Fine Structure of the Deinococcus radiodurans Nucleoid Revealed by Cryoelectron Microscopy of Vitreous Sections

Transmission electron microscopy revealed that the nucleoidof the extremely radioresistant bacteria Deinococcus radioduransmay adopt an unusual ring shape. This led to the hypothesisthat the tight toroidal package of the D. radiodurans genomemight contribute to radioresistance by preventing diffusionof ends of double-stranded DNA breaks. The molecular arrangementof DNA in the nucleoid, which must be determined to test thishypothesis, is not discernible by conventional methods of electronmicroscopy. We have applied cryoelectron microscopy of vitreoussections and found that the DNA arrangement in D. radioduransdiffers from toroidal spooling. Diffuse coralline nucleoidsof exponentially growing D. radiodurans do not reveal any particularmolecular order. Electron-dense granules are generally observedin the centers of nucleoids. In stationary-phase cells, thenucleoid segregates from cytoplasm and DNA filaments show locallyparallel arrangements, with increasing aspects of cholestericliquid crystalline phase upon prolonged starvation. The relevanceof the observed nucleoid organization to the radiation resistanceof D. radiodurans is discussed.

INTRODUCTION

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Introduction

Deinococcus radiodurans is a gram-positive, nonsporulating bacteriumwhich usually grows in tetrad form. This organism is interestingbecause of its extreme resistance to DNA damage induced by ionizingradiation (4). It exhibits detectable survival after irradiationof 15,000 Gy (4) and grows continuously at 60 Gy/h (25). Theunusual radiation resistance of D. radiodurans results fromits ability to repair the genome, containing more than a hundreddouble-stranded DNA breaks, without mutations and loss of genomeintegrity (4, 32). The double-stranded DNA break repair in D.radiodurans is recA-dependent (10), but an explanation for itsremarkable efficiency is yet to be found.

Initial analysis of D. radiodurans by transmission electronmicroscopy was performed in the 1970s (39, 40). Analysis focusedmainly on the structure of the cell wall, in which a periodicS layer was described and characterized in detail (5, 38, 40).Methods available at that time did not reveal an unusual structurewhich could contribute to the radioresistance of D. radiodurans.

Interest in the organization of D. radiodurans has reawakenedwith recent advances in transmission electron microscopy techniques.Levin-Zaidman et al. (29). applied rapid freezing and freeze-substitutionfor their study of D. radiodurans nucleoids. They found thatits nucleoid adopts an unusual ring shape. It is known thatin vitro DNA tends to form toroidal aggregates in the presenceof condensing agents (21). In these toroids, DNA molecules arecondensed in a closely packed hexagonal arrangement (20). Levin-Zaidmanat al. assumed that the ring-shaped nucleoid of D. radioduranshas basically the same structure. The hypothesis has been madethat the toroidal shape facilitates repair of the fragmentedgenome because its densely packed structure prevents double-strandedDNA break ends from diffusing (13, 29).

The molecular arrangement of the DNA in the D. radiodurans nucleoidmust be determined in order to test this hypothesis. Conventionalmethods of electron microscopy do not reveal the arrangementof DNA in bacteria, even when rapid freezing and freeze-substitutionstudy are used because the bacterial chromatin aggregates duringdehydration in the organic solvents, which are necessary insuch preparations (23). Obtaining the fine structure of bacterialchromatin requires that fully hydrated specimens be observed.

Cryoelectron microscopy of vitreous sections (CEMOVIS) enablestransmission electron microscopy observation of fully hydratedbiological material. This method relies on vitrification ofbiological samples by rapid cooling, similar to the techniqueused in freeze-substitution. Then, instead of replacing thesolidified water with an organic solvent and embedding the specimenin resin, native, unstained vitreous samples are cut at –140°Cinto thin sections which are observed in a cryoelectron microscopeat even lower temperatures (2). CEMOVIS has been successfullyapplied to different biological samples, including bacteria.A number of new structural details not preserved by other methodsof electron microscopy have been visualized in the cell envelopeand extracellular matrixes of bacteria (3, 30, 31).

Single isolated DNA molecules can be visualized in a thin layerof vitreous water (12), but tracing DNA inside the crowded environmentof a living cell remains a challenging task even if the structureis perfectly preserved by vitrification. Only when the DNA filamentsare ordered and favorably oriented it is possible to determinetheir arrangement. CEMOVIS resolved the local DNA package inhuman and horse spermatozoa (36). It therefore seems probablethat, if there is an ordered DNA package in D. radiodurans,it should be revealed in vitreous sections.

In this study we used CEMOVIS to examine the structure of nucleoidsin exponentially growing and stationary-phase D. radiodurans.We found that nucleoids of exponentially growing bacteria havediffuse coralline shapes and do not show a visible molecularorder. Electron-dense granules are generally observed in thecenter of nucleoids. In stationary-phase cells, the nucleoidsegregates from the cytoplasm and DNA filaments show locallyparallel arrangements, with increasing aspects of cholestericliquid crystalline phase upon prolonged starvation. The possiblerelevance of the observed nucleoid organization to the radiationresistance of D. radiodurans and to the hypothesis of Levin-Zaidmanet al. will be discussed.

MATERIALS AND METHODS

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Materials and Methods

D. radiodurans strain SARK was grown in TGY broth (0.8% Bactotryptone, 0.1% glucose, 0.4% yeast extract) at 30°C withvigorous shaking. Exponentially growing bacteria were collectedafter 10 to 12 h of growth at an optical density at 600 nm of0.4 to 0.6. Stationary-phase and long-stationary-phase bacteriawere collected after 4 days and 12 days of incubation, respectively.Bacteria were harvested by centrifugation with a Sorvall RS28Scentrifuge for 5 min at 3,000 rpm. A soft pellet of bacteriawas suspended with the same volume of 30% dextran (42 kDa, Sigma-Aldrich,St. Louis, MO), mixed by pipetting, and high-pressure frozenwithin 5 min in a Leica EMPACT (Leica, Vienna, Austria) apparatus.Thin frozen hydrated sections were obtained with a diamond cryoknife(Diatome, Biel, Switzerland) in a Leica FCS Ultracut S cryomicrotomewith a nominal cutting feed of 50 nm at –140°C asdescribed previously (3). For plunge freezing, 5 µl ofbacterial suspension was placed on a holey carbon grid and frozenby plunging into liquid ethane, as described (1).

Grids containing thin frozen-hydrated sections or vitrifiedthin layers were mounted in a Gatan 626 cryospecimen holder(Gatan, Warrendale, PA) and observed below –180°Cin Philips CM12 and CM100 cryoelectron microscopes (FEI, Eindhoven,The Netherlands) operating at an acceleration voltage of 80kV. Electron micrographs were recorded on Kodak SO-136 electronimage films or a 1K Multiscan charge-coupled device camera (Gatan,Warrendale, PA) or 2K TemCam charge-coupled device camera (TVIPSGmbH, Gauting, Germany). Negatives were digitalized using anImacon Flextight Precision III scanner (Imacon, Redmond, WA).The contrast of micrographs was adjusted with Adobe Photoshop.No other image correction was performed. Fourier transformsof images were made with the Image J program (National Institutesof Health [http://rsb.info.nih.gov/ij/]).

RESULTS

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Results

General morphology of D. radiodurans. The cell morphology of D. radiodurans was checked immediatelybefore vitrification by phase-contrast light microscopy. Inall cultures D. radiodurans cells were observed mainly in tetrads,but diplococci were also seen occasionally.

D. radiodurans was well visible in vitreous sections. Figure1 shows typical tetrads from exponentially growing (A), stationary-phase(B), and long-stationary-phase (C) cultures. The cytoplasm ofthe adjacent cells in the tetrad was either separated or interconnectedthrough incomplete septa. Cutting artifacts such as compression,knife marks (white arrows), and crevasses are seen in the sections.Compression appears as a shortening of cell dimensions alongthe direction of cutting, resulting in the elliptical shapeof the bacteria. The cutting direction is revealed by seriesof knife marks (Fig. 1A, B, and C, white arrows), which areirregularities in the thickness of sections originating fromunevenness of the cutting edge of the knife. Crevasses are fissuresperpendicular to the cutting direction. These cutting-inducedartifacts are still frequent in vitreous sections (3) and mustbe considered during the interpretation of images.

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FIG. 1. (A, B, C) Vitreous sections of typical D. radiodurans tetrads from exponentially growing (A), stationary-phase (B), and long-stationary-phase (C) cultures were imaged with high defocus (5 to 10 µm) to obtain strong contrast favorable for general morphology mapping. Knife marks (white arrows) and crevasses (Cr) are cutting artifacts. H, surface contamination with hexagonal ice. The cytoplasm of some adjacent cells in the tetrad is interconnected through incomplete septa (S). A large electron-dense granule (DG) is visible inside the exponentially growing cell. The small granules are ribosomes (R). In the central part of the exponentially growing and stationary-phase cells, ribosome-free areas (RFA) are seen and are outlined in one cell of the tetrad. Note the dispersed coralline shape of the ribosome-free areas in exponentially growing cells (A) and the compact roundish shape in stationary-phase cells (B). Some membranous structures (M) are the only distinguishable structures in the highly dense content of long-stationary-phase cells (C) in these imaging conditions. Irregularities in the structure of the outer part of the cell envelope (black arrowheads) are often observed in vitreous sections. (D) Magnified fragment of the cell envelope. The cytoplasmic membrane (CM), the diffuse dense layer (DL), the outer membrane (OM), and the outermost periodic S-layer (S) of the cell wall are distinguishable in vitreous sections. (E) Whole-mount exponentially growing tetrad plunge-frozen in culture medium. Note electron-dense granules (white arrows). Scale bars: A, B, and C, 500 nm; D, 100 nm; E, 1 µm.

A cell envelope is clearly visible around D. radiodurans cells.In regions of favorable orientation three sharp, high-contrastlayers and one diffuse dense layer are visible in the cell envelope(Fig. 1A, B, and C). Such organization of the cell envelopeis observed in all types of cultures. Figure 1D shows a magnifiedfragment of the cell envelope. The innermost sharp layer isthe cytoplasmic membrane, while the intermediate one is theouter membrane. The outermost layer is the S-layer in whicha periodic organization is seen on cross-sections and a hexagonalorder on tangential sections (not shown). The diffuse denselayer is seen in the periplasmic space. It is most pronouncedin long-stationary-phase cells. The outer membrane and the S-layersurround the entire tetrad and do not enter the septa. Irregularitiesin the structure of the outer part of the cell envelope, appearingas bubbles and wrinkles of the outer membrane and S-layer, areoften observed in vitreous sections (Fig. 1C, black arrowheads).

Internal organization of cells differs in exponentially growing, stationary-phase, and long-stationary-phase cells. Multiple small granules and one or several large electron-densegranules are visible inside exponentially growing cells (Fig.1A).The small granules are identified as ribosomes from theircharacteristic size and appearance. Ribosomes are distributedunevenly within the cell volume. The highest concentration ofribosomes is seen at the cell periphery. In the central partof the cell, individual ribosomes or small groups of ribosomesare spaced by ribosome-free areas. In a majority of exponentiallygrowing cells these ribosome-free areas have a dispersed corallineshape (Fig. 1A, outlined).

Electron-dense granules are located in the central part of exponentiallygrowing cells. Electron-dense granules have an elliptical shapein vitreous sections (Fig. 1A, dg). The large diameter of electron-densegranules rarely exceeds 400 nm. The small diameter of the ellipseis oriented along the cutting direction. This suggests thatelectron-dense granules are spherical bodies deformed into anelliptical shape by cutting-induced compression. In order tocheck whether electron-dense granules are an artifact of incubationwith the cryoprotector, we performed plunge-freezing of whole-mountbacteria directly in culture medium. The thickness of the bacteriafrozen in vitreous thin layers is too large for high-resolutionimaging with a normal-voltage microscope, but due to their highelectron density, electron-dense granules are nevertheless wellvisible (Fig. 1E, white arrows). This proves that electron-densegranules are a natural component of exponentially growing cells.

Vitreous biological material is prone to beam damage. Irradiationwith the electron beam changes the structure of the biologicalmaterial. The ultimate state of damage is apparent as bubbling.We found that electron-dense granules begin to bubble simultaneouslyor soon after cell membranes, whereas ribosome-free areas arethe most bubbling-resistant regions of cells.

In typical stationary-phase cells (Fig. 1B) electron-dense granulesare not observed and ribosomes are excluded from the centralpart of the cells, forming large unified ribosome-free areas.These ribosome-free areas have a roundish shape and homogenouscontents. Ribosome-free areas in adjacent stationary-phase cellscan be interconnected (Fig. 1B).

On a few occasions, in both exponentially growing and stationary-phasecells, we found ring-shaped ribosome-free areas with an islandof ribosome-rich cytoplasm in the center.

The content of a typical long-stationary-phase cell (Fig. 1C)has a high electron density, which makes it difficult to identifythe internal components. Ribosomes and electron-dense granulesare not distinguishable in long-stationary-phase cells. Occasionallysome membranous structures are seen inside long-stationary-phasecells.

Interestingly, the bacterial populations in stationary-phaseand long-stationary-phase cultures are not completely uniform.About 1% of the cells in a stationary-phase culture and fewcells in a long-stationary-phase culture present aspects typicalof exponentially growing cells; diffuse ribosome-free areasand electron-dense granules.

Fine structure of the nucleoid seen on vitreous sections. It is known from studies of freeze-substituted bacteria stainedwith osmium amine (19) that the bacterial nucleoid is locatedin ribosome-free areas. Hence, the ribosome-free regions visiblein vitreous sections of exponentially growing and stationary-phasecells of D. radiodurans can be identified with the nucleoid.

Contrary to what happens in stained embedded specimens, theaverage density of the various regions of D. radiodurans isabout the same everywhere, except in the dense granules. Imagingconditions favoring phase contrast over large areas must beused in order to obtain a significant contrast between the nucleoidand the rest of the bacteria.

Such conditions, implying high defocus (5 to 10 µm), havebeen used for recording Fig. 1 A to C. They are, however, notadequate for high-resolution observations. In order to revealthe molecular arrangement of the DNA in the nucleoid, a defocuscompatible with the expected distances of few nanometers hasbeen used (<3 µm).

Exponentially growing cells. Figure 2 shows a high-resolution view of the central regionof an exponentially growing cell. Electron-dense granules arerecognized because of the high contrast. As well as electron-densegranules, ribosomes and other granules, which can be transcriptionor replication complexes, are visible. The space between granulesis disordered, and Fourier transform of the image does not revealany predominant distance (Fig. 2, inset). Together these resultssuggest a dispersed, actively transcribed nucleoid without orderedcompaction.

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FIG. 2. High-resolution view of the central part of an exponentially growing cell (defocus, 1.8 µm). Electron-dense granule (DG), ribosomes (R), and other granules are visible. The space between the granules is disordered. Inset: Fourier transform of the outlined region. Scale bars: image, 100 nm; Fourier transform, (5 nm)^–1.

Stationary-phase cells. At high resolution, compact, roughly circular ribosome-freeareas of stationary-phase cells do not appear to be homogenousany more, and specific textures become apparent in them (Fig.3A). The "dotted" pattern (Fig. 3B) consists of highly contrastingdots and lines which are clustered and, in several cases, formregularly spaced arrays (Fig. 3B, underlined with white). The"stripy" pattern is formed from longer and partially parallellines (Fig. 3C) with lower contrast than the dots in the dottedpattern. Figures 3D and 3E show an intermediate case betweendotted and stripy patterns which simultaneously contains dotsand short and longer lines.

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FIG. 3. Fine structure of the nucleoid in stationary-phase cells. A, High-resolution view of stationary-phase cell (defocus, 2 µm). At this defocus the contrast of ribosomes (R) is faded, but fine structural details are visible in the nucleoid. Insets show Fourier transforms of the corresponding marked regions of the cell. CM, cytoplasmic membrane. B, C, D, and E, Different structural textures of the nucleoid. B, Dotted pattern. Regularly spaced arrays of dots are underlined in white. C, Stripy pattern. D and E, Intermediate case between dotted and stripy patterns which simultaneously contains dots and short and longer lines. The clusters of dots and lines are spaced by the regions without definable structure (asterisks). The aspects of the local order are less pronounced in some nucleoids (E). Scale bars: A, 100 nm; B, C, D, and E, 20 nm; Fourier transforms, (5 nm)^–1.

The high contrast of the dots suggests an accumulation of densityalong most of the section with a thickness averaging 70 nm.The diameter of the dots is in the range of 2 to 2.5 nm. Takentogether, these aspects are characteristic of a DNA fragmentseen along the viewing direction (6, 27). The dotted patterncan only be seen as a bundle of locally parallel DNA filamentswhose direction is perpendicular to the section plan. The stripypatterns confirm the local parallel orientation of the DNA.They appear when the bundle is tilted with respect to the viewingdirection. The Fourier transform reveals no reinforced dimensionsin the ribosome-rich area (cytoplasm, Fig. 3A, left inset),whereas distances corresponding to 4 to 6 nm are reinforcedin the ribosome-free regions. This characteristic order is bestseen in the dotted regions (maximum at ca. 4.8 nm; Fig. 3A,right inset) and in the stripy areas.

The regions with local order do not entirely occupy ribosome-freeareas. The clusters of dots and lines are spaced by regionswithout definable structure (Fig. 3B, C, and D, asterisks).The aspects of the local order are less pronounced in some nucleoids(Fig. 3E).

Long-stationary-phase cells. The crowded interior of long-stationary-phase cells does notallow us to distinguish the ribosome-containing area from theribosome-free area at low magnification. Nevertheless, the specificorganization of the central region of the cell revealed at higherresolution is similar to the dotted-stripy pattern observedin stationary phase (Fig. 4). The lines are frequently organizedin arches characteristic of a cholesteric arrangement (8). Thissuggests that the DNA in the nucleoids of long-stationary-phasecells has a higher degree of order than in stationary-phasecells. The Fourier transform shows that the average interfilamentdistance corresponds to 4 nm (Fig. 4, inset), which is slightlyshorter than in stationary-phase cells.

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FIG. 4. Nucleoid of long-stationary-phase cells shows the aspect of cholesteric arrangement: arches (dashed lines) formed by dots and lines (defocus, 1.6 µm). H, surface contamination with hexagonal ice. Inset: Fourier transform of the marked region. Scale bars: image, 100 nm; Fourier transform, (5 nm)^–1.

DISCUSSION

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Discussion

CEMOVIS revealed aspects of D. radiodurans structure which arenot resolved by conventional electron microscopy embedding andsectioning techniques. For example, the periodic structure andhexagonal order of the S-layer of the cell envelope are directlyseen in vitreous sections, whereas they were previously observedonly on biochemically isolated S-layers or on freeze-etchedcells (5, 37, 38).

Another characteristic aspect of native D. radiodurans structureis the dense spherical granules (electron-dense granules). Recentstructural studies did not report electron-dense granules (9,29), although early freeze-etching studies described sphericalstructures reminiscent of electron-dense granules in their sizeand location (37). Thornley et al. also observed dense granulesin resin sections on a few occasions, but their content wasoften lost during cutting (39). It seems, therefore, that electron-densegranules do exist in D. radiodurans but they are optimally preservedonly in frozen-hydrated material.

Electron-dense granules are located in the central part of exponentiallygrowing bacteria in the nucleoid region. This addresses a questionabout the possible chromatin nature of electron-dense granule.However, their high sensitivity to beam-induced bubbling suggeststhat they do not have a high nucleic acid concentration, sincearomatic-rich materials are radiation resistant (11). Due tostructural similarity, Thornley et al. (39) associated electron-densegranules with polyphosphate granules observed in other speciesof bacteria. Phosphate, in combination with proteins, wouldaccount for the high density and radiation sensitivity of thegranules. The precise composition of electron-dense granules,however, remains to be identified.

CEMOVIS successfully revealed aspects of the molecular arrangementof DNA within D. radiodurans nucleoids. The diffuse corallinenucleoids of exponentially growing cells do not show any particularorder. We attribute this to the high transcriptional and replicationactivity required for active growth. A local order first appearsin stationary-phase cells in the form of bundles of locallyparallel DNA filaments with an average interfilament distanceof 4.8 nm. Upon prolonged starvation the aspect of cholestericliquid crystalline order is observed and the average interfilamentdistance shortens to 4.0 nm. The gradual increase in the localorder together with reduction of the interfilament distancesuggests a liquid crystalline organization of the D. radioduransnucleoid.

It is known that liquid crystalline phases of DNA spontaneouslyassemble in vitro with increasing DNA concentration (26). Inbacteria, the increased DNA concentration leading to liquidcrystallization may result from accumulation of high-copy plasmids(34), but this is not the case for stationary-phase cells ofD. radiodurans, in which DNA content per cell is lower thanin exponentially growing cells (18). Since at stationary phasethe nucleoid segregates to a compact round domain excludingribosomes, the crowding of DNA can be originated by its redistributioninto the confined part of the cell volume. This effect can berelated to the fact that cholesteric liquid crystalline nucleoidswere found in starving Escherichia coli lacking Dps, an abundantstarvation-induced unspecific DNA binding protein (16). In contrast,wild-type E. coli and strains overexpressing Dps show nucleoidcompaction by formation of DNA-Dps cocrystals with a specificstructural appearance different from that observed in D. radiodurans(15, 16). It can therefore be concluded that the segregationand compaction of the D. radiodurans nucleoid can be drivenby a decrease of protein-DNA binding in the stationary phase.In addition, an unusually high concentration of Mn²⁺ ions foundin D. radiodurans (9, 28) can facilitate liquid crystallinecompaction by compensating for repulsive forces between DNAmolecules.

A minority of cells in stationary-phase cultures of D. radioduranshave the typical morphology of exponentially growing cells.We attribute this morphological polymorphism to the presenceof mutants gaining a growth advantage during the extended stationaryphase (14).

Nucleoid shapes that can be interpreted as rings have been foundin only a few cases. This observation argues against the resultsof other researchers showing an abundance of ring-like nucleoids,which were considered DNA toroids (29). We suspect that conventionalstudies have misinterpreted the dispersed nucleoids as DNA toroidswhen the central electron-dense granule is lost during samplepreparation and the resolution is insufficient to define themolecular arrangement. Our high-resolution analysis revealsthat the DNA arrangement in nucleoids of D. radiodurans differsfrom toroidal DNA spooling both in actively growing and in stationary-phasecultures.

Diffuse coralline nucleoids without a specific molecular orderobserved in exponentially growing D. radiodurans are similarto those of nonradioresistant bacteria (7, 19, 30). Nevertheless,exponentially growing cells of D. radiodurans are known to tolerate5,000 Gy (33). Stationary-phase cultures have a radioresistanceapproximately three times higher, but this increase alreadyoccurs at the beginning of the stationary-phase (24) and istherefore independent of the ordered genome compaction whichappears with the aging of the culture. This suggests that thearrangement of the nucleoid does not play a key role in theradioresistance of D. radiodurans. Even if it does, the mechanismmust differ from the one proposed by Levin-Zaidman et al. (29),in which the dense toroidal package prevents separation of double-strandedDNA break ends, thus favoring efficient repair.

We have found that the dense toroidal package probably doesnot exist in D. radiodurans. Furthermore, we observed that theordered condensation of DNA, leading to cholesteric organization,always remains changing and dynamic. It might well be that thediffusibility of DNA fragments is reduced in liquid crystalsbut that is the nature of this type of order; they still remainmobile. This is confirmed by experiments in ${lambda}$ phage DNA cyclizationin the presence of polyamines, which show that DNA ends remainsmobile in most condensed liquid crystalline phases (22). Thehypothesis that the liquid crystalline order of DNA is not directlyrelated to radioresistance is also supported by the fact thatdinoflagellata, whose genome is normally in the form of cholestericliquid crystal (35), are not unusually radioresistant.

The fact that the segregation of the nucleoid from the ribosome-richcytoplasm is already complete at the early phase of DNA orderingsuggests that nucleoid separation is the basic structural changeaccompanying the transition to the stationary phase. Thereforeit could be relevant to the increase in radioresistance at thebeginning of stationary phase. This idea is supported by factthat the nucleoids of radiation-sensitive bacteria remain corallineand dispersed at stationary phase (17, 41), whereas the nucleoidsof radioresistant bacterial species are more localized (41).We speculate that the segregation of nucleoids reduces the damagecaused by free radicals generated in the cytoplasm by radiation(17).

The molecular arrangement of DNA revealed in the nucleoid ofD. radiodurans cannot directly serve as a structural supportfor DNA repair. We believe that the unusual efficiency of thelatter in D. radiodurans is more likely to have a physiologicalthan a structural basis. Nevertheless, nucleoid segregationat the stationary phase can be protective and requires furtherstudy.

ACKNOWLEDGMENTS

This work was supported by the 3D-EM Network of Excellence withinResearch Framework Programme 6 of the European Commission.

We are grateful to H. Engelhardt for supplying bacteria andto A. Minsky for stimulating discussions.

FOOTNOTES

* Corresponding author. Mailing address: Laboratoire d'Analyse Ultrastructurale, B $a$ timent de Biologie, Université de Lausanne, CH-1015 Lausanne, Switzerland. Phone: 41 21 6924289. Fax: 41 21 6924285. E-mail: Mikhail.Eltsov{at}unil.ch.

REFERENCES

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