Electron Spectroscopic imaging of the centrosome in cells of the Indian

muntjac

J. B. RATTNER* and D. P. BAZETT-JONES

Departments of Medical Biochemistry and Anatomy, Faculty of Medicine, The University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta,T2N4NI, Canada

•Author for correspondence

Summary

Specific antibody labelling indicates that phospho-proteins are present at microtubule-organizingcentres, including the centrosome. We haveemployed electron spectroscopic imaging tech-niques that permit high-resolution elemental analy-sis of thin sections of intact cells to investigate theprecise distribution of phosphorus and thereforephosphoproteins at the centrosome of Indian munt-jac cells. We report that these proteins are localizedto both the pericentriolar matrix and the centriole.The matrix contains an abundance of phosphorusand is associated with microtubule elements.

Within the mature centriole, major structures in-cluding the nine triplet blades and linking elementsthat connect adjacent blades are composed of phos-phorylated proteins. In addition, phosphoproteinsare abundant at the ends of the centriole, at theinterface between the centriole lumen and the peri-centriolar environment. From these observationswe suggest that phosphoproteins may play both astructural and a functional role within the centro-some region.

Key words: centriole, centrosome, phosphorylation.

Introduction

The centrosome is the anchoring site for microtubulesthroughout the cell cycle and is composed of two com-ponents, the centriole and an associated electron-densematrix, the pericentriolar matrix, that surrounds thecentrioles. The centrosome can act as the site of micro-tubule polymerization-depolymerization and can, forexample, specifically influence the number of protofila-ments that form individual microtubules (Evans et al.1985). Most microtubules are inserted directly into thematrix about the centrioles and it is generally believedthat it is this region and not the centriole itself that isimportant to microtubule-related events (Gould & Bor-isy, 1977; Peterson & Berns, 1980). However, the cen-triole may play a critical role in both the temporal andspatial organization of the matrix (Peterson & Berns,1980).

Centrioles are duplicated during early S-phase and arefound in pairs throughout the remainder of the cell cycle.In thin section the mature centriole has a cylindricalshape and the walls of the structure are composed of nineblades. Each blade is in turn formed from three micro-tubules that are fused together into a single group. Thepresence of tubulin within the walls of the microtubuleshas been detected with anti-tubulin antibodies (Pepper &

Journal of Cell Science 91, 5-11 (1988)Printed in Great Britain © The Company of Biologists Limited 1988

Brinkley, 1976). The lumen of the centriole often con-tains vesicles and a cartwheel structure at the proximalend of the cylinder has been reported (Brinkley &Stubblefield, 1970). It has recently been discovered thatsera from patients with progressive systemic sclerosishave autoantibodies to the centriole (Osborn et al. 1986;Moroi et al. 1983). Analysis of the distribution of theseantigens in thin section using an immuno-peroxidasestaining protocol has shown that these autoantibodies arelocalized at the outside of centrioles and do not recognizetubulin, a component of the blades (Osborn et al. 1986).It is possible that these antigens are located in either thematrix or a structure called the centriolar rim thatsurrounds the blade microtubules and can be visualizedindependently of the microtubules (Fais et al. 1986). Theblades of the centriole appear to represent a unique classof microtubules, because they are not disrupted by agentsor conditions that dissociate cytoplasmic microtubules.In the daughter centriole the timing of blade microtubulepolymerization is cell-cycle-dependent; however, onceformed, the microtubules retain their structure andlength throughout the life of the cell (Rattner & Phillips,1973).

The centrosome plays an important role in the organiz-ation of microtubule arrays at cell division and proteinphosphorylation has been suggested as a possible control

mechanism for events associated with the initiation andcompletion of mitosis. Vandre et al. (1984) have demon-strated that monoclonal antibodies that recognize a classof phosphoproteins react with microtubule-organizingcentres, including the centrosome, kinetochore and mid-body. Further, these antibodies are capable of blockingmicrotubule growth at the centrosome, suggesting thatthe site of phosphorylation or a site adjacent to it isrequired for microtubule nucleation (Centonze et al.1986). Dephosphorylation of these proteins appears con-comitant with anaphase (Vandre & Borisy, 1987). Whileindirect immunofluorescence techniques have providedinformation concerning the general localization of phos-phoprotein within the cells, this technique has limitedresolution. It has now become possible to obtainincreased resolution through the application of an elec-tron microscopic technique, electron spectroscopicimaging (ESI) (Bazett-Jones & Ottensmyer, 1981). Thistechnique makes it possible to obtain simultaneous ultra-structural and elemental information from molecularspreads and from thin sections of intact cells. Thetechnique has been used to examine the organization ofnucleic acid in nucleoprotein complexes by selectivelyimaging phosphorus, an element in high abundance innucleic acid but not in the associated proteins (Bazett-Jones et al. 1988; Bazett-Jones, 1988). The resolution issuch that it is possible to resolve the path of a single DNAdouble helix as it passes around the histone octomerassociated with each nucleosome. This technique hasrecently been used to investigate the composition andultrastructural localization of heavy metal deposits inembedded and sectioned Escherichia colt cells (Taylor etal. 1988). In addition, electron-spectroscopic imaging hasalso been applied to the examination of physical speci-mens in both imaging and diffraction modes (Reimer etal. 1988). Electron spectroscopic imaging takes advantageof the fact that as incident electrons pass through aspecimen they cause ionization and excitation of thespecimen's atoms. This information can be analysed interms of X-ray emission spectra or with the energyspectra of electrons that have passed through the sample,reflecting its chemical composition. An electron micro-scope equipped with a parallel electron spectrometer canrecord electron energy-loss spectra and images at aparticular energy loss. The two-dimensional distributionmap of a particular element produced in this mannerdisplays both high spatial resolution and high sensitivity(Bazett-Jones et al. 1988). In this report we describe theapplication of electron spectroscopic imaging techniquesto the study of the distribution of phosphorus andtherefore phosphorylated proteins within the centro-some.

Materials and methods

Cell culture and electron microscopyFibroblast cells of the Indian muntjac (Muntjac muntjacus), anasiatic deer, were grown in monolayer culture in RPMI mediasupplemented with 10 % foetal calf serum. Cells were harvestedafter brief treatment with trypsin and fixed for 1 h in 3 %glutaraldehyde in a phosphate-free buffer (1 mM-sodium barbi-

tol buffer, pH7-4). The cells were pelleted and washed threetimes in buffer and post-fixed for 1 h in 1 % OsO.( buffered in asimilar manner. The specimens were then washed in water andpassed through a graded ethanol series and embedded in Spurr'sresin. Sections of 30-40 nm in thickness were cut from blocksand placed on 1000-mesh copper grids.

Electron spectroscopic imaging was performed on a ZeissEM902 electron microscope operated at 80keV equipped with aprism-mirror-prism type electron imaging spectrometer. Aknife-edge slit aperture with a width that corresponds to anenergy window of 12eV, a 600/<m condenser and 40jzmobjective aperture were used. Electron micrographs wererecorded at X10000-20000 and to an optical density ofapproximately 0-4. The electron exposure required for theUOeV image and the 150eV image were 0-7xl04-2-5X 104

electrons nm2 and 16xlO 4 -6x 104 electrons nm~2 respectively,depending on the magnification. Images were recorded onScientia (Agfa) 70 mm roll film and developed for 12 min in full-strength D-19 (Kodak) developer.

The phosphorus Ln.ni ionization edge is superimposed on aplasmon-like background. To eliminate the background contri-bution two images were recorded, a reference image at anenergy loss below the ionization edge (UOeV) and a phos-phorus-enhanced image near the maximum of the ionizationspectral peak (ISOeV). Areas of micrographs containing thecentrioles were digitized using a linear, high-resolution videocamera (Bosch). An image analysis computer was used tocalculate a normalization factor to compensate for differingexposures calculated over regions of plastic embedding matrixbetween cells that are known to be devoid of phosphorus. Theimage pairs were then aligned to within one pixel and thereference image was subtracted from the phosphorus-enhancedimage to produce a net phosphorus distribution map.

Comparison of net phosphorus images derived using only oneparameter to correct for the background in the energy-lossspectrum were made with net phosphorus images derived fromone-parameter and two-parameter extrapolation methods. Thetwo-parameter method can more effectively overcome thepossibility of generating a false signal from mass-density effects,non-linearities arising in image intensities from multiple scatterand local changes in the energy-loss spectrum giving a net falsesignal (Bazett-Jones et al. 1988). However, regions that showthe greatest mass-density often produce an extremely lowphosphorus signal (see Results). Nevertheless, to determine theseriousness of this potential problem in these specimens, tworeference images were used to extrapolate a more exact value atany point in the image, thereby producing a corrected referenceunder the ionization edge. The extrapolation of the energy-lossspectrum under the ionization edge was based on the relation-ship:

I=AETH,

where I represents the intensity of the energy-loss spectrum, Eis the energy loss, and A and R are parameters that can becalculated for any point in the image (Egerton, 1975). Resultswith the one-parameter fit, requiring only one reference image,were qualitatively the same as those obtained with a two-parameter fit, although the net phosphorus signal obtained wasat the most 10 % greater using the one-parameter extrapolationmethod.

Specimens examined by standard transmission electron mi-croscopy were prepared as described above except that aMillonigs' phosphate buffer was used and the cell pellet was pre-stained with 2% uranyl acetate prior to dehydration andembedding. Sections post-stained with uranyl acetate and leadcitrate were examined in a Hitachi H-500 microscope operatedat60kV.

J. B. Rattner and D. P. Bazett-Jones

Results

The ultrastructure of the centrioles in Indian muntjaccells is much like that found in other mammals. In cross-section it is apparent that they are composed of ninetriplet blades that are interconnected by a series of armsor linking elements extending between tubules A and C ofadjacent blades (Fig. 1A, small arrow). The surfaces ofthe blades are coated with an electron-dense matrix that isconfined to the outer surface of the blades (Fig. 1A, largearrow). Additional pericentriolar matrix material ofmoderate electron density is often seen in the cytoplasmadjacent to the centrioles. Microtubules can be seenassociated with both types of matrices (Fig. IB and C).In interphase other components of the cytoskeleton arealso associated with the matrix (Fig. 1C). One unusualfeature of the Indian muntjac centrioles is noticed duringmitosis. During this period it is common to see spindlemicrotubules inserted directly into the lumen of thecentriole (Fig. 1A and B) and in cross-section it ispossible to detect direct connections between thesemicrotubules and the blades of the centriole (Fig. 1A,double arrows). These microtubules occupy only thedistal third of the centriole lumen and are most com-monly found in association with the daughter centriole.

To evaluate the distribution of phosphorylated pro-teins within the centriole and its immediate environment,electron spectroscopic analysis was performed on thinsections taken from cells at several points in the cell cycle.A cross-section of a centriole from an interphase cell isillustrated in Fig. 2A. This image is comparable to theconventional image illustrated in Fig. 1A; the low con-strast in Fig. 2A is due to the absence of stains. However,resolution was facilitated by the extremely thin sectionthickness (30— 40nm) used, a physical requirement forelectron spectroscopic imaging. To obtain informationconcerning the distribution of phosphorus two imageswere recorded for each area of interest, a reference image(Fig. 2A) and a phosphorus-enhanced image (Fig. 2B).The cross-sectional profile of the centriole is seen in thelower portion of the micrographs while a cluster of matrixmaterial is located adjacent to it in the upper portion ofthe micrographs. Computer-aided subtraction of theimages shown in Fig. 2A and B results in the productionof a net phosphorus image illustrated in Fig. 2C. In thismicrograph the phosphorus-rich regions appear blackwhile those poor in phosphorus appear white.

By comparing the net image illustrated in Fig. 2C withthe original reference image in Fig. 2A, the precisedistribution of phosphorus can be ascertained. Althoughthe cross-section of the centriole is slightly oblique, it ispossible to localize a positive phophorus signal to thewalls of the microtubules (Fig. 2C, lower large arrow). Inaddition, the arms that interconnect adjacent blades alsodisplay a strong positive signal (Fig. 2C, small arrow).The lumen present in this section is devoid of phos-phorus, while the cytoplasm outside the pericentriolarregion shows a diffuse distribution of phosphorus. This ischaracteristic of the cytoplasm in general and to a largedegree reflects the distribution of ribosomes. The matrixmaterial around the surface of the centriole prominent in

Fig. 1. A. Cross-section of a centriole from a stained thinsection of an Indian muntjac cell illustrating the nine tripletblades that form the walls of this structure. Adjacent bladesare linked by small projections between the A and Cmicrotubules (small arrow) and each blade is coated with anelectron-dense matrix (large arrow). Cytoplasmicmicrotubules extend into the lumen of the centriole (doublearrow). Bar, 0-2/Jm. B. Longitudinal section through acentriole doublet. Microtubules are inserted along the walls ofthe centrioles as well as extending into the lumen of thisstructure (arrows). Bar, 0-25^m. C. Longitudinal sectionthrough two centrioles in an interphase cell. An amorphouselectron-dense matrix can be seen adjacent to the centriolesand associated with components of the cytoskeleton (arrow).Bar, 0-25 Jjm.

Electron spectroscopic imaging of centrosome

Fig. Z. A reference image (A), a phosphorus-enhanced image(B) and a net phosphorus image (C) of a centriole cut incross-section (lower arrows). Matrix material is denoted bythe arrow at the top of each micrograph. Phosphorus-richregions are denoted by the black areas in C. Note that thereis not a simple enhancement of density bewteen A and C.Bar, 0-15 Jim.

Fig. 2A is not as apparent in Fig. 2C, indicating thatphosphorus is not a major component of this area. Thisregion also illustrates that electron density is not acriterion for enhancement. The matrix material locatedabove the centriole did appear to contain abundantphophorus, suggesting that this region is probably par-ticularly rich is phosphoproteins.

The localization of a strong phosphorus signal at thecentriole and its component microtubules and the peri-centriolar matrix can also be detected in longitudinalsections of a centriolar pair from a metaphase cell(Fig. 3). It is possible to invert the image contrast in thecomputer to facilitate interpretation. Therefore, thecontrast of images in Fig. 3 is inverted in comparisonwith those presented in Fig. 2. Three images are illus-trated: the reference image (Fig. 3A), the phosphorus-enhanced image (Fig. 3B) and the net phosphorus image

(Fig. 3C). A positive phosphorus signal is denoted inFig. 3C by the white areas. The centriole on the left isshown in full longitudinal section whereas the one on theright is cut obliquely. It is clear that phosphorus ispresent along the entire length of the microtubulescomposing the blades and in the pericentriolar matrixmaterial (Fig. 3C, small arrow). The matrix materialcoating the centriole blades did not provide a majorcontribution to the net phosphorus image. It should benoted that whereas the lumen is devoid of phosphorus, ahigh phosphorus level is seen across both the proximaland distal ends of the centriole. This distribution is alsofound in a longitudinal section of a centriole from a latetelophase cell (Fig. 4). This region marks the boundarybetween the lumen of the centriole and its environment.In Fig. 4 the reference image is illustrated in A, thephosphorus-enhanced image in B and the net phosphorusimage in C. An oblique section through one centriole ofthe pair appears in the upper portion of the micrographswhile a longitudinal section appears in the lower portionof the micrographs. Note that the chromatin (Fig. 4C,c/i)within the condensing nucleus surrounds the centriolepair and contains a high level of phosphorus. Thisoriginates from the phosphate backbone of the DNA(Bazett-Jones & Ottensmeyer, 1981; Bazett-Jones et al.1988). The images of the mature centrioles illustrated inFigs 2, 3 and 4 are comparable, suggesting that thelocalization of phosphoproteins within the centriole isunchanged during the cell cycle.

Discussion

Electron spectroscopic imaging produces images withelectrons that have lost characteristic amounts of energyfrom ionizing or exciting specific elements in the speci-men. Using this approach a detection sensitivity of theorder of 2x 10~21 g or about 50 atoms of phosphorus canbe achieved at a spatial resolution of 0-5 nm, dependingon the stability of the specimen (Bazett-Jones & Ottens-meyer, 1981; Ottensmeyer et al. 1981). The resolutionlimits are determined primarily by the sensitivity of thespecimen to evaporation and migration of its atoms underthe influence of the electron beam. Recent studiesindicate that the conditions used in the present study aresufficient to minimize the effects of radiation so thatbiologically relevant structural information is preserved.Thus, in the study of DNA preparations, the netphosphorus image has been used to determine relativestoichiometric levels of this element in different regionsof an image and thereby determine the relative amountsof nucleic acids in those regions (Bazett-Jones et al.1988). In addition to the high contrast produced by thistechnique with unstained specimens, the parallel electronspectrometer is also capable of providing elementaldistributions at a spatial resolution of 2-0 nm or better(Bazett-Jones et al. 1988). These features indicate thatelectron spectroscopic imaging can be used with a highdegree of precision in the mapping of phosphorus andhence phosphoproteins. While there is no indication thatour preparative procedure permits the redistribution of

8 J. B. Rattner and D. P. Bazett-Jones

Fig. 3. A reference image (A), a phosphorus-enhanced image (B) and a net phosphorus image (C) of a centriole pair cut inlongitudinal section (large arrows). Matrix material is denoted by the small arrow. The arrowhead in C denotes a highphosphorus level at the proximal end of the centriole. Bar, 013/im.Fig. 4. A reference image (A), a phosphorus-enhanced image (B) and a net phosphorus image (C) of a centriole pair, one cut inoblique section (upper arrow) and one cut in longitudinal section (lower arrow). The centrioles are surrounded by the formingnucleus containing condensing chromatin (ch). Bar, 014/im.

phosphorus and phosphoproteins, we cannot rule out thispossibility. The comparison of information obtainedfrom samples prepared by alternative protocols such asfrozen-dried cryosections may in future clarify this point.

Electron spectroscopic imaging of the centrosome ofIndian muntjac cells reveals the precise distribution of

phosphorus within this cellular domain. Phosphoproteinsare a major component of the pericentriolar matrix butare not as abundant along the outer surface of thecentriole. In addition they are a major form of theproteins that compose the microtubule blades ofthe centriole and the accessory structures that inter-relate

Electron spectroscopic imaging of centrosome

adjacent blades. The ends of the centriole lumen alsoappears to be foci for phosphoproteins. These obser-vations are in agreement with the observation thatantibodies to phosphorylated proteins recognize micro-tubule-organizing sites, including the centrosome(Vandre et al. 1984). However, the present study extendsthese observations by defining the precise ultrastructuraldistribution of these proteins.

Protein phosphorylation and phosphorylation cascadeshave been found to play a role in the control of manymetabolic processes. In addition, phosphorylation ofstructural proteins such as actin (Steinberg, 1980), inter-mediate filaments (Gard & Lazarides, 1982), tubulin(Eipper, 1974) and microtubule-associated proteins (Slo-boda et al. 1975) have been observed. It has beensuggested that such post-translational modificationsmight provide a mechanism for the regulation of cytoskel-etal assembly and function.

Immunoperoxidase detection of anti-tubulin anti-bodies indicates that tubulin is present both in themicrotubules forming the blades of the centriole and inthose in the pericentriolar region (Pepper & Brinkley,1976). Although little is known about the relationshipbetween tubulin phosphorylation and microtubule as-sembly and function in vivo, recent evidence suggeststhat there is a correlation between phosphorylation of )3tubulin and increased microtubule assembly in mouseneuroblastoma cells (Gard & Kirschner, 1985). If phos-phorylation modulates the rate at which tubulin subunitspolymerize or is related to a shift in the equilibriumtowards whole microtubules, then such phosphorylatedforms should be abundant at active sites of microtubuleassembly (the centriole matrix) and perhaps microtubulestructures that are highly stable (the centriole blades).

Microtubules and microtubule-organizing centres areassociated with a specific subset of proteins in addition totubulin. Some of these proteins may play specific roles inthe maintenance of microtubule structure, whereasothers may be involved in the regulation of assembly anddisassembly (Gard & Kirschner, 1987). At least in somecases the functionality of these types of proteins appearsto be correlated with their level of phosphorylation (Gard& Kirschner, 1987). Thus the pericentriolar matrix mayrepresent a focus for many proteins whose functions areregulated by phosphorylation. Within the centriole a fewphosphoproteins could also play a role in ensuring thestability of its complex structure. However, the func-tionality of the proteins localized at the centriole rim doesnot appear to depend, to a large degree, on phosphoryl-ation. We have found it difficult to recognize procen-trioles in the unstained sections prepared for electron-spectroscopic imaging. In future it will be of interest todocument the relationship between the level and distri-bution of phosphorus and the genesis of a new centriole.

The detection of a phosphorus-rich region at both theends of the centriole is of particular interest. Theproximal end of the centriole is occupied by a cartwheelstructure and this component, like much of the centriole,may be composed of phosphorylated proteins. However,no such structure exists in the distal end of the centriole.The abundant phosphorus signal in this region may

indicate that this region has a unique but unknownfunctional role. Proteins in this region could, forexample, be responsible for specifying or directing theinteractions of the centriole with spindle microtubulessimilar to those detected in our study. This organizationmay in turn play a role in the movement and placement ofthe centrioles during cell division. Many authors havesuggested that nucleic acids are present in the lumen ofthe centriole (see, e.g., Brinkley & Stubblefield, 1970).We cannot rule out the possibility that all or part of thesignal detected at the apex of the centriole lumen, forexample, is due to nucleic acid. We are currently develop-ing procedures for the labelling of antibodies so that theirintercellular localization can be detected by electron-spectroscopic imaging. This technology should allow usto address the question of the nucleic acid composition ofthe centriole in a more direct way.

The authors thank T. H. Wang for her excellent technicalassistance. This work was supported by grants from the MedicalResearch Council (to D.B.-J.) and the Natural Sciences andEngineering Research Council (to J.B.R.).

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(Received 16 Februan> 1988 - Accepted 13 May 1988)

Electron spectroscopic imaging of centwsome 11