Journal of Structural Biology 129, 48–56 (2000) doi:10.1006/jsbi.1999.4204, available online at http://www.idealibrary.com on

Cryo-electron of Neurospora Mitochondria

Daniela Nicastro,1 Achilleas S. Frangakis, Dieter Typke, and Wolfgang Baumeister

Abteilung Molekulare Strukturbiologie, Max-Planck-Institut fu¨ r Biochemie, 82152 Martinsried, Germany

Received October 11, 1999, and in revised form November 22, 1999

the three-dimensional volume by weighted back- Cryo-electron tomography was used to study the projection or algebraic reconstruction. Electron to- structural organization of whole frozen-hydrated mography is—in terms of resolution—the most prom- mitochondria from Neurospora crassa. Unlike mito- ising method for investigating pleomorphic structures chondria from many other species and tissues, in such as large supramolecular assemblies (Dierksen this case the cristae form a three-dimensional net- et al., 1995; Moritz et al., 1995; Grimm et al., 1997), work of interconnected lamellae. Basically, the three- dimensional structure of ice-embedded mitochon- organelles (Ladinsky et al., 1994; Mannella et al., dria from this species is consistent with previous 1994; Shillito et al., 1997; Perkins et al., 1997a, descriptions of mitochondria prepared by chemical 1998), or whole cells (Grimm et al., 1998). Unlike fixation and resin embedding. Nonetheless, ice- other 3-D visualization methods such as serial ultra- embedded mitochondria display some important thin sectioning or stereo imaging, electron tomo- differences: the outer surface of the mitochondria graphic studies of cellular structures have already was found to be rather smooth, the intermembrane attained resolutions in the range of 10 nm (Perkins space was constant in width, and distinct contact et al., 1997a; Baumeister et al., 1999). With the sites between the membranes were clearly revealed. automation of tomographic imaging procedures, low- Furthermore ATP synthase particles on the outer dose conditions can be implemented that allow the surface of an ‘‘inside-out vesicle’’ were visible in 3-D cumulative dose to which the biological specimen is reconstructions. Thus, cryo-electron tomography exposed to be limited to subcritical levels (2000–5000 can provide detailed insights into these organelles electrons/nm2) at magnifications that allow resolu- with minimal perturbations of the physiological tions well below 10 nm to be attained (Typke et al., state. This indicates that it is a realistic goal to achieve ‘‘molecular resolution’’ with rather large 1991; Dierksen et al., 1992, 1993; Koster et al., 1992; biological specimens in the near future, ultimately Braunfeld et al., 1994). This development opened up allowing the identification and localization of mac- new perspectives and applications for electron tomog- romolecules in their cellular context. ௠ 2000 Academic raphy, since it became feasible to investigate biologi- Press cal specimens embedded in vitreous ice. Key Words: cryo-electron microscopy; cellular to- To date, most electron microscopic and tomo- mography; frozen-hydrated specimens; contact sites; graphic studies of cellular structures are based on ATP synthase. conventionally prepared specimens, i.e., specimens that are subjected to chemical fixation and staining before being dehydrated and embedded in resin. INTRODUCTION However, these procedures may give rise to severe Conventionally recorded electron micrographs structural artifacts (see, for example, Gilkey and provide two-dimensional projections of a three- Staehelin, 1986; Kellenberger et al., 1992). One of dimensional object. In order to retrieve 3-D struc- the major goals of cellular structural biology is to tures, specimens must be tilted with respect to the elucidate the supramolecular organization in an electron beam and series of 2-D images at different undisturbed cellular context (Koster et al., 1997; projection directions recorded. This data set is then Baumeister et al., 1999). Structural investigations used to calculate a reconstruction or ‘‘tomogram’’ of that aim at ‘‘molecular’’ resolution cannot rely on conventional preparation techniques; only cryo- electron microscopy offers a prospect of maintaining 1 To whom correspondence should be addressed at Abteilung close-to-life structural organization (Dubochet et al., Molekulare Strukturbiologie, Max-Planck-Institut fu¨ r Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany. Fax: ϩϩ49ϩ89 1988). In addition to the structure-preserving effect 8578 2641. E-mail: [email protected]. of fast-freezing, the specimens are kept at tempera-

1047-8477/00 $35.00 48 Copyright ௠ 2000 by Academic Press All rights of reproduction in any form reserved. CRYO-ELECTRON TOMOGRAPHY OF Neurospora MITOCHONDRIA 49 tures near Ϫ185°C during microscopy, which consid- zero-loss mode of the energy filter. For further details of the erably reduces radiation damage to the specimen. experimental set-up, see Grimm et al. (1997). The angular range of the tilt series was about Ϯ65° with 1° increments (120–130 Specimens to be investigated by transmission elec- projection images). Data acquisition was carried out under low- tron microscopy should not exceed a thickness of dose conditions using automated procedures. The projection im- approximately 1 µm, even when intermediate accel- ages were recorded at 14 700ϫ magnification with a 1024 ϫ 1024 erating voltages are used and the microscope is pixel CCD camera; the pixel size at the specimen level was 1.7 nm. equipped with an energy filter removing the inelasti- Because of the increased specimen thickness at higher tilt angles, the exposure time was varied according to (cos ␣)Ϫ1. The defocus cally scattered electrons. Since most eukaryotic cells was set to 10 µm; thus the first zero of the phase-contrast transfer measure several micrometers in size, electron tomog- function was at a spatial frequency of (5.8 nm)Ϫ1, about half the raphy will not allow investigation of these cells Nyquist frequency. The cumulative dose of incident electrons for frozen in toto. However, organelles like mitochon- recording the tilt series was below 2000 electrons/nm2. dria are sufficiently small and can be embedded in Image processing. Image processing of the tilt series was carried out a posteriori on a Silicon Graphics workstation using toto in vitreous ice. Mitochondria are the cellular the EM software package (Hegerl, 1996). Projections were interac- organelles of energy conversion and sites of essential tively aligned with respect to a common origin, using 10-nm metabolic reactions. There is a growing number of colloidal gold particles distributed in the ice film as markers. diseases that have been linked to malfunctions of Subsequently 3-D reconstructions were calculated by weighted mitochondria (Wallace, 1999). Mitochondria are easy backprojection. In order to assess the resolution achieved in the reconstructions, 1-D line-plots were fitted through the geometric to isolate in a state that maintains the full physiologi- centers of repetitive and regularly arranged structural features. cal activity. They can display a remarkable polymor- From these line-plots the power spectra were calculated and phism, but some of the basic features such as the averaged (Fig. 6D). For three-dimensional visualization of mito- dual-membrane system composed of a smooth outer chondria, particularly of the architecture of the cristae, surface- membrane (OM) and an inner membrane (IM) with rendering techniques provided by the AVS software package were used. Although most of the mitochondrial membranes were clearly many invaginations that form the cristae are com- visible in reconstructed volumes, the inherent variable density mon to all forms. Most of these features were estab- distribution due to a noisy background prevented the use of lished by early electron microscopic studies (Palade, automatic techniques such as density thresholding for surface- 1952; Sjøstrand, 1953). Recent electron tomographic rendering. Therefore, contours of the cristae were manually studies of conventionally fixed mitochondria have enhanced prior to visualization similar to procedures described by Marko et al. (1988) and Perkins et al. (1997b). led to a revision of some traditional views of the internal organization of these organelles (Mannella RESULTS et al., 1994, 1997; Perkins et al., 1997a, 1998). Although these studies provided high-resolution mi- Here we present 3-D reconstructions from two crographs, it is essential for addressing some of the single-axis tilt series, each showing a single mitochon- still controversial issues to use frozen-hydrated drion embedded in vitreous ice (Fig. 1). In addition, a samples and thus to minimize structural perturba- specimen area containing a frozen-hydrated ‘‘inside- tions. In the present study we employed automated out vesicle’’ has been reconstructed (see Figs. 6A– cryo-electron tomography to investigate the struc- 6C). Such vesicles were frequently found in mitochon- ture of whole frozen-hydrated mitochondria from drial preparations; they are formed by released Neurospora crassa. mitochondrial inner membrane fragments and show a reverse organization with the former matrix side of the inner membrane outward. MATERIALS AND METHODS The size of the ellipsoidal mitochondria of Neuros- Specimen preparation. Mitochondria from N. crassa were pora was found to vary between 600 and 1500 nm. In isolated by differential centrifugation, basically following the most cases mitochondria were compressed to a thick- standard procedure as described in Sebald et al. (1979). Mitochon- ness of 200–450 nm in the direction perpendicular to drial sediment (obtained by centrifugation at 7600g) was sus- pended in an isotonic 0.25 M sucrose medium containing 0.1 mM the ice film. This compression is most likely the glucose in order to maintain mitochondria in a metabolically result of capillary forces exerted onto mitochondria active state. To prevent deleterious effects of anoxia on mitochon- in the short period of time between blotting of excess dria, the solution was gassed with air and stored on ice (4°C) for a fluid and plunge freezing. short time before fast-freezing. A droplet of the mitochondria The reconstructions provide detailed information suspension was applied to a copper grid covered with Quantifoil (holey carbon support film; Ermantraut et al., 1998) and pread- about the overall membrane organization and some sorbed 10- and 20-nm gold clusters. After being blotted, mitochon- specific features such as crista junctions and contact dria were embedded in vitreous ice by plunging the grid into liquid sites. Unlike in many other species and tissues, in ethane. The grid was then transferred to liquid nitrogen and Neurospora mitochondria the folded part of the inner Ϯ inserted into the microscope using a 70° tilt cryoholder (Gatan, membrane (crista membrane) forms a three-dimen- Pleasanton). Electron tomography. Several single-axis tilt series were ac- sional network of interconnected lamellae rather quired from different ice-embedded mitochondria using a CM 120 than an array of regularly spaced, parallel tubes Biofilter TEM (Philips, Eindhoven) operated at 120 kV in the (Figs. 2 and 3). Lamellar cristae extend throughout 50 NICASTRO ET AL.

FIG. 1. Results of two single-axis tilt series each showing a mitochondrion of Neurospora crassa embedded in toto in vitreous ice. (A, C) Electron micrographs of zero-degree projections of tilt series I (A) and series II (C). Both mitochondria are situated close to the edge of a hole in the carbon film that is visible in the bottom region of the images. (B, D) Central x–y slices through the reconstructed volume of the corresponding tilt series I (B) and II (D). Note the inside-out vesicle (V) in proximity to the mitochondrion of series I (B). OM, outer membrane; IM, inner membrane; C, crista; CJ, crista junction. the interior of the mitochondria (matrix) and branch arbitrary density distribution in the perpendicular predominantly in a tripartite fashion (Figs. 1B, 2, direction will remain invisible in 3-D reconstructions and 3). While the lateral extension of lamellar if the normal to it points into the ‘‘missing wedge’’ in cristae shows considerable variations (50–250 nm), reciprocal space. This means that such a layered their thickness was found to be remarkably uniform; structure is visible only if its inclination against the when the opposing membranes are included, it is x–y plane exceeds a certain angle. This limiting approximately 30 nm (Fig. 2). In reconstructions, the angle depends on the azimuthal orientation of the cristae appear to be predominantly oriented at steep layer and may vary between the complement of the angles, roughly perpendicular to the x–y plane; it is maximum tilt angle to 90° (approximately 30° in our very likely though that lamellae at small angles to case) and almost 90° in the worst azimuthal orienta- the x–y plane are also present. These, however, tion. remain invisible due to the limited tilt angle range At junctions, the crista membrane merges with and, as a consequence, the lack of information in the inner boundary membrane. The visible junctions Fourier space (‘‘missing wedge’’). As noted by Dierk- are rather extended laterally (Fig. 4), up to about 200 sen et al. (1995), any laterally smooth layer with an nm. Also, in the vicinity of crista junctions the CRYO-ELECTRON TOMOGRAPHY OF Neurospora MITOCHONDRIA 51

FIG. 2. Collage of x–y slices spaced by approximately 15 nm through the 3-D reconstructed volume of the tomographic tilt series I. The tilt angle ranged from Ϫ66° to ϩ59° with 1° increments (126 projections). In the reconstruction the smooth outer membrane, regularly sized intermembrane space, and the inner membrane forming interconnected lamellar cristae are clearly visible. The cristae project through the interior of the mitochondria and branch in a predominantly tripartite fashion (arrows). The matrix appears remarkably electron-dense in comparison to the intermembrane and intracristal spaces. intermembrane distance of cristae appears to be the inner volume of mitochondria is filled with slightly reduced in some cases. The outer membrane granular and remarkably electron-dense material is smooth and contiguously visible with the excep- (Figs. 1B, 1D, and 2). Large-scale inclusions such as tion of its upper and lower caps, which remain highly electron-dense intramitochondrial granules, invisible due to the ‘‘missing wedge’’ (Figs. 2 and 3). crystals, or glycogen particles were not found. The outer membrane and inner boundary membrane Although many small particles with sizes close to 10 of frozen-hydrated mitochondria are separated by a nm appeared to be visible, the resolution and the rather uniform intermembrane space with a mean signal-to-noise ratio were too low and the matrix too inner distance of 8 nm or a 20-nm OM-IM width crowded to extract any structural features. including both membranes (Figs. 1, 2, 4A, and 5). In contrast to the crowded interior of the mitochon- The intermembrane space between the outer and the dria, the reconstructed ‘‘inside-out vesicle,’’ located inner membranes is bridged at a number of clearly near one of the mitochondria, exhibited improved visible contact sites, and the OM-IM width is re- contrast and provided information at nearly ‘‘molecu- duced to approximately 12 nm (Fig. 5). The contact lar’’ resolution (Fig. 6). Electron-dense particles with sites seem to be randomly distributed and are not the shape of ‘‘stalked heads’’ were clearly visible on associated with crista junctions. The exact number the outside of the vesicle (Figs. 6A–6C). The diam- of contact sites per mitochondrion cannot be speci- eter of the heads measured about 8 nm and the thin fied, although approximately 10 contact sites per stalk appeared to be approximately 5 nm long origi- mitochondrion could be identified unambiguously nating from the membrane. At this region the mem- (Fig. 5). There are additional sites where the outer brane was thickened, indicating that the basal part and inner membranes appear to be in close contact, of the particles spans the membrane. Also notable is but a clear identification is not possible. that most particles are regularly arrayed, forming Compared to the homogeneous, electron-lucent contiguous rows with a mean distance of approxi- lumen of the intermembrane and intracristal spaces, mately 18 nm between neighboring particles (Figs. 52 NICASTRO ET AL.

FIG. 3. Surface-rendered representation of the mitochondrial reconstruction of tilt series I. (A) Orientation of mitochondrion as in Fig. 2. (B) In order to provide a three-dimensional impression of the complex mitochondrial architecture the reconstructed volume was tilted about the y-axis. The crista membrane that forms a three-dimensional network of interconnected lamellae is continuous with the inner boundary membrane (both shown in yellow). The outer membrane (magenta) is separated from the inner boundary membrane by a narrow intermembrane space of remarkably constant width. FIG. 4. A crista junction from the mitochondrial reconstruction of tilt series II. (A) An x–y slice of the reconstructed 3-D volume; a rectangle outlines the detail visualized in (B, C). (B, C) Surface-rendered representations showing the broad juncture between the inner boundary and the crista membrane (both yellow); outer membrane is shown in magenta.

6A–6C). Several rows of these particles were used to cristae have been described previously, particularly estimate the resolution to 7 nm (Fig. 6D). for fungal (e.g., Nunnari et al., 1997) and plant cells (e.g., Schieber et al., 1994). Prince (1999) recently DISCUSSION found tripartite branching, lamellar cristae in mito- The complex network of interconnected lamellar chondria from human steroid-producing Leydig cells, cristae in mitochondria of the fungus Neurospora is indicating that this type of ultrastructural organiza- obviously at variance with the more common text- tion is not restricted to plant and fungal cells. While book ultrastructure of mitochondria. However, it is the lateral extension of cristae varies considerably in known that mitochondria exhibit a great deal of our reconstructions, their thickness was found to be structural variability, dependent on the organism remarkably uniform. A similar thickness has been and type of cell. Mitochondria with interconnected reported for cristae in many other mitochondria, CRYO-ELECTRON TOMOGRAPHY OF Neurospora MITOCHONDRIA 53

FIG. 5. Contact sites. Selected slices of 3-D reconstructions of tilt series II (A) and I (B); rectangles outline clearly visible contact sites between outer and inner membranes. Boxes: zoom-in of the corresponding images showing the contact sites at higher magnification. regardless of their shape (e.g., Mannella et al., 1994; of the ice-embedded mitochondria is in good agree- Perkins et al., 1997a, 1998). Thus, one may ask how ment with the ‘‘orthodox’’ morphology (expanded this uniform intracristal space is determined in matrix) described by Hackenbrock (1968). However, ultrastructural terms. Possibly, the cristae are stabi- it is questionable whether the so-called ‘‘condensed’’ lized by molecular structures anchored in both oppos- conformation or contracted matrix of chemically ing membranes, similar to Omp␣, the coiled-coil fixed mitochondria really exists under physiological protein spanning the periplasmatic space of the conditions. bacterium Thermotoga maritima (Engel et al., 1992). Contact sites that are thought to be import path- However, no such structures have been reported for ways for destined for the matrix (for a mitochondria yet, and, at the present resolution, no review, see Schatz and Dobberstein, 1996) have been such structures are detectable in the tomograms. described for conventionally prepared mitochondria The lateral extension of membranous junctions by Hackenbrock (1966, 1968) and later authors (e.g., between crista membrane and inner boundary mem- Rassow et al., 1989; Brdiczka, 1991). Although some brane was found to be similar to that of the cristae of these studies provided detailed images, the pres- terminating at these sites. The observation of later- ence of obvious preparation artifacts such as dis- ally extended crista junctions in Neurospora mito- rupted, ‘‘scalloped’’ membranes, or swollen intermem- chondria is in contrast to the multiple tubular junc- brane spaces prevented a definite verification for the tions recently described in mitochondria of brown existence of contact sites. Clearly visible contact adipocytes from rat that also contain lamellar cris- sites between well-preserved and regularly spaced tae (Perkins et al., 1998). membranes of ice-embedded mitochondria now con- The smooth mitochondrial surface and the uni- firm the morphology and punctuate nature of these form intermembrane and intracristal spaces seen in functionally important membrane regions. our reconstructions are clear indicators that ice- The reconstructed inside-out vesicle illustrates embedding maintains mitochondria in a near- the potential of cryo-electron tomography for an in physiological state. A morphology devoid of swellings situ analysis of macromolecular and supramolecular or membrane disruptions was invoked as a criterion structures. At present an identification of specific for a close to in vivo preservation of mitochondria molecular structures is impossible due to the crowded (Malhotra and Tewari, 1991). The smooth surface nature of the matrix, the low signal-to-noise ratio, and close apposition between the outer membrane and limited resolution. However, the inside-out and the inner boundary membrane seem to be vesicle is considerably thinner (thickness about 70% typical for cryofixation even when it is followed by of organelle) and much less ‘‘crowded.’’ Obviously, freeze-substitution and resin-embedding (Malhotra these factors sufficiently improve the chances for and Tewari, 1991; Dalen et al., 1992; Pfanner et al., visualizing specific macromolecules. Since these 1992; Perkins et al., 1998). However, in mitochondria vesicles are formed by fusion of inner membrane prepared by the latter technique, collapsed cristae fragments of disrupted mitochondria, they contain and intermembrane spaces have been observed proteins associated with the membrane, such as (Dalen et al., 1992; Perkins et al., 1998); no such certain tricarboxylic acid cycle enzymes and respira- artifacts were found in the ice-embedded mitochon- tory chain components. Considering the quite dis- dria described in this communication. The structure tinct structural features of the stalked heads ob- 54 NICASTRO ET AL.

FIG. 6. (A) Collage of x–y slices of reconstructed inside-out vesicle. (B, C) High-magnification views of the periphery of the vesicle. The depicted details show membrane-bound particles on the outside of the vesicle; these electron-dense particles are apparently composed of a head (black arrows), a more or less visible stalk (arrowheads), and a basal region anchored in the membrane (white arrows). (D) Averaged power spectrum of 10 one-dimensional (1-D) plots that were fitted through the geometric head centers of the rather regularly arranged particles; a peak (arrow) could be detected at (6.8 nm)Ϫ1, indicating significant information in the 3-D reconstruction at this resolution; note that the amplitude is displayed in a logarithmic scale. served on the outside of the inside-out vesicle, these tions. It is our goal to further improve resolution to a particles are most likely ATP synthases. These pro- level that macromolecules and supramolecular as- tein complexes, also referred to as ‘‘inner membrane semblies in intact cells or organelles can be revealed; spheres’’ or F0F1 particles, were first visualized by this will undoubtedly provide new insights into their negative staining of inside-out vesicles (Fernandez- functional organization. In order to attain molecular Moran et al., 1964), and meanwhile the structure of resolution, further improvements of the instrumen- the F1 part has been determined by X-ray crystallog- tation (higher accelerating voltage, field-emission raphy (Abrahams et al., 1994). gun, energy filter, larger tilt angle range, and im- The results of this study demonstrate that cryo- proved tilt geometry) and image analysis (improved electron tomography is a promising tool for investi- feature detection, noise reduction, 3-D visualization) gating the three-dimensional organization of large are required and, hopefully, can be realized in the biological structures under near-physiological condi- near future. Then, cryo-electron tomography will be CRYO-ELECTRON TOMOGRAPHY OF Neurospora MITOCHONDRIA 55 able to bridge the ‘‘resolution gap’’ between confocal Grimm, R., Ba¨rmann, M., Ha¨ckl, D., Typke, D., Sackmann, E., and light microscopy, which allows investigation of dy- Baumeister, W. (1997) Energy-filtered electron tomography of ice-embedded actin and vesicles, Biophys. J. 72, 482–489. namic cell processes in situ, and techniques such as Grimm, R., Singh, H., Rachel, R., Typke, D., Zillig, W., and X-ray crystallography or NMR spectroscopy that Baumeister, W. (1998) Electron tomography of ice-embedded provide atomic resolution of isolated molecular struc- prokaryotic cells, Biophys. J. 74, 1031–1042. tures. Hackenbrock, C. R. (1966) Ultrastructural bases for metabolically linked mechanical activity in mitochondria. I. Reversible ultra- We thank W. Neupert, S. Nussberger, P. Heckmeyer, and M. structural changes with change in metabolic steady state in Braun for supplying us with conidia of Neurospora crassa and isolated liver mitochondria, J. Cell Biol. 30, 269–297. introducing us to their isolation protocol of mitochondria as well Hackenbrock, C. R. (1968) Chemical and physical fixation of as for helpful discussions. We are also grateful to D. 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