests that detail and resolution of Ta replicas are similar to those estimated for 3D maps currently obtained by electron tomography of viruses and cells.

Keywords: Cell architecture, Freeze-fracture, Freeze-etching, 3D analysis, Ta replicas.

*Corresponding author. Tel.: +34 91 5854507; fax: +34 91 5854506.

1 Introduction

Since the introduction of metal replicas for studying the surface of biological structures in combination with transmission electron microcopy, resolution has been limited by a number of factors such as the size of the metal grain overlaid on the structures and the shadowing conditions (Egelman et al., 1989; Ruben and Yurchenco, 1994). Platinum/carbon (Pt/C) has been the metal of choice for many years when visualizing surfaces of cellular structures, because it provides very stable replicas and images of high contrast (Fujimoto and Pinto da Silva, 1988; Risco and Pinto da Silva, 1995; Severs, 1991). The importance of a number of factors in the resolution and quality of metal replicas, such as the shadowing angle, the performance of rotary versus unidirectional shadowing, the outgassing times, or the heating of the specimens during metal deposition has been explored in the past to obtain the highest resolution with Pt replication of macromolecular complexes, either isolated or integrated into more complex biological structures (Costello and Escaig, 1989; Ruben, 1989; Slayter, 1976).

All these studies provided very valuable knowledge that was consequently incorporated as technical improvements in the freeze-fracture/freeze-etching units and protocols. Looking for alternative metals that could provide better resolutions, the mixture of platinum/iridium/carbon (Pt/Ir/C) or tantalum (Ta) have been tested (Bachmann et al., 1985; Peters, 1986; Wepf et al., 1991). Tantalum is a biocompatible inorganic material that has been used to support adequate reconstruction of tissues and functional three-dimensional cell cultures (Kokubo et al., 2004; Poznansky et al., 2000; Rosenzweig et al., 1997). In the structural biology field, tantalum has been used for making replicas of macromolecular complexes. Since Ta forms a thinner layer of fine grain when compared to Platinum, it distorts the true dimensions of particles and structures to a lesser extent. Thus, Ta replicas showed a detectable increase in resolution of individual particles, either isolated or integrated in membranes (Gross et al., 1985; Schnyder et al., 1991; Studer et al., 1981).

Sendai virus nucleocapsids, for example, present different helical states, one of them observed only in Ta preparations (Egelman et al., 1989). Optimal results were also obtained with membrane proteins reconstituted into proteoliposomes. Purified lac permease and cytochrome o oxidase were examined by freeze-fracture EM with Pt/C or Ta replicas (Costello et al., 1987). The intramembrane particle diameters in tantalum replicas were about 20–25% smaller than those observed in conventional platinum/carbon replicas, indicating that the dimensions of the particles revealed with tantalum more accurately reflect the sizes of the lac permease and cytochrome o. One important problem that is particularly difficult to resolve with integral membrane proteins is their state of oligomerization. The dimensions of intramembrane particles (IMPs) observed with tantalum replicas were sufficient to contain monomers of each protein and too small to contain dimers. Careful inspection of these IMPs reveals two major domains of unequal size surrounding a notch or cleft. This type of analysis can be particularly useful to study the structure of macromolecular complexes that have not been susceptible of isolation in homogeneity for subsequent single particle analysis, such as many membrane proteins.

The advantages of tantalum have also been demonstrated using Scanning Electron Microscopy (SEM) (Peters, 1985). Isolated nuclear envelopes were coated with a continuous film of chromium or tantalum. Surface studies may be important in understanding how the NPC (nuclear pore complex) interacts with other structures such as the lamina, chromatin, membranes and cytoplasmic structures and how this interaction is related to function. In addition to the NPC baskets and features of the cytoplasmic face of the NPCs, which have been shown by others, tantalum replicas showed a previously unknown structure, the NEL (nuclear envelope lattice) that appears to be distinct from the nuclear lamina (Goldberg and Allen, 1992). In addition, the nucleoplasmic filaments that make up the baskets were seen attached to the outer periphery of the coaxial ring at a position between each of its subunits. These filaments extend into the nucleoplasm and insert at the distal end to the smaller basket ring (Goldberg and Allen, 1993).

Our main objectives in this work have been exploring the potential of Ta after freeze-fracture and freeze-etching for 3D analysis of viral and cellular structures, and estimating the potential use of these replicas as a support in the interpretation of 3D maps of cells obtained with other techniques that reach a molecular resolution. Viruses are very adequate structural models for these studies since as isolated particles they exhibit a variable level of complexity and inside cells they produce a high number of identical structures, facilitating their detection and study within the very complex intracellular environment.

2 Materials and methods

2.1 Viruses and cells

Bunyamwera virus particles were purified from supernatants of infected BHK-21 cells by centrifugation in Optiprep gradients as described (Novoa et al., 2005a). Vaccinia virus (strain Western Reserve) was propagated in HeLa cells and intracellular mature viruses (IMVs) that were purified in sucrose gradients following well established methods (Esteban, 1984), were kindly provided by Dr. Mariano Esteban (CNB-CSIC, Madrid), as well as control and VV-infected HeLa cells. Cultures of Entamoeba histolytica were supplied by Dr. Rosana Sánchez (Instituto de Biotecnología, UNAM, Cuernavaca, Mexico).

2.2 Freeze-etching of isolated particles

Purified viral particles were adsorbed onto recently exfoliated mica sheets, fast-frozen in liquid ethane and transferred to a BAF 060 freeze-fracture unit (BAL-TEC, Liechtenstein) previously cooled at −150°C. When the vacuum reached 10−9mbar, the temperature was switched to −80°C and maintained for 3h. When etching was completed, shadowing was done at 45° for Pt or Ta and at 90° for carbon (Risco and Pinto da Silva, 1993). The thickness of the Pt layer was 2nm as controlled by a crystal quartz measuring device. For Ta, layers of 1.5, 2, 2.5 and 3nm were tested, 2.5nm being the thickness that provided the best results in terms of detail and contrast. Carbon layers of 20nm were made in all cases. A previous outgassing step of 120s for both Pt and Ta guns and 150s for C was applied. Evaporation started when the vacuum in the chamber was at least 5×10−6mbar. Pt was evaporated at 1.6kV and 60mA, Ta at 1.85kV and 110mA, and C at 1.85kV and 80mA. Replicas were floated in commercial bleach and maintained overnight before intensive washing in distilled water. Special care was taken when washing Ta replicas that bend easily. Replicas were then picked up in uncoated copper EM grids of 400 mesh (Taab Laboratories, Aldermaston, Berkshire, UK) and dried at room temperature before electron microscopy. Platinum, tantalum and carbon rods were supplied by BAL-TEC.

2.3 Negative staining of isolated particles

Viruses were adsorbed onto EM grids covered by formvar and carbon and made hydrophilic by glow-discharge. Adsorbed viral particles were washed and stained with 2% uranyl acetate using standard procedures (Novoa et al., 2005a).

2.4 Freeze-fracture and surface replication

For conventional freeze-fracture, cell cultures were fixed with aldehydes and cryoprotected with glycerol as described (Risco and Pinto da Silva, 1998; Risco et al., 2002), fast-frozen in liquid ethane and transferred to the BAF 060 unit at a temperature of −150°C and a vacuum of 10−7mbar. Fracture was done at the mentioned temperature (Risco and Pinto da Silva, 1993; Risco et al., 1994) and shadowing was immediately done on the exposed surfaces as described above. For surface replication, monolayers were frozen in the absence of cryoprotectants, transferred to the freeze-fracture unit and submitted to etching at −90°C for 10min before shadowing with Pt or Ta and carbon as described above.

2.5 Freeze-substitution

Infected cells were fast-frozen in liquid ethane and subsequently substituted (at −90°C in pure acetone containing 1% osmium tetroxide) in a dedicated automated freeze-substitution unit (AFS; Leica-Reichert-Jung, Vienna, Austria) as described (Risco et al., 2002; Salanueva et al., 2003). Samples were embedded in the epoxy-resin EML-812 (Taab laboratories, Aldermaston, Berkshire, UK). Ultrathin sections (30–40nm) were stained with uranyl acetate and lead citrate.

2.6 Image analysis

All samples were studied in a Jeol 1200-EX II electron microscope operating at 80kV. Conventional micrographs at 0° were taken at magnifications between ×10,000 and ×40,000. Stereo-pairs, that were taken after adjusting the z-axis at a magnification of ×25,000, consisted of pairs of images at +6° and −6°. Electron micrographs were scanned at a resolution of 400ppi using an Epson Perfection 2450 PHOTO scanner and Picture Publisher 8 software.

3 Results

Viruses and eukaryotic cells were processed by freeze-etching or freeze-fracture before making replicas of Pt or Ta. We have studied two enveloped viruses of different sizes and structural complexity (Fig. 1): Bunyamwera virus (BUNV), a Bunyavirus of approximately 100nm in diameter and vaccinia virus (VV), one of the largest viruses known to date with more than 300nm in its larger dimension. Isolated BUNV particles visualized by freeze-etching followed by Pt replication exhibit hexagonal and pentagonal contours (Fig. 1A, B) which suggest an icosahedral symmetry for the whole viral particle, not yet demonstrated for these viruses (Novoa et al., 2005a). No features are distinguished on the surface of the particles. However, a close packing of globular particles is seen on their surface when Ta is used (Fig. 1C).

Fig. 1

Visualizing isolated large macromolecular complexes: the surface of isolated viruses. Bunyamwera virus (A–C) and vaccinia virus (VV) (D and E) as visualized by freeze-etching and metal replication with Pt/C or Ta/W, as indicated. F and G show isolated Bunyamwera virus (F) and VV (G) as visualized by negative staining. Arrow in F points to the thick envelope of Bunyamwera virus while arrows in G point to deformed envelope parts that collapsed during drying of the particle. Bars: 100nm.

These globular particles, of an approximate diameter of 9nm, correspond to the heterodimers of viral Gc and Gn glycoproteins that form the mature spikes when the virus exits the cell (Salanueva et al., 2003). In the case of VV, Pt replicas show the characteristic elongated shape and smooth surface (Fig. 1D), widely described in the literature from images of cryo-SEM (Griffiths et al., 2001). Ta replicas, however, show numerous very fine particles that form linear arrays on the surface of the virus (Fig. 1E). These elongated “stripes”, of an approximate diameter of 8nm, do not seem to have a preferential orientation and build a rather complex pattern that has not been solved in the recently reported 3D reconstruction of this virus by electron tomography (Cyrklaff et al., 2005).

To make a comparison with a simple, widespread method for studying isolated particles, viruses were also visualized by negative staining (Fig. 1F,G). This method provided a different type of information such as the visualization of a rather thick envelope in Bunyamwera virus particles (Fig. 1F) or the soft, deformable nature of VV envelope (Fig. 1G). However, fine, well-preserved details on the surface of viruses were appreciated only in Ta replicas. The reason for this is that although negative staining is a very useful method that provides medium resolution when dealing with non-enveloped viruses and macromolecular complexes, it can be too disrupting for non-fixed membranous structures, such as viral envelopes (Dubochet et al., 1994; Harris and Scheffler, 2002; Kiselev et al., 1990).

Looking for large macromolecular assemblies inside cells, we have visualized viruses within the intracellular environment. We have studied the areas of assembly of vaccinia virus in infected HeLa cells (Fig. 2), in particular the perinuclear regions where viral factories and spherical immature viruses (IVs) assemble (Novoa et al., 2005b). The typical aspect of these areas with spherical immature viruses, as seen in two dimensions, is shown in thin sections of cells processed by freeze-substitution (inset on top of Fig. 2A). Pt replicas of these regions show rather flat areas (asterisks) where fractured IVs exhibit a rough texture in cross-fractured particles, as well as in concave and convex views of IVs (white stars in Fig. 2A).

Fig. 2

Visualizing large viruses inside cells: structures in the cytoplasm of VV-infected eukaryotic HeLa cells. Immature spherical VV particles (white stars) in the cytoplasmic areas of assembly, as visualized by freeze-fracture and metal replication with Pt/C or Ta/W (A and B, respectively). Inset on top of A shows an equivalent area of an infected cell processed by freeze-substitution (FS) and thin sectioning. Asterisks mark representative areas of the cytoplasm. Arrows in B point to linear arrays of particles in the direction of the alignment, a feature that is only distinguished in Ta/W replicas. Insets on the bottom show characteristic spherical viral particles. (C) Stereo-pair of the area shown in (B) and rotated 90° counterclockwise. Bars: 200nm.

Ta replicas show that IVs are formed by linear elements, observed in all views (arrows in Fig. 2B). Their width, around 30nm, is compatible with the thickness calculated for the viral crescents, membranous pieces that build the spherical viral particles. Their lateral arrangement has been hypothesized but not clearly visualized (Risco et al., 2002). Cytoplasm around viral particles is also filled with structures, mainly particles of a variety of sizes (asterisks). Insets show higher magnification views of IVs where it can be seen that while Pt replicas do not show a defined pattern for the particles, Ta reveals a preferential orientation of the particles in linear arrays (marked by the arrow). Stereo-pairs of Ta replicas show that lateral attachment of linear membranes is clearly seen in all views of fractured particles (Fig. 2C). This feature is compatible with the formation of IVs by lateral association of viral crescents (modified membranes derived from the tubular pre-Golgi elements), as previously suggested by serial sections and freeze-etching studies (Risco et al., 2002).

Looking for smaller macromolecular-containing structures inside cells, we have studied the nuclear envelope (Fig. 3). There are 3D maps of medium resolution of nuclear pores recently obtained by electron tomography (Beck et al., 2004). After freeze-fracture and Pt replication, the nuclear envelope of E. histolytica cells shows a regular distribution of nuclear pores on a rather flat sheet (Fig. 3A). Nuclear pores have particles with no defined pattern (Fig. 3A, inset). When these cells are replicated with Ta, the pores have a well-defined ring inserted in a cavity (Fig. 3B). The particles of the ring and the surrounding areas are more patent (inset in Fig. 3B). In both types of replicas we show images generated when the fracture plane goes along the outer membrane of the envelope. Nuclear pores with or without a central mass are not clearly distinguished in Pt replicas (Fig. 3A). However, Ta replicas clearly show the presence of an additional mass in different locations within the central area of the pores or on their periphery (arrows in Fig. 3B) while some pores are clearly devoid of this additional mass (arrowheads in Fig. 3B). A mass of variable size, shape and position, known as the central plug/transporter (CP/T), has been recently visualized by electron tomography, and its analysis supports the notion that it makes up, at least in part, cargo complexes arrested during translocation (Beck et al., 2004).

Fig. 3

Details of cellular macromolecular complexes in situ: nuclear pores within the nuclear envelope of Entamoeba histolytica cells visualized by freeze-fracture and metal replication with Pt/C or Ta/W, as indicated. Nuclear pores with or without a central mass (arrows and arrowheads, respectively) are distinguished in Ta/W replicas. Bars: 250nm in main fields and 75nm for the insets.

A direct demonstration of the increase in detail of large cellular regions with Ta replicas is shown in Fig. 4. Regions of the plasma membrane of E. histolytica cells are here visualized after freeze-fracture and replication with Pt (Fig. 4A) or Ta (Fig. 4B). In addition to individual particles randomly distributed and representing integral membrane proteins, as documented in the literature (Verkleij and Ververgaert, 1978), Pt replicas show some linear structures of unknown nature (Fig. 4A, arrows). Ta replicas reveal that these straight elements are in fact composed of individual particles that exhibit a linear arrangement in the bilayer (Fig. 4B, arrows). Thus, the rod-like structures seen in Pt replicas are in fact “decoration artifacts” (Peters, 1979). In this sense, we have recently demonstrated by immunogold detection and label-fracture techniques that certain receptors have a propensity to form linear arrays in the plasma membrane (Schamel et al., 2005). A variety of diameters are observed for the particles visualized in the plasma membrane replicated with Ta, some of them apparently below 5nm (Fig. 4B).

Fig. 4

Intramembrane particles (IMPs) in plasma membrane. Entamoeba histolytica cells were processed by freeze-fracture and metal replication with Pt/C or Ta/W, as indicated. Arrows in (A) point to linear, stick-like elements of the membrane that in fact correspond to individual particles with a linear arrangement, as visualized in replicas of Ta/W (arrows in B). Bars: 100nm.

4 Discussion

The development of methods for the three-dimensional characterization of cells with molecular resolution is nowadays a real breakthrough in structural biology since it offers multiple possibilities and applications (Lučić et al., 2005). In this field, electron microscopy methods are showing new achievements every day. The transition from single particle analysis, a method that requires the computational combination of identical particles (either macromolecular complexes or viruses) to the structural characterization of unique, non-symmetrical objects (such as cells and their parts) is being made possible through the development of approaches such as electron tomography. This is providing 3D maps of organelles and whole cells with unprecedented detail (He et al., 2003; Kürner et al., 2005; Marsh et al., 2000; McIntosh et al., 2005; Messaoudi et al., 2006; Murk et al., 2003; Soto et al., 1994; Steven and Aebi, 2003). Interpretation of tomograms is, however, a complex task and new image processing methods are now being developed (Bajaj et al., 2003; Fernández and Li, 2003; Subramaniam and Milne, 2004). Understanding the very complex tomograms originating from interesting areas of eukaryotic cells will be a considerable challenge, in particular with vitreous sections (Leis et al., 2005). In this sense, the assistance of methods that provide more familiar 3D images with an adequate resolution can be of considerable help.

Freeze-fracture and related techniques have considerably helped to understand the organization of biological membranes (Pinto da Silva and Branton, 1970; Pinto da Silva, 1987). Metal replication has considerably helped to characterize many biological structures in three dimensions (Cetonze et al., 1986; Fujimoto and Pinto da Silva, 1988). Technical developments and knowledge accumulated in this field over the years allow the fabrication of high quality replicas following standard procedures, with improved molecular resolutions for complex cellular structures. The fact that Ta provides higher resolution when visualizing macromolecular complexes and cellular structures, as shown in the present study, is not a new concept. However, we believe that our images provide an intuitive idea of the usefulness and new applications that these methods can have today in the field of 3D EM. In fact new data such as the small masses visualized in the pore complexes, the surface covered by small particles in vaccinia mature virus and the organization of vaccinia immature virus as linear attached pieces, are new details visualized in the images presented in this study. Replicas can also be easily combined with cytochemical techniques for a specific detection of components (Pavan et al., 1990; Pinto da Silva et al., 1981; Robenek et al., 2005; Visco et al., 2000). This detection can be of considerable help for localizing functions in complex 3D maps. Technical advances in EM cytochemistry will also be important in this field (Bendayan, 2000; Mayer and Bendayan, 2001).

In terms of detail, our results suggest that we can be visualizing rather small individual monomers in macromolecular aggregates integrated in cellular structures with a resolution close to the results recently obtained by electron tomography of cellular organelles and large viruses (Baumeister, 2005; Grünewald et al., 2003). With the limitations coming from the fact that we are not visualizing the structures themselves but a replica and the final size is modified, we can estimate that with Ta replicas of the surface of complex structures we are reaching resolutions similar to the values calculated for electron tomograms of cell parts. In fact, the surface of VV mature virus in Ta replicas shows small structures that were not solved by electron tomography of vitrified virus (Cyrklaff et al., 2005). When dealing with whole cells we believe that in combination with high-preservation methods for large structures, such as high pressure freezing (Muller-Reichert et al., 2003; Studer et al., 2001; Walther, 2003), stereo-pairs of Ta replicated surfaces can provide complementary information. This can help us in the process of segmentation, particularly through the assignment of dimensions to certain cell parts and macromolecular assemblies in 3D, when analyzing the very complex tomograms of eukaryotic cells.

Acknowledgments

We are very grateful to Dr. Rosana Sánchez (Instituto de Biotecnología, UNAM, Cuernavaca, Mexico) and Dr. Mariano Esteban (CNB, Madrid) for providing some of the samples used in this study. This work has been supported by grants 07B/0039/2002 and GR/SAL/0671/2004 from the Comunidad de Madrid and BMC2003-01630 from the Ministerio de Educación y Ciencia of Spain (to C.R.).

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Received 21 March 2006/4 April 2006; accepted 11 May 2006

doi:10.1016/j.cellbi.2006.05.006
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