Characterization of Pales spermatocyte spindles, with reference to an

MTOC-associated protein

MARTIN BASTMEYER* and DAVID G. RUSSELLf

Max-Planck-lnstitut fiir Biologie, Spemannstrasse 34, D74OO Tubingen, FRG

•Author for correspondencef Present address: Department of Pathology, NYU Medical Center, 550 First Avenue, New York, NY 10016, USA

Summary

Pales ferruginea spermatocytes, because of theirhigh proportion of dividing cells, offer manyadvantages in the study of the meiotic spindle. Inthe present study we have combined ultrastruc-tural and immunofluorescence techniques withbiochemical analysis of constituent spindle com-ponents. Using a centrosome-reactive serum,which recognizes an antigen in the centrosomaiand midbody regions of dividing cells, we have

identified an MTOC-associated polypeptide of112000Mr with a pi of 5-6. The relative abun-dance of this protein in cells and isolated cyto-skeletons indicates that it is present in otherregions of the cell and is either more abundant,or is only recognized by the antibody in associ-ation with MTOCs.

Key words: Pales, spermatocyte, spindle, MTOC-associated protein.

Introduction

Since the original coining of the phrase 'microtubule-organizing centre' (MTOC) by Pickett-Heaps (1969)this term has been virtually universally adopted todescribe the structures, usually associated with theminus or slow-growth end of microtubules, thatapparently impart information to microtubular arrays.Subsequent years have seen a vast number of ultra-structural studies of MTOCs in a wide range of celltypes. The structure of MTOCs is highly diverse, as isthe degree of organization they confer on microtubularaggregates (see review by Tucker, 1984). Despite theamount of attention MTOCs have attracted very littleis known about their biochemical composition and themolecular basis of their activity.

The most extensively studied MTOC is undoubt-edly the centrosome, which, because of its role inorganizing the interphase cytoskeleton and as thepolar body of most eukaryotic dividing spindles, is themost ubiquitous of all MTOCs. The centrosomeconsists of a pair of centrioles and a cloud of electron-dense material designated the pericentricolar material(PCM). With respect to proteins present in MTOCs,

Journal of Cell Science 87, 431-438 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

Lin et al. (1981) reported finding anti-centriolar ac-tivity in normal sera, which was shown, by immuno-blot analysis, to react with two polypeptides of Mr

14000 and 17 000. These antigens were detected in awide variety of cell types. Monoclonal antibody studiesof centrosomes have also been reported. Kuriyama &Borisy (1985) identified proteins of 190000, 180000,20000 and 50000Mr as part of the centrosome in sea-urchin eggs. Vandre et al. (1984), using a monoclonalantibody against phosphoproteins, decorated specificregions in mitotic mammalian cells. These regionscorresponded to the sites normally associated withMTOC function, namely the centrosomes, kineto-chores and midbodies. Such immunochemical studiesare gradually building a more complete picture of thebiochemical constituents of MTOCs and will, it is to behoped, lead to the identification of the functionallysignificant components.

In this study we report the characterization of anMTOC-associated polypeptide in the spindle of Palesspermatocytes. Crane-fly spermatocytes have beenshown to be a good experimental system for examiningspindle formation because it is possible to producefunctional acentric spindles that lack a centrosome atone of their poles (Steffen et al. 1986; Bastmeyer et al.1986). Despite the absence of a centrosome the cells go

431

on to build an apparently normal spindle, capable oftransporting the chromosomes to the poles. Such asystem offers considerable potential, particularly inclarifying the role of the centrosomal components inspindle formation. In a recent immunofluorescentstudy with the anti-PCM (pericentriolar material)reactive serum 5051 (Bastmeyerei al. 1986), the serumstained the two 'normal' poles but failed to interact withthe aster-free pole, indicating that it lacked any detect-able PCM. In this study we have started biochemicalcharacterization of isolated spindles and, with the useof another MTOC-reactive antiserum, we have beenable to demonstrate the existence of a high molecularweight protein that is particularly abundant in thecentrosomes of dividing cells.

Materials and methods

Cell preparation for live observationTestes from IV instar larvae were disrupted under a drop ofliquid paraffin on a polylysine-coated coverslip, as describedby Steffen et al. (1986). Cells were dispersed by drawing thetestis sheath along the coverslip with a needle. The coverslipwas inverted and placed on a microscope slide to which twostrips of coverslip glass had been glued. This procedureresulted in a 'hanging-drop' preparation to which differentlysis buffers could be applied from one side of the coverslip.

Light microscopy

Cytoskeletal preparations were made by lysing cells pre-pared as outlined above in a buffer containing 90mM-Pipes(pH6-8), 30mM-fructose, 3mM-MgClz, 20mM-NaCl, 0-5%Triton X-100 and lO^lgml"1 taxol (kindly provided by DrM. Suffness, National Cancer Institute, Bethesda, MD).Following extraction for 5 min coverslips were transferred toa fixative solution consisting of 2% glutaraldehyde in 0 1 M-Pipes (pH6-8), 1 mM-MgClz and 1 mM-EGTA, and incu-bated for 10 min. Prior to microscopy the lysed cells werestained with Coomassie Blue, dehydrated and embedded inEuparal, as described (Steffen et al. 1986).

Electron microscopyCytoskeletons were processed for electron microscopy follow-ing lysis in a buffer (modified from Capco & Penman, 1983),containing lOmM-Pipes (pH68) , 300mM-sucrose, 3 mM-MgCl2, lOOmM-NaCl, 0-5% Triton X-100, and 10/igmr1

taxol. Fixation, and subsequent processing, have been de-scribed (Steffen el al. 1986).

Immunofluorescence

Rabbit anti-tubulin antibody was prepared by inoculation oftubulin isolated from Crithidia fasciculata (Russell et al.1984), together with Freund's complete adjuvant. After twobooster injections serum was collected. The antibody wasaffinity purified by passage over a pig brain tubulin-Sepharose column. Centrosome-reactive serum (CR serum)was isolated from one of us (D.G.R.) and used directly.

Cells to be examined by immunofluorescence were ex-tracted for 5 min in either 16% hexylene glycol, 1 % Triton

X-100 in 0-1 M-Pipes, pH 6 8 , or in the lysis buffer describedin the 'Light microscopy' section. Preparations were thenwashed in buffer A, SOmM-phosphate buffer, 140mM-salinewith 1 % BSA, 1 mM-EGTA, 1 mM-MgCl2 and 0-1 % TritonX-100, and fixed for 10 min in methanol at -20°C. Cells werethen rehydrated in buffer A prior to incubation in antisera.Cells were double-labelled by co-incubation in rabbit anti-tubulin (1:200) and CR serum (1:100) in buffer A for 30 minat room temperature. Following washing, cells were then co-incubated in FITC-conjugated anti-rabbit immunoglobulinG (IgG) (1:100) and rhodamine-coupled goat anti-humanIgG (1:100) (both from Jackson Immunoresearch Labora-tories). Preparations were washed and mounted in 80%glycerol in SOmM-Tris-HC1, p H 7 3 , containing 0 1 /<gml~'DAPI.

Sodium dodecyl sulphate-polyacrylamide gelelectrophoresis

Testes from IVth instar larvae were disrupted in 1 ml of theextraction buffer described in the 'Light microscopy' section.The resulting cytoskeletons were collected by centrifugationat 10000 £ for 5 min and prepared for sodium dodecylsulphate (SDS) or two-dimensional polyacrylamide gel elec-trophoresis (PAGE). SDS-PAGE was performed as de-scribed by Laemmli (1970) in 10 % separation gels with a 3 %polyacrylamide stacking gel. Two-dimensional PAGE wascarried out according to O'Farrell (1976) with modification(Russell et al. 1984), in a pi gradient containing 1-2% pH3-5-10 Ampholines and 0-8 % pH 5-7 Ampholines (LKB).

"Western' blotting and immunolabellingProtein transfer to nitrocellulose membranes was performedby the technique of Batteiger et al. (1982) utilizing aTris-HCl/Tween-20 blocking buffer. After overnight incu-bation at 4°C in blocking buffer, filters were probed withanti-tubulin antibody (1:1000) or CR serum (1:250). Boundantibody was detected with peroxidase conjugated anti-rabbitor anti-human IgG (Jackson Immunosearch Laboratories)developed using chloronapthol as substrate.

Results

Structural observations

The cells isolated from Pales testes consist of approxi-mately 45 % in early meiotic stags, 45 % in cell divisionand 10% mature spermatids. Cells were attached tocoverslips and cytoskeletal preparations were made byextraction in Triton X-100 detergent in a microtubulesupport buffer containing Taxol. Under these con-ditions the spindles retained their birefringence forseveral hours at 0°C. Light-microscope observationrevealed a well-defined structure (Fig. 1A,B) consist-ing of three autosomal bivalents and two sex chromo-somes with associated chromosomal fibres. Thecentrosomes, with very clearmicrotubule asters, lie atthe opposing poles of the spindle. In Pales, thecentrioles are highly developed, each possessingaxonemes that extend several micrometres.

432 M. Bastmeyer and D. G. Russell

Fig. 1. Light micrographs of an isolated metaphase Ispindle in phase-contrast (A) and differential-interference-contrast (B). X1500.

Ultrastructural observations of isolated spindle prep-arations indicate that many spindle structures wereretained during extraction of the cells with detergent(Fig. 2). The microtubule bundles of the spindleappeared slightly squeezed together, a similar obser-vation to that of Albertini et al. (1984) on isolatedcytoskeletons. The MAPs (microtubule-associatedproteins) may be seen on the spindle microtubules(Fig. 2B) although their appearance differs from thatfound in intact cells. Previous researchers have attri-buted microtubule bundling and changes in MAPstructure to the inclusion of taxol in the isolation buffer(Albertini et al. 1984; Foisner & Wiche, 1985). At thespindle pole the centriolar pair, with associated axon-emes, is clearly visible (Fig. 2A). The centrioles aresurrounded by a diffuse mass of electron-dense ma-terial, usually referred to as PCM (pericentriolarmaterial). The aster microtubules and some ofthe spindle microtubules appear to emanate from the

\rax

Fig. 2. A. A thick section (about 0-15^m) from an isolated prometaphase I spindle. The microtubules of the spindle andtwo chromosomes (c), one of them with a kinetochore (k), are visible. The polar complex consists of pericentriolar material(pan), two centrioles (white arrows) and axonemes (ax). Xl l 550. Bar, 2j*m. B. A thin section from the same cell.Microtubules, associated with proteins, and a part of a chromosome (c) are visible. X42000. Bar, 0-5 f.lm.

MTOC-associated proteins 433

PCM. Chromosomes are directly attached to thespindle fibres via their kinetochores (Fig. 2A).

lmmtinofluorescence observations

Detergent-extracted cell cytoskeletons were treated bydouble immunofluorescence with rabbit anti-tubulin

antibody in conjunction with CR (centrosome-reac-tive) serum. The light- and electron-microscope stud-ies indicated that structural preservation was good andthis conclusion was further supported by anti-tubulinantibody immunofluorescence, which indicated that atleast the majority of microtubular elements were pre-served (Fig. 3F,J). Fig. 3A,B shows a cytoskeletal

Fig. 3. Immunofluorescence staining of different division stages. A,C,E,I. Phase-contrast. B,D,G,K. Labelling of PCMwith CR-serum. F,J. Anti-tubulin labelling. H,L. Chromatin staining with DAPI. A,B. Late diakinesis with pronouncedstaining of the centrosomes. C,D. Telophase. Note staining of the midbody (arrow in D). E-H. Metaphase I. I-L. Oneanaphase II spindle and some spermatids. Note the CR-reacting spot in the spermatids (arrows in K). X1500.

434 M. Bastmeyer and D. G. Russell

Fig. 4. Metaphase I spermatocyte showing an aster-free spindle pole {afp) and a dislocated centrosome (arrow in C).A. Phase-contrast. B. Anti-tubulin labelling. C. Labelling of centrosomes with CR-serum. D. Chromatin staining withDAPI. X1500.

preparation from a cell in diakinesis; this is the earliest

stage in which a strong reaction with CR serum wasdetected. The CR serum (Fig. 3B) labels the centro-somes, which can be seen in Fig. 3A to be situated inthe centre of the forming asters. Later during division,in telophase, the CR serum reacted not only with thecentrosomes but also with an area of the spindlecorresponding to the midbody (Fig. 3D). Double im-munofluorescence labelling with anti-tubulin antibodyand CR serum, in addition to staining the chromo-somes with DAPI, was used for Fig. 3E-L. The cellshown in Fig. 3E-H is in metaphase I. Fig. 3E showsthe normal spindle structure with centrally situatedchromosomes (Fig. 3H). Staining with anti-tubulinantibody is shown in Fig. 3F. Staining with CR serumis restricted to the centrosomes (Fig. 3G). Fig. 3I-Lshows a cell in anaphase II and also some maturespermatids. In this preparation the CR serum identifiesan antigen(s) in both the spindle centrosomes and inthe region of the basal body in spermatids (Fig. 3K).The centrosome labelling (Fig. 3B,G,K) is certainlynot restricted to the centrioles and appears to corre-spond in location to the pericentriolar material.

In Pales spermatocytes one of the centrosomes canbe displaced prior to spindle formation by flatteningthe cells (Dietz, 1966; Steffen et al. 1986). Theproduction of acentric spindles facilitates further studyof the distribution of centrosome-associated antigenswith respect to the centrosome or aster-free pole (AFP)of the spindle. Although lacking a centrosome, theaster-free pole is still associated with a normally struc-tured half-spindle (Fig. 4A,B), capable of transportingchromosomes to that pole. Labelling with CR serumindicates that the antigen(s) recognized by this serum

remain associated with the centrosomes and are re-duced, at least beneath the level of detection, at theaster-free pole (Fig. 4C). Control preparations incu-bated with normal human serum, or in the absence ofprimary serum, were both negative.

B D

205

116,

Fig. 5. SDS-PAGE of Pales testes cytoskeletonpreparations run on 10% polyacrylamide gels, and theircorresponding immunoblots. A,C. Coomassie Blue-stainedgels run with either whole cells (A) or detergent-extractedcytoskeletons (C). B,D. Corresponding sampleselectrotransferred to nitrocellulose membranes and probedwith CR serum. This antiserum identified an antigen ofcomparable molecular weight (112000), in both whole cells(B) and cytoskeletons (D). Mv values (X 10~3) are on theleft.

MTOC-associated proteins 435

Immunochemical identification of CR serum antigenPales testis and cytoskeletal preparations were sub-jected to SDS-PAGE and two-dimensional PAGE,transferred to nitrocellulose filters and probed witheither anti-tubulin antibody or CR serum. Immuno-blots from SDS-PAGE preparations from whole cells(Fig. 5A,B) and from detergent-extracted cytoskel-etons (Fig. 5C,D) probed with CR serum reacted witha single antigen of 112000iV/r. Control preparationsprobed with either normal human serum or the

CREST scleroderma syndrome serum, 5051 (Calarco-Gillam et al. 1983) were both negative (not illustrated).

Two-dimensional PAGE analysis of whole testisrevealed a complex array of spots (Fig. 6A), as onewould expect, whereas the cytoskeletal preparationsshowed a much simpler pattern (Fig. 6B). The mostabundant proteins in the cytoskeletal preparations arethe tubulins, shown by their reaction with anti-tubulinantibody (Fig. 6C), they can also be detected in thewhole-cell sample (Fig. 6A). Both the cytoskeleton

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B

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Fig. 6. Two-dimensional gel electrophoresis of Pales testes cytoskeleton preparations and corresponding immunoblotsprobed with anti-tubulin and CR antiserum. A. The whole-cell samples show a complex array of polypeptides as expected,this gel was stained with Coomassie Blue. B. In contrast, the detergent-extracted cytoskeletons reveal a much simplerpattern. The identity of the tubulins (arrowheads in C), and of the 112000Mr MTOC-associated protein (arrow in D), aredemonstrated by immunoblotting similar two-dimensional gels. Both the tubulins and the 112000MT polypeptide could bedetected in both whole cells and detergent-extracted cytoskeleton.

436 M. Bastmeyer and D. G. Russell

preparations (not illustrated) and the whole cells(Fig. 6D) were transferred to nitrocellulose andprobed with CR serum. In both cases the antiserumrecognized an antigen with a molecular weight of112 000 and a pi of 5-6. As this polypeptide was presentin both whole cells and isolated cytoskeletons it isprobably, in part at least, a constituent of the spindleapparatus. These findings are in agreement with theimmunofluorescence results (Figs 3, 4) that indicatethat the CR serum antigen is associated with thecentrosomes. Control preparations run with normalhuman serum and 5051 serum were again negative.

Discussion

The role played by the centrosome in the organizationof a cell's cytoskeletal elements, in particular its activityduring cell division, still remains to be elucidated. For,although the centrosome is generally regarded as thestructure most directly implicated in cytoskeletalorganization, it should be remembered that manyeukaryotic cells divide without any centrioles. Forexample, mouse oocytes lack centrioles; however, thespindle poles possess PCM, which apparently fulfils anMTOC function (Callaro-Gillam et al. 1983; Maro etal. 1985). The same is also true in higher plant cells(Clayton et al. 1985), where PCM-like material per-sists, despite the loss of centrioles, to which is attri-buted the nucleation of spindle microtubules (Lloyd,1984). More recently, experiments on Pales spermato-cytes (Steffen et al. 1986; Bastmeyer et al. 1986)involving the production of acentric, tripolar spindles,one pole of which lacks centrioles and any detectablePCM, indicate that the centrosome may not be anabsolute requirement for the assembly of a functionalhalf-spindle. However, despite this controversy, theubiquitous nature of the centrosome as a cellularstructure makes the study of its composition of generalinterest.

In the present study we report finding an antiserum,CR serum, which reacts specifically with an antigensituated in the centrosomes and midbodies of Palesspermatocytes, and near to the basal bodies of maturespermatids. The antigens that give rise to this stainingremain associated with the spindle structures even inthe presence of lysis buffer. Immunoblotting studiesindicate that the antigen recognized by CR serum is asingle polypeptide with a molecular weight of 112 000and an isoelectric point of 5-6. These results differfrom the staining pattern obtained in Pales spermato-cytes with the CREST scleroderma syndrome serum,5051. The 5051 serum has been shown to be PCM-specific in a variety of cell types (Calarco-Gillam et al.1983; Clayton et al. 1985; Schatten et al. 1986). InPales spermatocytes, in common with CR serum, itrecognizes antigen in the centrosomes. However, in

contrast to CR serum, it also interacts with thekinetochore regions of the cell and it fails to label themidbody or mature spermatids (Bastmeyer et al.1986). In addition, immunoblots probed with 5051serum failed to detect the antigen. This is in agreementwith previous studies conducted with 5051 serum(Maro et al. 1985), which is produced by an autoim-mune response and reacts with many cells. In contrast,preliminary experiments with CR serum to dectect theantigen in other cell types, including various mam-malian cell lines and some protozoan cells, have allbeen negative. This suggests that the occurrence of theanti-centrosome activity is not due to any autoimmunemechanism but more probably to a chance cross-reactivity of antibody.

Our data indicate that the 112000A/r antigen isassociated with the centrosome and midbodies ofdividing cells; however, its relative abundance in wholecells and isolated cytoskeletons is certainly not consist-ent with it occurring only in these sites. In whole cells(Fig. 6A) this antigen is very common, having ap-proximately half the cellular concentration of that ofa'-tubulin. In contrast, in isolated cytoskeletons(Fig. 6B) the concentration of antigen has fallen withrespect to the ar-tubulin concentration. This anomalymay be due to the presence in the cytoplasm of the112000Afr protein, which is lost on extraction, butexists in higher concentrations at the MTOC sites. Afurther explanation may be suggested from the work ofVandre et al. (1984), who, using monoclonal anti-bodies, demonstrated the appearance of phosphory-lated antigenic determinants in sites of microtubulenucleation in mammalian culture cells. This indicatesthat some proteins are post-translationally modified, orhave existing modifications unmasked, on incorpor-ation into MTOCs. If our CR serum recognizes anantigenic determinant on the 112000Mr polypeptidethat is only expressed, or accessible, when the proteinis MTOC-associated, this could also explain why theimmunofluorescence labelling was so MTOC-specificwhilst the protein itself is, by its relative abundance inwhole cells compared with cytoskeletons, unlikely to berestricted to MTOCs.

Previous studies on isolated centrioles or by immu-nochemistry of centrosomes have identified a range ofprotein components. Anderson & Floyd (1980) demon-strated that centrioles isolated from chicken oviductcontained, in addition to tubulin, polypeptides of17 000, 19000 and 180000Mr. Turksen et al. (1982)purified basal bodies from the ciliate Tetrahymena andfound major polypeptides of 35 000, 45 000-47 000 and55 000Mr, the last being tubulin. They also found thata batch of normal rabbit serum recognized a 50 000 A/r

antigen, which occurred, not only in protozoan basalbodies but in a wide range of culture cell types. Lin etal. (1981) showed, using another'non-immune'serum,

MTOC-associated proteins 437

two polypeptides of 14000 and 17000Mr present in thecentrioles of many cell types. Finally, Kuriyama &Borisy (1985) identified, using monoclonal antibodies,proteins of 190000, 180000, 20000 and 50000iWr inthe centrosome of sea-urchin eggs.

In conclusion, although the centrosome is widelyregarded as fulfilling major roles in cell division,cytoskeletal organization and cell locomotion (Mcln-tosh, 1983; Tucker, 1984; Mazia, 1984) the exactnature of its involvement has not been clarified. Withthis end in mind it is hoped that continuation ofbiochemical studies into the constituents of the centro-some will yield further information as to its cellularfunctions.

The authors thank Drs R. Dietz and W. Steffen for theirhelp during the course of this study. We are also grateful toHeike Wilhelm and Gerda Miiller for their expert technicalassistance, and to Ursula Hieke for typing the manuscript.

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(Received 17 November 1986 -Accepted 5 January' 1987)

438 M. Bastmeyer and D. G. Russell