Definition of individual components within the cytoskeieton of

Trypanosoma brucei by a library of monoclonal antibodies

ANGELA WOODS, TREVOR SHERWIN, ROSEMARY SASSE*, THOMAS H. MacRAEf,

ANTHONY J. BAINES and KEITH GULL*

Biological laboratory, University of Kent, Canterbury, Kent CT2 7NJ, UK

•Present address: Biochemical Sciences, Wellcome Research Laboratories, Langley Court, Beckenham, Kent CT3 3BS, UK•(•Present address: Biology Department, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J1% Present address: Department of Biochemistry & Molecular Biology, University of Manchester, School of Biological Sciences, StopfordBuilding, Oxford Road, Manchester M13 9PT, UK

Summary

The detergent-insoluble T. brucei cytoskeieton con-sists of several morphologically distinct regions andorganelles, many of which are detectable only byelectron microscopy. We have produced a set ofmonoclonal antibodies that define each structuralcomponent of this highly ordered cytoskeieton. Themonoclonal antibodies were selected by cloning ofhybridomas produced from mice injected withcomplex mixtures of proteins of either the cytoskei-eton itself or salt extracts thereof. Four antibodiesdefine particular tubulin isotypes and locate themicrotubules of the axoneme and sub-pellicular

array; two antibodies recognize the flagellum at-tachment zone; one recognizes the paraflagellar rodand another the basal bodies. Finally, one antibodydefines a detergent-insoluble component of the nu-cleus. The antigens detected by each monoclonalantibody have been analysed by immunofluor-escence microscopy, immunogold electron mi-croscopy and Western blotting.

Key words: Trypanosoma brucei, cytoskeieton, monoclonalantibodies.

Introduction

The plasma membrane forms the boundary of a cell andso defines its form: to achieve this the fluid mosaic mustbe moulded to the required shape and stabilized to permitselective positioning of membrane components and pro-vide resistance to mechanical stress. This is achieved, atleast in part, by an underlying cytoskeletal apparatus thatinteracts with the plasma membrane. In cells of highereukaryotes, each of the three major cytoplasmic filamenttypes (microfilaments, microtubules and intermediatefilaments) can contribute to cell shape and stability: onlyrarely does one filament system perform this functionexclusively. By contrast, in some eukaryotic microbes,particularly the free-living and parasitic flagellates, asingle filament type (often microtubules) forms the'membrane skeleton'.

African trypanosomes belonging to the Trypanosomabrucei complex are the causative agents of sleepingsickness in man and of nagana in livestock. This organismforms an ideal system for the investigation of the micro-tubule-based membrane-associated cytoskeieton. Procyc-lic forms are easily cultured in vitro and have a cytoskel-

Journal of Cell Science 93, 491-500 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

etal structure that is highly ordered but simple enough tobe experimentally accessible. In T. brucei there arediscrete cytoskeletal structures in both the cell body andflagellum. In the cell body, a precisely ordered array ofmicrotubules is closely associated with the plasma mem-brane (Angelopoulos, 1970). Electron microscopy hasrevealed the presence of cross-bridges connecting micro-tubules to the membrane and to each other (Vickerman,1985). Curiously, there are no observable cytoskeletalmicrofilaments within the cytoplasm, even though trypa-nosomes apparently possess a functional actin gene(Amar et al. 1988). A specialized region of the membraneskeleton exists close to the site where the flagellum meetsthe cell body, namely, the flagellum attachment zone(FAZ). This appears as a zip-like structure in the electronmicroscope and has not yet been isolated or characterizedbiochemically. In the flagellum, in addition to a 9+2microtubule axoneme, a paraflagellar rod is present. Theparaflagellar rod of trypanosomes is antigenically relatedto that of Crithidia and Euglena, the major cross-reactingprotein component appearing on SDS-containing gels asa doublet of Mr68000 and 76000 (Russell et al. 1983;Gallo & Schrevel, 1985).

491

It appears that only a few cytoskeletal organelles,primarily based on microtubules, control the cell's shapeand stability. Understanding the generation and mainten-ance of cytoskeletal order requires a detailed descriptionof the use and positioning of particular gene productswithin the cytoskeleton. Although trypanosomes possessmultiple tubulin genes (Seebeck et al. 1983), only onetype of a- and one type of j8-tubulin mRNA can bedetected; nevertheless, a number of protein isoforms areproduced by post-translational modification of the a-chain (acetylation of lysine 40 and detyrosination/tyrosination of the C terminus; (Steigere/oV. 1984; Sasse& Gull, 1988). Their cellular distribution and modu-lation throughout the cell cycle has now been described(Sherwin et al. 1987; Sasse & Gull, 1988). The com-ponents of the cross-bridges between the cell bodymicrotubules and the plasma membrane have not beenbiochemically defined (Bramblett et al. 1984; Vickerman& Preston, 1976); they do not seem to be transmembraneproteins and two lipid- and tubulin-binding proteins(yV/r 40 000 and 60 000; Seebeck et al. 1988) seem to be thebest candidates. The cross-bridges between microtubuleshave not yet been defined.

A major limitation on attempts to determine thebiochemical cytology of the cytoskeleton is the avail-ability of some of the components; the FAZ, for example,comprises only a small fraction of the cytoskeletal pro-tein. Nevertheless, detergent-insoluble cytoskeletons thatretain the shape and order observed in vivo may beisolated from trypanosome cells. Using these as compleximmunogens, it should be possible to prepare monoclonalantibodies specific for individual components. Thesewould identify components of structures otherwise inac-cessible to standard biochemical analysis. Concomitantly,they would provide probes for the analysis of individualcomponents during the cell cycle and their detectionduring attempts at purification.

In this paper we describe the preparation of a panel ofnine monoclonal antibodies to cytoskeletal antigens.They provide novel probes for components of the FAZ,basal body, paraflagellar rod and nucleus as well ascertain isotypes of tubulin.

Materials and methods

Cell linesProcyclic liypanosoma brttcei, stock 427 were grown in tissue-culture flasks in semi-defined medium 79 (Brun & Schonen-berger, 1979). Vero cells were grown in Dulbecco's modifiedEagle's medium supplemented with 10 % (v/v) foetal calf serum(PCS) and non-essential amino acids (NAA). PtK2 cells weregrown in Eagle's minimal essential medium supplemented with10% (v/v) PCS and NAA.

Production of monoclonal antibodiesMice were either immunized with detergent-insoluble cytoskel-etons or salt extracts containing depolymerized pedicular micro-tubules. For the preparation of cytoskeletons cells were har-vested by centrifugation, washed in phosphate-buffered saline(PBS) and then incubated on ice for 5 min in 0 5 % NonidetP40, lOOniM-Pipes, pH6-9, 2mM-EGTA, 1 mM-MgS04,

0-lmM-EDTA (NP40/PEME). Cytoskeletons harvested froml-5x 108 cells, washed twice in PBS (014M-NaCl, 2 5 mM-KCl,8mM-Na2HPO4, 1 mM-KH2PO4) and resuspended in 02mlPBS, were used per injection per mouse.

For salt extracts cytoskeletons were resuspended in PEME,1 M-KC1 and left on ice for 40 min before centrifugation for10 min at 11 600gav. The supernatant was then dialysed againstPBS. The extract from 5-5X 108 cells was used per injection permouse.

Balb/c mice were inoculated. For the initial inoculation theantigen was emulsified with an equal volume of Freund'scomplete adjuvant and applied intraperitoneally. Repeat injec-tions were made in Freund's incomplete adjuvant. Final inocu-lations were done in PBS alone and administered intravenously.Splenectomy was performed 4 days after the final injection andthe splenocytes were fused with approximately a third of theirnumber of Sp2-O/Ag myeloma cells using polyethylene glycol(PEG) 1500, essentially as described by Galfre et"al. (1977).Normal mouse splenocytes were used as feeder cells. A 100 jt/lsample of medium was taken from each well between 14 and 21days postfusion and screened for the presence of anti-cytoskel-eton antibodies by immunofluorescence. Cloning of positivewells was performed by limiting dilution three times. Typing ofthe subclasses of each antibody was carried out by doubleimmunodiffusion against specific antisera from ICN, Immuno-biologicals, UK.

ImmunofluorescenceTrypanosomes were washed in PBS and settled onto poly-L-lysine-coated slides. Slides were fixed in cold methanol for30 min and rehydrated in PBS. Isolated flagella complexes wereprepared according to Sasse & Gull (1988). Slides wereincubated in culture supernatant for 1 h in a moist atmosphereand washed three times in PBS. The slides were then incubatedwith fluorescein-conjugated second antibody (rabbit anti-mouseimmunoglobulins, Dakopatts) for 1 h and washed in PBS beforemounting in Mowiol 488 (Harco, Harlow, UK) containing1 mgrnl"1 />-phenylene diamine as an antifade agent.

Vero and PtK2 cells (grown on coverslips) were washed inwarmed PBS, fixed in cold methanol and incubated in anti-bodies, washed and mounted as above.

SDS—polyacrylamide gel electrophoresisThe method of Laemmli (1970) was used, with SDS obtainedfrom Sigma (Clayton et al. 1980). The polypeptides were eitherstained with Coomassie Blue R250 or transferred to nitrocellu-lose paper.

ImmunoblottingWith the exception of gels to be blotted and probed with CD10,proteins were transferred to nitrocellulose paper (45 ,um poresize, Schleicher & Schuell) probed with primary and secondaryantibody (Dakopatts) as described by Birkett et al. (1985).Antibody supernatants were diluted with an equal volume ofeither lOrnM-Tris-HCI (pH7-4), 140mM-NaCl containing0-1 % (v/v) Tween 20 (TBS-Tween) in the case of Alphabet,C3B9and PFR, or lOmM-Tris-HCl (pH7-4), 1 M-NaCl, 0-5%(v/v) Tween 20 (HST) in the case of TAT1, CD10 and CA12.

Proteins to be probed with CD10 were transferred tonitrocellulose according to Small (1988).

ImniunogoldThe method of Sherwin & Gull (1989) was used. Cells wereharvested and washed in PBS. After settling onto Formvar-filmed, carbon-coated grids, they were then extracted usingNP40/PEME. Cytoskeletons were then fixed in 3-7% parafor-

492 A. Woods et al.

maldehyde, 0-01 % glutaraldehyde, followed by washing in20niM-glycine in PBS. Grids were then blocked with 1 % BSA(bovine serum albumin) in PBS and incubated in first antibodydiluted in 1 % BSA in PBS for 1 h at room temperature. Afterwashing in 1% BSA in PBS (3 times, 5 min) the grids wereincubated in second antibody (rabbit anti-mouse 10 nm goldconjugate, Biocell) diluted in 1 % BSA in PBS for 1 h. Washeswere then done in 1 % BSA in PBS, followed by 0-1 % BSA inPBS and then by a wash in PEME. Cells were then relaxed in2-5% glutaraldehyde and negatively stained using 2% am-monium molybdate, pH7-0.

Synthetic peptidesPeptides corresponding to the Physannn polycephalum myx-amoebal a'-tubulin sequence residues 37-45 and the mammaliantt'-tubulin sequence residues 36-46 were made and kindlydonated by Adrian Blindt and Paul Walden (this laboratory). A2mg sample of peptide (8 ing ml"1 in water) was acetylatedusing acetic anhydride (Means & Feeney, 1971): 250,1(1 ofsaturated sodium acetate was added to the peptide solution,then six additions, each of 4ftl of acetic anhydride (10% in 1,4-dioxane), were made during a 1-h incubation on ice. Theacetylated peptides were desalted on a G15 column using 50%acetic acid. The peak fractions containing each peptide werepooled. The acetic acid was removed by rotary evaporation,followed by methanol washes and the peptide residues wereeach resuspended in 1 ml of water to give a final concentrationfor each of approximately 1-5 ing ml"1.

C3B9 culture supernatant was diluted 20-fold inTBS-Tween. The following combinations of antibody andpeptides were then set up: (1) 2-5 ml C3B9 + 500,ul acetylatedpeptide solution; (2) 2-5 ml C3B9 + approx. 95 ,ul unacetylatedpeptide solution + 405 j.i\ water; (3) 2-5 ml C3B9 +500^1water. The pH of each was adjusted to approximately 7-5 using2M-Tris-HCl and they were incubated overnight at 4CC withgentle agitation. The incubated antibody solutions were thenused to immunostain identical Western blots of total T. bmceiproteins separated by the normal one-dimensional PAGEmethod.

Preparation of erythrocytes and erythrocyte ghostsHuman or porcine blood was anticoagulated with EDTA.Washed erythrocytes and ghosts were prepared by the methodof Bennett (1983).

Results

The T. brucei cytoskeletonDetergent extraction of T. brucei cells reveals a cytoskel-eton that retains the original cell shape and form. Themajor structural components of this cytoskeleton can bevisualized by negative-stain electron microscopy. Fig. 1shows such a cytoskeleton and acts as an ultrastructuralreference for the light-microscope images described later.There are seven major features in the cytoskeleton of T.brucei. The major component of the cell body cytoskel-eton is a helical arrangement of microtubules that areextensively cross-linked. The flagellum, subtended by abasal body located at the posterior of the cell, extendsalong the cell body, eventually overlapping the anteriorend by a short distance. There are two major componentswithin the flagellum: the microtubule axoneme and theparaflagellar rod, and both are clearly distinguished inthese cytoskeletal preparations (Fig. 1). Also a probasal

body is located close to the flagellar basal body. Negative-stain electron microscopy of the T. brucei cytoskeletonpermits the visualization of a complex periodic structuretermed the flagellum attachment zone. This structureserves to link the flagellum to a specific region of thecortical microtubule array. Finally, the cytoskeletonpreparations contain a specific identifiable remnant of thenucleus retained in its original cellular position (Fig. 1).

Selection of a panel of monoclonal antibodiesPurification of all the individual components of thecomplex T. bmcei cytoskeleton is unrealistic. Conse-quently, in order to produce a panel of monoclonalantibodies recognizing specific individual components weused a complex immunogen and then used immunofluor-escence subsequently to assay and categorize the anti-bodies. We used two separate immunogens: first, thecomplete, intact cytoskeleton; and second, the super-natant from a high-salt extraction of the cytoskeleton inorder to produce a complex yet soluble immunogen. Weselected and characterized hybridoma cell lines produc-ing nine monoclonal antibodies. Table 1 surveys each ofthese antibodies and provides an overall summary of theirnomenclature, class and specificity. This panel of anti-bodies provides probes for each of the major structuralcomponents of the cytoskeleton of T. brucei (Table 1 andFig. 2).

MicrotubulesThis study produced four monoclonal antibodies (Alpha-bet, TAT1, C3B9 and CA12), each of which gave ageneral immunofluorescence recognition of the cell bodytogether with the flagellar axoneme (Fig. 2). Immuno-gold electron microscopy shows that each recognizes boththe pellicular and axonemal microtubules (Fig. 3). West-ern blotting revealed that all four monoclonal antibodiesrecognize the tubulin component of the microtubules, yetthere are interesting and unusual differences in their

Table 1. Survey of monoclonal antibodies

Antibodyname

Alphabet

TAT1

C3B9

CA12

NUC

BBA4

ROD1

CD10

DOT1

Immunogen

Cytoskeletons

Cytoskeletons

High-saltsupernatant

High-saltsupernatant

High-saltsupernatant

High-saltsupernatant

Cytoskeletons

High-saltsupernatant

Cytoskeletons

Antibodyclass

IgM

IgG2b

lgG2b

IgG3

IgG3

IgM

IgM

IgG3

IgM

Specificity

o'- and /^-tubulin

a'-tubulin

Acetylated a'-tubulin

/3-tubulin(trypanosome-specific)

Nucleus

Basal body

Paraflagellar rod(doublet,A/r180+200K)

Flagellar attachmentzone (jWr>300K)

Flagellar attachmentzone and distal tipof flagellum

Trypanosoma brucei cytoskeletal components 493

Posterior Anterior

A

Fig. 1. Negatively stained electron micrograph of a detergent-insoluble cytoskeleton of T. brucei. Showing nucleus (n), basalbody (bb), probasal body (pbb), the flagellar attachment zone (faz), the paraflagellar rod {pfi"), pellicular microtubules (pint)and the microtubular axoneme (a).

recognition epitopes. The monoclonal antibody Alphabetdetects both a- and /3-tubulin polypeptides of both T.bnicei and mammalian brain microtubules (Fig. 4).TAT1 specifically recognizes a'-tubulin and again cross-reacts with a wide range of species. This contrasts withCA12, which recognizes T. bnicei, but not mammalianbrain, /3-tubulin. Thus, these three monoclonal anti-bodies illustrate the conservation of epitopes between thea- and /3-tubulins and the evolutionary changes in thetubulin polypeptides leading to conserved and non-conserved regions. These anti-tubulin monoclonalsshould serve as useful probes for both the T. bniceicytoskeleton and the tubulin polypeptides.

The fourth monoclonal antibody that gave recognitionof microtubules by immunogold electron microscopy,C3B9, recognized the a'-tubulin of T. bnicei when used inWestern blotting experiments but detected mammalianbrain a'-tubulin to a limited extent only (Fig. 4). Thisresult might be explained by a reduced general avidity formammalian a'-tubulin or, rather, that C3B9 recognizes anisotype of a'-tubulin that is well-represented in the T.brucei microtubules but only poorly so in the mammalianbrain tubulin. This latter explanation was supported bythe results of immunofluorescence studies with the anti-

body. When Vero cells in culture were stained with theanti-a'-tubulin monoclonal TAT1, the expected complexpattern of cytoplasmic microtubules was detected. How-ever, C3B9 gave a very restricted immunofluorescencepattern of microtubules in Vero cells (Fig. 5). Thisantibody detects a sub-population of microtubules thathave wavy profiles and a general perinuclear distribution.C3B9 also detects specific microtubule structures such ascentrioles and mid-bodies. This immunofluorescencepattern is extremely similar to that of acetylated a'-tubulin distribution in cells detected by other monoclonalantibodies (e.g. 6-11-B1; Piperno & Fuller, 1985). Wehave shown previously that the T. brucei cytoskeleton isrich in acetylated a'-tubulin (Sasse & Gull, 1988). Inorder to assess whether C3B9 recognizes acetylated a'-tubulin we subjected a preparation of mammalian braintubulin to chemical acetylation. This resulted in a largeincrease in the detection of a'-tubulin by C3B9 incomparison to an unacetylated control. The only knownpost-translational acetylation site on a'-tubulin is a lysineresidue at position 40 in the polypeptide. In order toassess whether this particular lysine is acetylated in T.bnicei tubulin and to provide the recognition epitope forC3B9, we synthesized two peptides that span this region.

494 A. Woods et al.

These corresponded to a consensus mammalian o'-tubu-lin sequence (residues 36-46) and that of P. polycepha-lum cr-tubulin (residues 37-45). Both peptides includedthe single lysine at residue 40.

We then used these peptides in both the unacetylatedand acetylated forms in competition experiments with theC3B9. Fig. 6 shows the result of this experiment and

illustrates the fact that the peptide can compete out thebinding of C3B9 to T. brucei a'-tubulin, but only whenthe lysine in the peptide is in the acetylated form. Thus,C3B9 recognizes an epitope that includes an acetylatedlysine at residue 40 in the a'-tubulin polypeptide and isthus able to detect acetylated a'-tubulin in an evolution-arily diverse range of organisms. Moreover, it provides

B

D

Fig. 2. Fluorescence (above) and phase-contrast (below) images of cells probed with different monoclonal antibodies.A. Alphabet; B, TAT1; C, C3B9; D, CA12; E, NUC; F, BBA4; G, R0D1; H, CD10; 1, D 0 T 1 .

Trypanosoma brucei cytoskeletal components 495

B

Fig. 3. Electron micrograph of immunogold staining of acytoskeleton probed with TAT1. B shows enlarged region ofcell, shown in a box in A.

A B C D1 2 1 2 1 2 1 2

Fig. 4. Western blots using anti-tubulin monoclonalantibodies. Lanes 1, T. bnicei whole-cell lysate; lanes 2,mammalian brain tubulin probed with Alphabet (A), TAT1(B), CA12 (C), C3B9 (D). Note: gels run with buffers madeusing sigma SDS, therefore trypanosome a-tubulin migratesFurther than (5, but in mammalian brain tubulin the reverse istrue (Clayton el al. 1980).

direct confirmation of the presence of acetylated a-tubulin in subpellicular and axonemal microtubules inthe T. bnicei cytoskeleton.

The nucleusThe monoclonal antibody NUC is a representative anti-body that detects the nucleus of T. bnicei cells. Thisspecific detection of the nucleus is shown in Fig. 2 andthe immunofluorescence pattern does not appear tochange with the position of the nucleus within the cellcycle. The antibody does not appear to be directedagainst DNA, since the large amount of kinetoplast DNAin the mitochondrion is not seen by immunofluorescence(Fig. 2). However, we have been unsuccessful in ourattempts to identify a polypeptide recognized by NUCusing various Western blotting protocols.

The basal bodiesBBA4 represents a monoclonal antibody that, when usedin immunofluorescence assays, detects two very smallstructures towards the posterior portion of the T. bruceicell (Fig. 2). Phase/immunofluorescence images of cyto-skeletons and isolated flagella stained with this mono-clonal clearly show that it detects the flagellar basal bodyand pro-basal body (Fig. 7). We have been unable todetect recognition of a polypeptide using BBA4 byWestern blotting experiments. It may be that recognitionof the epitope is dependent on conformation or it ispossible that the antigen is present in very small amounts.Nevertheless, BBA4 represents a specific probe for thebasal bodies of the T. bnicei cytoskeleton. The pattern offluorescence detection emphasizes that the antigendetected by BBA4 becomes associated with the basalbody very early during its biogenesis as a pro-basal body.Moreover, the antigen has an extremely restricted lo-cation within the cell and does not appear to be associatedwith either the flagellar axoneme or other cellular organ-elles. From staining patterns observed using the DNAintercalating dye, DAPI, it is apparent that BBA4staining is distinct from the kinetoplast. Moreover,immunogold studies suggest that BBA4 does not reactwith a specific mitochondrion [kinetoplast]/basal bodyconnection. BBA4 does not stain the basal bodies in PtK2

cells, Vero cells or Physanim amoebae in fluorescencestudies.

The paraflagellar rodOne monoclonal antibody, ROD1, when used in immu-nofluorescence studies provides a specific detection of theT. brucei flagellum (Fig. 2). Immunogold electron mi-croscopy shows that ROD1 binds to the paraflagellar rod(Fig. 8C). Further, the antigen detected by ROD1 islocated on the area of the paraflagellar rod closest to thezone of attachment to the cell body (Fig. 8). The majorconstituents of the paraflagellar rod are polypeptides withmolecular weights of 76K and 68K (K = 10 Mr) (Russellet al. 1983). One monoclonal antibody, 5E9, has alreadybeen shown to recognize both of these polypeptides(Gallo & Schrevel, 1985). Western blot analysis of ROD1revealed that this monoclonal did not detect the majorPFR1 and PFR2 proteins but, rather, a doublet of two

496 A. Woods et al.

Fig. 5. Immunofluorescence staining pattern of Vero cells probed with TATl (A) and C3B9 (B).

A B C

7AFig. 7. Immunofluorescence/phase-contrast pattern ofBBA4 on a post mitotic cell (A), and an isolated flagellum(B).

Fig. 6. Competitive binding of C3B9 and peptides onidentical Western blots. A. C3B9 supernatant; B, C3B9supernatant previously incubated with unacetylated peptide;C, C3B9 supernatant previously incubated with acetylatedpeptide.

polypeptides with molecular weights of around 180K and200K (Fig. 8).

When used to probe Western blots of human andporcine erythrocyte polypeptides, R0D1 detects bandsof slightly higher molecular weight that comigrate withboth human and porcine |3-spectrin. R0D1 also gives a

general whole-cell staining pattern when used in immu-nofluorescence assays with human erythrocytes (notshown).

The flagellum attachment zoneTwo monoclonal antibodies, CD 10 and DOT, gaveimmunofluorescence staining of T. bnicei cells that indi-cated that they both detected components of the flagel-lum attachment zone (Fig. 2). CD 10 detects an intermit-tently punctate line along the flagellum attachment zone.Immunogold electron microscopy revealed that the anti-gen detected by CD 10 is located along a complex filamentsystem that comprises the FAZ (Fig. 9). Further, immu-nofluorescence studies show that this filament system is

Trypanosoma brucei cytoskeletal components 497

B

PFR1.PFR2L

Fig. 8. A and B show corresponding Coomassie Blue-stained gel and Western blot probed with RODl. A. Coomassie Blue-stained gel of T. brucei whole-cell lysate; and B, corresponding Western blot probed with RODl. D shows enlarged area of thecytoskeleton, shown by a box in C, which has been immunogold labelled with RODl.

A B

Fig. 9. Electron micrograph of immunogold staining patternusing CD10. B shows enlarged area of the cytoskeleton,shown in a box in A.

elaborated during the process of flagellar growth. Cellslate in the cell cycle, which possess two flagella, have twoflagellum attachment zones revealed by the CD 10 mono-clonal. Western blotting of T. brucei proteins using CD 10(Fig. 10) revealed that this monoclonal detects a polypep-tide of very high molecular weight (>300K). The detec-tion of the CD 10 antigen relies upon using a particularblotting protocol that was developed for use with integralmembrane proteins.

The DOTl monoclonal antibody gives an immuno-fluorescence image on T. brucei cells that is superficiallysimilar to that of CD 10. There are, however, subtledifferences in that DOTl gives a more punctate pattern

Fig. 10. A shows the total protein of whole-cell lysate,Amino Black-stained. B shows an identical blotimmunostained with CD 10. C shows the fluorescence stainingpattern of a post-mitotic cell stained with CD10 (shown inphase-contrast in D).

and also detects material at the distal tip of the flagellum.So far we have been unable to detect an antigen for theDOTl monoclonal antibody by exhaustive Western blotanalyses. Immunogold electron microscopy of T. bruceicytoskeletons using DOTl reveals that the antigendetected is located in regions associated with the unique

498 A. Woods et al.

11A

BFig. 11. Immunogold staining pattern revealed by DOT1 onisolated flagellum. B shows enlarged boxed area in A.

filament systems of the flagellum attachment zone(Fig. 11).

Discussion

The T. brucei cytoskeleton is very well defined ultrastruc-turally but the correlative biochemistry is under-developed. We have extensive information pertaining tothe abundant molecules in the cytoskeleton, i.e. a- and /3-tubulin and the PFR proteins (Sasse & Gull, 1988;Steiger et al. 1984; Schneider et al. 1987; Gallo &Schrevel, 1984). However, the biochemical identificationof other cytoskeletal components is more problematical,since they resist classical approaches because of theirinsolubility or low abundance. Thus, we have adoptedthe approach of producing probes for structures andconstituent molecules in the form of monoclonal anti-bodies produced against antigenic determinants presentin a complex mixture of proteins, i.e. the cytoskeletonitself.

This has a number of advantages. Preparation of theimmunogen is very easy, merely involving the productionof detergent-insoluble cytoskeletons or salt extractsthereof. The use of immunofluorescence as an assay hasenabled us to select antibodies that elicit a specific patternin the cell, giving us the polypeptide localization fromsimple primary screens. This method of screening alsopermits detection of very small signals, which maypresent only minor components of the whole structureand may not be detectable by other screening methodssuch as ELISA or blotting techniques. This is exemp-lified by the BBA4 antibody.

The variety of tubulin antibodies obtained is notsurprising, as tubulin is the most abundant single proteinin the cytoskeleton of T. brucei. The fact that the fourtubulin antibodies cross-react to differing extents be-tween both species and tubulin isotypes demonstratesseveral properties of tubulin. TAT1 emphasizes the

evolutionary conservation of tubulin between species inthat it cross-reacts with mammalian, Physanou andtrypanosomal tv-tubulin. The overall sequence homologybetween mammalian and trypanosomal a'-tubulin is84-85% (Kimmel et al. 1985). Alphabet shows that a-and /3-tubulin share homologous antigenic regions, pre-sumably corresponding to a portion of the approximately40 % of their amino acid sequence that is homologous.C3B9 is an example of an isoform-specific antibody,which enables the distribution of such post-trans-lationally modified tubulin as well as its role in the cellcycle and tubulin dynamics to be studied. This antibodyhas also enabled us to demonstrate the evolutionaryconservation of lysine 40 as an acetylation site. CA12,however, appears to be specific. It does not cross-reactwith tubulin from any other species including Physanim(plasmodial or amoebal), Chlamydomonas or PtK2 cellson Western blots. It was even found not to cross-reactwith tubulin from Trypanosoma lewisi, implying diver-gence of /3-tubulin even within Trypanosoma. Each ofthese anti-tubulin antibodies will be useful probes infuture experiments.

The flagellar attachment zone observed by thin-sectionelectron microscopy appears as a complex, filamentoussystem and a series of punctate connections between theflagellar and pellicular membranes (Sherwin & Gull,1989). There are invariably four microtubules associatedwith a portion of the smooth endoplasmic reticulum,which lie immediately to the left-hand side of the flagellarattachment zone when viewed from the posterior of thecell (Gallo & Precigout, 1988). Our investigation, usingmonoclonal antibodies, has revealed two novel proteinsexhibiting differing properties, which are located in thiscomplex structure. These antibodies used in immunoflu-orescence and immunogold studies show that the flagellarattachment zone acts to connect the flagellum to the cellsurface as soon as a new flagellum has emerged from theflagellar pocket. Thus, development of the flagellumattachment zone appears to be concomitant with bothaxoneme and paraflagellar rod elongation.

The ROD1 monoclonal has revealed a doublet ofpolypeptides (Mr 180-200K), in addition to the pre-viously described doublet, PFR1 and PFR2 (Mr68-76K;Russell et al. 1983), to be present in the paraflagellar rod.These proteins are seen to be only minor components ofthe paraflagellar rod in comparison to PFR1 and PFR2.ROD1 has been shown to cross-react with a band thatcomigrates with /3-spectrin in human and porcine eryth-rocyte ghosts. Schneider et al. (1988) reported that apolyclonal antibody raised against human erythrocytespectrin cross-reacted with a doublet of 180-200K in T.brucei. It has been shown that spectrin is a protein notrestricted to vertebrates but may indeed be ubiquitous,having been found in many tissue types and evolution-arily diverse organisms (Baines, 1984; Virtanen et al.1984; Pollard, 1984). A recent report by Schneider et al.(1988) has shown that antibodies raised against mam-malian spectrin cross-react with a trypanosome protein inthe paraflagellar rod. Conversely, we have now raised anantibody to trypanosome proteins that cross-reacts withmammalian spectrin.

Trypanosoma brucei cytoskeletal components 499

The trypanosome cell undergoes a variety of changes incell shape and form during its life cycle. These changesare likely to be due to the direct modulation of itsintracellular cytoskeleton. Moreover, the complex flagel-lum and flagellar attachment zone is undoubtedly in-volved in the establishment of connections involved inattachment of the T. brucei cell to host cell surfaces in theGlossina stage of its life cycle. Our library of monoclonalantibodies, which defines each component of the cyto-skeleton, should prove useful in future studies of theseintra- and inter-cellular interactions.

This work was supported by grants from the ScienceEngineering Research Council, Medical Research Council (andThe Royal Society). This investigation received financial sup-port from the UNDP/World Bank/WHO Special Programmefor Research and Training in Tropical Diseases. We thankAdrian Blindt and Paul Walden for very kindly providing thepeptides.

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(Received 27 January 1989 - Accepted 6 April 1989)

500 A. Woods et al.