STRUCTURE AND CHEMICAL COMPOSITION OF INSOLUBLE ... · components are only approximate, c. Enlargement of another decomposed central pair of microtubules, showing the remaining stable

J. Cell Set. 58, 1-22 (1982)Printed in Great Britain © Company of Biologists Limited 1982

STRUCTURE AND CHEMICAL COMPOSITION

OF INSOLUBLE FILAMENTOUS COMPONENTS

OF SPERM FLAGELLAR MICROTUBULES

R. W. LINCK AND G. L. LANGEVINDepartment of Anatomy, Harvard Medical School,25 Sfull tuck Street, Boston, Massachusetts 02115, U.S.A.

SUMMARY

By progressive solvent extraction, we have obtained a series of subfragments of flagellarmicrotubules. Mild treatment gives rise to ribbons that contain longitudinally arrangedprotofilaments. Further extraction leaves a distinctive residue containing thinner ribbons, ofthree and eventually two protofilaments. Finally, filaments 2-3 nm in diameter and fibrousribbons apparently containing 6 or more 2 nm subfibrils are found. This latter solvent-resistantmaterial is consistently enriched in a characteristic set of polypeptides, which are found inflagella of several different species, including echinoderms and a mollusc. These polypeptidesappear different from a- and /9-tubulin on the basis of their solubilities, isoelectric points andelectrophoretic mobilities in sodium dodecyl sulphate/polyacrylamide gels; these conclusionsare reinforced by peptide mapping after limited proteolytic digestion, although the latter methodreveals certain similarities between these unique flagellar proteins, tubulin, chicken gizzarddesmin and rabbit actin. A remarkable feature of the protein in the final fraction is the higha-helical content: 71 % as measured by circular dichroism. We consider the possible origins ofthese filaments in the microtubule, in particular the possibility that microtubule protofilamentsare heterogeneous in protein composition, and we discuss some of the implications of ourfindings.

INTRODUCTION

Flagellar microtubules are uniquely suited for the study of microtubule structureand chemistry, since they can be isolated in their native state and free of contaminationfrom other cellular elements (Gibbons, 1965; Stephens & Edds, 1976). It has pre-viously been shown that flagellar doublet microtubules can be fractionated intochemically resistant ribbons of protofilaments, suggesting microheterogeneity withinthe walls of microtubules (Behnke & Forer, 1967; Linck, 1976; Meza, Huang &Bryan, 1972; Witman, 1970; Witman, Carlson, Berliner & Rosenbaum, 1972a;Witman, Carlson & Rosenbaum, 19726). Our own studies suggested a preliminaryexplanation for the stability of these protofilament ribbons; i.e. the ribbon fraction,when compared to doublet microtubules, was found to be composed of the classicala- and /?-tubulins, together with a unique set of polypeptides specifically associatedwith the ribbon moiety (Linck, 1976; Linck & Langevin, 1981). It was suggested atthe time that these unique proteins might account for the stability of the protofilamentribbons.

More recently we have studied the arrangement of subunits in the walls of flagellar

2 R. W. Linck and G. L. Langevin

microtubules (Linck & Langevin, 1981; Linck, Olson & Langevin, 1981; Woodrum &Linck, 1980). Our analyses indicated that structurally or chemically unique 'seams'may exist between certain protofilaments in native central-singlet microtubules andsuggested that singlet microtubules might also possess stable protofilament ribbons intheir walls. We report here the results of further investigations on the stable proto-filament ribbons isolated from flagellar axonemes and discuss the possible interpreta-tions and implications of our results. Preliminary reports of this work have beenpublished elsewhere (Linck et al. 1981; Linck, 1982; Linck, Albertini, Kenney &Langevin, 1982).

MATERIALS AND METHODS

The sperm flagellar microtubules used in this study were from sea urchins (Strongylocentrotusdroebachiewis and purpuratus) and clams (Spisula tolidissima). Axonemes and doublet tubuleswere purified as previously described (Linck & Langevin, 1981); they were fractionated withvarying concentrations of NaSCN or urea and analysed according to the quantitative pellet-assay procedure of Linck (1976). For controls, axonemes were resuspended in a final concentra-tion of 0-15 M-KC1, 10 mM-Tris, 5 mM-MgClj, 0-5 mM-ethylenediaminetetra-acetate (EDTA),1 mM-ATP, 1 mM-dithiothreitol (DTT) (pH 8-3), and doublet tubules in a final concentrationof 0-15 M-KC1, 10 mM-Tris, o-i mM-EDTA, 1 mM-DTT, pH 8-3. Fractionated samples wereresuspended to a final protein concentration identical to the controls, in 10 mM-Tris, 1 mM-EDTA, 1 mM-DTT (pH 83) and varying concentrations of NaSCN or urea. Samples wereextracted for 0-5 h and centrifuged at 100000 # for 90 min. The supernatants were discardedand the pellets freeze-dried. Duplicate fractions were prepared in parallel for negative-stainelectron microscopy (EM).

Flagellar B(a/?)-tubulin was purified from S. purpuratus by thermal fraclionation (Stephens,1970) and subsequent polymerization in vitro as previously described (Linck & Langevin,1981). Desmin was purified from chicken gizzards by the procedure of Geisler& Weber (1980);the final preparation contained filaments 10-12 nm in diameter, as judged by negative-stainEM, and was composed principally of 55000 M, polypeptides. Actin was purified from rabbitskeletal muscle (see Wilson, 1982). Bovine serum albumin was purchased from Sigma ChemicalCompany.

Sodium dodecyl sulphate (SDS)/polyacrylamide gel electrophoresis (PAGE) was carriedout according to the procedure of Laemmli (1970) with a o-6 mm thick slab gel apparatus.For analysis of flagellar microtubules the freeze-dried pellets described above were dissolvedin identical volumes of SDS sample buffer, boiled for 2 min and then dialysed against the samefor 12-18 h. Identical sample volumes were applied to each lane. Electrophoresis was carriedout at a constant 100 V, until the dye front moved through a 10 mm 3 % stacking gel and a130 mm 7 % running gel. A series of protein standards were electrophoresed for molecularweight estimations (Linck & Langevin, 1981). Gels were stained in 0-025 % Coomassie BrilliantBlue R in 25 % isopropanol/10 % acetic acid and destained in 10 % acetic acid.

Two-dimensional electrophoresis was performed according to O'Farrell (1975), with somemodifications. The isoelectric focusing (IEF) gel was composed of 4-0 % acrylamide, 0-2 %bisacrylamide, i -6% LK.B ampholines (range 5 to 7), 0-4% ampholines (range 3 5 to 10),9-5 M-urea and 2-0 % Non-Idet NP-4O, cast in a 1 • 5 mm x 100 mm tube. The protein sampleswere dissolved in 9-5 M-urea, 2 % Non-Idet, 5 % 2-mercaptoethanol. Afier prefocusing of thegel, a sample was applied to the basic end and focused for 22 h at 400 V and 1 h at 500 V. ForSDS—PAGE, an IEF gel was equilibrated with SDS sample buffer, placed across a slab gel(Laemmli, 1970) and electrophoresed as described above. To determine the isoelectric pointsof relevant polypeptides, a duplicate IEF gel was cut into 5 mm lengths and eluted with o-5 mldeionized water; the pH of the eluted ampholines was measured with a pH meter. These valueswere plotted with respect to gel length. The isoelectric points were then correlated with thestained bands on another duplicate gel after correction for expansion of the stained/destainedgel.

Microtubule structure and composition

Fig. i. Transverse sections of intact and decomposed S. purpuratus flagellar micro-tubules (tannic acid fixation), A. Flagellar axoneme. After exposure to low ionicstrength, the central-pair singlet microtubules decompose; one tubule decomposesfirst to yield a stable ribbon of four protofilaments (arrow). B. The doublet micro-tubule is composed of A- and B-subfibres (A, B). The A-tubule is composed of anouter 13 protofilaments and an inner 'adluminal component' (arrow), and the A-tubulepossesses at least six other circumferentially arranged binding sites specific for inner(ia) and outer (oa) dynein arms, radial spokes (1), the B-subfibre junctions and nexinfibres (see Linck & Langevin, 1981; Stephens & Edds, 1976); the A-tubule is thereforein some manner highly asymmetric (see Linck, 1976). Indicated positions of thecomponents are only approximate, c. Enlargement of another decomposed centralpair of microtubules, showing the remaining stable ribbons, one of which is composedof four protofilaments (arrow). Bars, 50 nm (A); 20 nm (B, C).

Peptide mapping was conducted according to the limited proteolytic digestion procedure ofCleveland, Fischer, Kirschner& Laemmli (1977). Initially, proteins were separated by electro-phoresis on a 06 mm thick slab gel as above (Laemmli, 1970). Relevant lanes were cut out fromthe unstained gel and stored at — 30 °C until needed. Individual bands containing ~ 10 fig ofprotein were then located and cut out, after comparing unstained lanes with adjacent stainedlanes. The cut-out bands were then incubated for 20 min in 2 ml of SDS sample buffer andplaced side-by-side in contact with a 1-5 mm thick Laemmli (1970) slab gel (3-0 % stacking gel,15 mm high; 15 % running gel, 115 mm high). The gel slices were overlaid with 1 ml of 1 %agarose in stacking gel buffer. Over the hardened agarose was layered a 1 ml solution containing15 fig of Staphylococcus aureus protease A (V 8 strain obtained from Miles Laboratories) inSDS sample buffer free of mercaptoethanol. Samples were electrophoresed at 100 V for 0-5 h,after which the voltage was turned off for a 05-h incubation period. Following the incubationthe gel was electrophoresed at 50 V until the dye front was 10 mm from the bottom of the gel.Gels were st-ined in 0-0175 % Serva Blue R in 25 % 2-propanol/io % acetic acid and destainedin 10% acetic acid.

2AR. W. Linck and G. L. Laneevin

Fig. 2. Negatively stained preparations from S. droebachiensis sperm flagellar axonemes.A. Representative example from purified axonemes showing an array of nine outerdoublet microtubules; the 24 ran axial periodicity of the dynein arms can be seen onthe lower tubule. B. Representative field of ribbons composed of two or three proto-filaments, after extraction of axonemes with 0-45 M-NaSCN. The polypeptide com-position of this exact same preparation is shown in Fig. $b, 0-45. Note that there aretwo classes of ribbons: clean and straight ribbons that consist mostly of two proto-filaments (i), or ribbons coated with flocculent or globular material and apparentlytwisted so that the number of protofilaments is difficult to determine (2). c. Representa-tive field of material after extraction of axonemes with o-8 M-NaSCN. The polypeptidecomposition of this exact same preparation is shown in Fig. 56, o-8. The material seenin the EM apparently consists of 2-3 nm fibrils existing singly, in pairs or formingtwisted or coiled sheets similar to those shown at higher magnification in Figs. 3D and4. Bars, isonm (A); 150 nm (B, C).

For thin-section electron microscopy flagellar microtubules were fixed with 6 % tannic acid/1 % glutaraldehyde (Tilney et al. 1973). Specimens were prepared for negative staining analysisusing 1 % uranyl acetate (Huxley, 1963). Electron microscopy was conducted on JEOL-100CXand JEOL-100S instruments. The axial periodicities of the microtubule surface lattice (Amos &Klug, 1974; Grimstone & Klug, 1966) and that of the dynein arms (Amos, Linck & Klug,1976) were used as internal calibration standards of the magnification factor.

Circular dichroism (CD) measurements were made in a Cary 60 spectrophotopolarimeterwith a 6001 CD attachment. For this work flagellar doublet microtubules were extracted with0-5 % sodium dodecyl sarcosinate (Sarkosyl), 3-5 M-urea, 10 mM-Tris, 1 mM-EDTA (pH 8)at 4 °C, and washed free of extracting solution with 1 mM-Tris, o-i mM-EDTA (pH 7-8).Sample concentrations ranged between 01 mg/ml and 0-5 mg/ml. The CD results were eval-uated in terms of the mean residue ellipticity (0) in units of (deg cms)/dmol. Data were fittedby a non-linear least-square routine using the spectra of Chen, Yang & Chau (1974) as basisfunctions for the determination. The percentages of a, /? and other forms are constrained to


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sum to ioo %. The CD analysis was conducted with the assistance and cooperation of DrThomas Rounds of the Department of Biological Chemistry, Harvard Medical School.

RESULTS

Decomposition of flagellar microtubules into chemically resistant filaments

Of the microtubules comprising the 9 + 2 flagellar axoneme, the central-pair singletmicrotubules are the most labile, and frequently one central tubule is more labile thanthe other (cf. Behnke & Forer, 1967; Stephens & Edds, 1976). In response to briefwarming, ageing at 4 °C or low ionic strength conditions, both of the central-singletmicrotubules decompose, each singlet tubule yielding one stable ribbon of four ormore protofilaments in S. droebachiensis (Fig. 1).

Doublet microtubules also decompose into ribbons of protofilaments (Linck, 1976)and into single filaments, 5-7 nm in diameter, that are relatively resistant to solubiliza-tion (Figs. 2, 3). Extraction of either purified flagellar axonemes or purified doubletmicrotubules with increasing concentrations of NaSCN or urea causes a stepwisesolubilization of protofilaments and the production of ribbons of gradually fewer andmore uniform numbers of protofilaments. For S. droebachiensis 0-35 M-NaSCN or2-5 M-urea produces ribbons of three protofilaments (Fig. 3 A) contaminated slightlyby ribbons of two and four protofilaments; 0-45-0-5 M-NaSCN or 3 M-urea producestwo kinds of ribbons composed of two and three protofilaments (Figs. 2B, 3B); andfinally, extraction with o-8 M-NaSCN or 4 M-urea yields populations of single ordouble 2-3 run filaments and twisted sheets of fine subfibrils. In the Discussion wewill consider the exact relationship of these remaining filaments to the microtubulewall; for the present we will refer to them as the population of chemically resistantfilaments. The negative-stain EM analysis was conducted in a semi-quantitativemanner; i.e., constant amounts of axonemes were suspended either in control bufferor in extracting solutions, and identical amounts of the samples were applied to grids.The chemically resistant filaments are never seen in the original preparations ofaxonemes, and they appear only following the decomposition of the ribbons of threeprotofilaments; thus the stable filaments are not contaminants but originate from thedecomposition of the microtubule wall.

Fig. 3. Representative examples of chemically resistant ribbons and filaments fromS. droebachiensis flagellar microtubules, negatively stained, A. Ribbon of three proto-filaments (arrows), 18 nm wide, from 0-35 M-NaSCN. B. Ribbon of two protofilaments(arrows), 12 nm wide, from 0-5 M-NaSCN. c. Stable filament (arrow), 6 nm wide,from 4 M-urea. D. A chemically insoluble filament, apparently formed from a twistedor coiled sheet of six subfibrils spaced at 2-9 nm intervals (arrows), from 5 M-urea.Note that the preparations of insoluble filaments from 4 M and 5 M-urea are essentiallydevoid of a- and ^-tubulin; compare with Figs. 4, 5. Some of the protofilaments inA and B and the insoluble filament in c appear either to be hollow or to be subdividedby a longitudinal cleft, and the middle protofilament of A appears to have a helicalsubstructure with a pitch of 96 nm: details are best seen by tilting the page and sight-ing along the axes of the filaments. Bar, 20 nm.


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The ribbons, and protofilaments thereof, produced by the milder treatments, varysomewhat in their appearance in negative stain. Some are relatively clean and appearto be composed exclusively of protofilaments: these 'clean ribbons' almost alwaysappear straight and rigid (Fig. 2 B). Most of the ribbons, however, are heavily coatedor decorated with amorphous or globular material (Fig. 2 B), which occasionally is seenperiodically arranged along the ribbons (see Linck, 1976; Meza et al. 1972); these'coated ribbons' are usually curvilinear and frequently twisted. The individualprotofilaments, either free or within a ribbon, vary in diameter in negative stain from


5 to 7 nm; they frequently appear to be hollow or to be subdivided by a longitudinalcleft (Fig. 3A-c), and occasionally they appear to possess a helical substructure(Figs. 3 A, D).

After the harsher treatments, such as extraction with 4 M-urea or o-8 M-NaSCN,few 5-7 nm wide protofilaments or filaments can be identified; those that are seenappear to consist of two fibrils measuring less than 3 nm wide (Figs. 2 c, 3 c). Thefibrils sometimes appear to fray apart into individual 2-5-3 n m subfibrils. Occasionallyin o-8 M-NaSCN and frequently in 5 M-urea, sheets of six or more longitudinally orhelically arranged subfibrils are seen (Figs. 3 D, 4). As many as seven or eight subfibrilshave been seen within a sheet, and within a sheet the centre-to-centre spacing ofthese elements measures 29 nm or less.

Morphological results similar to those reported above have been obtained withother species (e.g., S. purpuratus and S. solidissima).

Electrophoretic analysis of fractionated flagellar microtubules

SDS/polyacrylamide gel electrophoresis was used to identify the polypeptidesforming the chemically resistant filaments. Flagellar axonemes and doublet microtu-bules were fractionated as above with NaSCN or urea and the insoluble components(isolated as 9 x io6 g-min pellets) were analysed in a quantitative manner by SDS-PAGE. Fig. 5 summarizes these results. Fig. $a-b compares the results for thefractionation of S. droebachiensis axonemes with urea and NaSCN. Fig. 5 c shows theresults for the fractionation of S. solidissima doublet tubules with urea. For a givenspecies, urea and NaSCN have similar effects, except in the solubilization of certainminor polypeptide components; however, the effective concentrations of NaSCNand urea are different. In general the pattern of extraction is similar for differentspecies. Increasing concentrations of urea or NaSCN solubilize increasing amountsof a- and /J-tubulin and most other proteins. At higher concentrations of urea, only afew major proteins are recovered quantitatively in the insoluble fraction, including avariable number of proteins in the molecular weight range of 46000 to 62000 and afraction of the polypeptides in the dynein region. In 4-5 M-urea, essentially all of thea- and /?-tubulins are solubilized and two principal polypeptide components of lowerapparent molecular weights remain, constituting approximately 80% of the filamentprotein as measured by gel densitometry (see also Fig. ja). It is important to note thatthe o-8 M-NaSCN- and the 4-5 M-urea-insoluble samples correspond to the resistantfilaments with a fibrous substructure (Figs. 2C, 3D, 4). Although a considerablefraction (approximately 80%) of the polypeptides in the dynein region is extracted

Fig. 4. A. Typical example of a chemically resistant filament, after extraction ofS. droebachiensis axonemes with 5 M-urea and negative staining. This filament prepa-ration is devoid of a- and /9-tubulin (see Fig. 5a, 5). This unique kind of filamentappears to have the form of a twisted ribbon of subfibrils, with inflection points atarrowheads. B. Portion of A between stars, shown at higher magnification. The sheetappears to be composed of seven (or possibly eight) subfibrils, with a lateral centre-to-centre spacing of 20. nm. In examples not shown the sheets of subfilaments separateinto individual strands less than 2-5 nm in diameter. Bars, 100 ran (A); 50 nm (B).

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in 2 M-urea (and 0-25 M-NaSCN), the extraction of these high molecular weightpolypeptides quickly levels off, and thereafter a relatively constant amount(approximately 10%) of this material remains in the insoluble fraction.

The polypeptides comprising the protofilament ribbons were also analysed byisoelectric focusing and two-dimensional electrophoresis. Fig. 6 shows the resultsobtained with ribbons isolated by extraction of S. droebachiensis axonemes with 0-35 M-NaSCN; such ribbons contain approximately three protofilaments. By independentmarkers (sea-urchin sperm flagellar B-tubulin) two of the polypeptides were shown tohave the same molecular weights and isoelectric points as a- and /?-tubulins. Othermajor ribbon polypeptides have molecular weights ranging from 46000 to 55000 andisoelectric points ranging upwards from 57. In the samples extracted with higherconcentrations of NaSCN or urea the quantities of a- and /9-tubulin are significantlyreduced, while the quantities of certain of the other polypeptides remain unchanged(refer to Fig. 5). In S. droebachiensis the insoluble filament fraction isolated fromo-8 M-NaSCN (Fig. 2c) is composed predominantly of polypeptides 10, 11, 14, 16, 20and 23-25 (refer to Fig. 6), which are present in approximately equal amounts. The5 M-urea-insoluble filament fraction (Figs. 3C-D, 4) is composed of polypeptides12-14 and 16-18 (refer to Fig. 6), and these polypeptides constitute approximately80% of the filament protein. Similar results as above have been obtained with S.pwpuratus (see Fig. ja).

Peptide mapping analysis

Insoluble filament fractions were prepared by the extraction of S. purpuratusflagellar doublet microtubules with 3 and 5 M-urea; the 5 M-urea-insoluble filamentswere found by negative staining to be composed of multistranded fibrils, as in S.droebachiensis (Fig. 2D, 3 D, 4). From these two urea fractions three major polypeptides

Fig. 5. SDS—PAGE analysis of different species of flagellar microtubules afterextraction with varying concentrations of NaSCN or urea. Sample loading wasquantitative and stoichiometric: within each gel set (a, b, c) the samples applied toeach lane (except A*) represent the insoluble material (9 x 10' g min) obtained fromthe identical amount of sample in the first control lane of each set. Apparent mole-cular weights are given (x io~3). a. Urea extraction of S. droebachiensis flagellaraxonemes. Lane A, 50 fig axonemes; lane A#, 10 fig axonemes; lanes 2-5, insolublematerial after extraction of 50 fig axonemes with 2-5 M-urea, respectively, b. NaSCNextraction of S. droebachiensis flagellar axonemes. Lane A, 50 fig axonemes; lanes0-25-0-80, insoluble material after extraction of 50 fig axonemes with 0-25-0-8M-NaSCN, respectively, c. Extraction of 50 fig of S. solidissima doublet microtubules(lane D) with 3 and 4 M-urea. D (Mr ~ 330000) indicates the dynein region on the SDSgels; a. and /? arrows, a- and ^-tubulin. Examples of these exact same preparationsare shown in Figs. 2-4. Note that the o-8 M-NaSCN and the 4-5 M-urea-insolublesamples are depleted or devoid of a.- and /9-tubulin and correspond to the mostresistant filaments with fibrous substructures. In the quantitative studies of thefractionated tubule3 presented here visually, it is important to realize that at higherconcentrations of NaSCN or urea, the solubility points of tome of the filamentproteins are approached and recovery of the polypeptides composing the stablefilaments may not be complete. More heavily loaded gels of 5 M-urea-insolublefilaments are shown in Fig. 7.

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Fig. 6. Two-dimensional isoelectric-focusing (IEF)/SDS-PAGE analysis of theinsoluble protofilament ribbon fraction isolated by the extraction of S. droebachiensisaxonemes with 0-35 M-NaSCN. The protein sample was applied to the basic end ( —)of the IEF gel and focused toward the acidic end ( + ). Stars indicate the ends of theIEF gel on top of the SDS (Laemmli, 1970) slab gel. Apparent molecular weights( x i o ~ s ) are given on the left and right margins. Isoelectric points (regarded aspreliminary), determined from a duplicate IEF gel, are indicated at the bottom.All Serva Blue-stained spots that are reproducibly seen in these preparations areindicated by reference numbers. Spots io(a) and u(/?) were shown independently tocomigrate with flagellar a- and /?-tubulin, respectively. This preparation can becompared with that in Fig. 56, 0-45. The major polypeptides composing the o-8 M-NaSCN-insoluble filaments (Fig. 2C and Fig. $b, o-8) correspond to the polypeptidesmarked io(a), u(/5), 14, 16, 20 and the group 23-25. The major polypeptides com-posing the 5 M-urea-insoluble fraction are numbers 14 and 16. Chicken gizzarddesmin has the same isoelectric points as polypeptides 20-22.


were purified for peptide mapping analysis (Fig. ja). Comparisons of these poly-peptides were made with S. purpuratus flagellar B(a/?)-tubulin, rabbit muscle actin,chicken gizzard desmin and bovine serum albumin, using the S. aureus proteaselimited digestion technique of Cleveland et al. (1977). The results of the peptidemapping are best described by referring to the actual data (see Figs. 76, 8); briefly,the results are as follows. (1) Two of the chemically resistant flagellar filament proteins,which are electrophoretically quite different from a- and /?-tubulin, are neverthelesssimilar in their peptide maps to a- and /?-tubulin. (2) One of the flagellar filamentproteins, which migrates closely with /?-tubulin on SDS-PAGE gels, is quite differentfrom a- and /?-tubulin. (3) Certain of these flagellar filament proteins and the tubulinsshare some peptide-map similarities with chicken gizzard desmin. (4) Finally, /9-tubulin and actin yield rather similar size-related peptide fragments (Fig. 8).

Determination of helical content by circular dichroism

The chemically resistant filament fraction was isolated from S. purpuratus byextraction of doublet microtubules with 0-5 % sodium dodecyl sarcosinate, 3-5 M-urea,and the secondary structure of the filament polypeptides in solution was measured bycircular dichroism (CD). By negative-stain EM this material is composed of 2 runfilaments and bundles thereof (Fig. 9 A) and is composed of two principal polypeptidesthat account for greater than 80% of the protein (Fig. 9B) and comigrate with thetwo 5 M-urea-insoluble polypeptides (Fig. ja). Preliminary CD spectra of suchmaterial (Fig. 10) reveals this filament fraction to have an a-helical content of approxi-mately 70%, compared to approximately 30% reported for tubulin (Ventilla, Cantor& Shelanski, 1972; Ponstingl, Krauh, Little & Kempf, 1981). A more extensive CDanalysis of these filaments is in progress and will be presented elsewhere.

DISCUSSION

Identity of the chemically unique filaments

The results presented here demonstrate that under certain conditions flagellarouter-doublet and central-singlet microtubules disassemble to a point at whichribbons of protofilaments remain stable. While each central-singlet tubule appears toyield only one such protofilament ribbon (see also Meza et al. 1972), our previouswork indicated that doublet tubules possess at least two probably different ribbonmoieties (Linck, 1976). As shown here, doublet microtubules can be subfractionatedfurther into populations of structurally and chemically unique filaments, following thesolubilization of most if not all of the normal a- and /?-tubulin. Our correlative EM/biochemical analysis indicates that these unique insoluble filaments must be presentin the stable ribbons resulting from the milder treatments and must therefore form orbe associated with certain of these ribbon protofilaments.

The more chemically resistant of these filaments and/or protofilaments (isolatedfrom o-8 M-NaSCN or 4-5 M-urea) are composed predominantly of a heterogeneousset of proteins in relatively equal amounts, which are different from (but not neces-sarily unrelated to) a- and /?-tubulin in their solubility properties, apparent molecular


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Microtubule structure and composition i5

weights (on discontinuous SDS-PAGE gels), and isoelectric points. The generalelectrophoretic properties of these unique proteins vary slightly for differentanimal species (compare Fig. 5 a and c; and see Linck, 1976). Each of thesepolypeptides on SDS-PAGE gels represents approximately 3-4-5% of the micro-tubule protein as measured by quantitative gel densitometry (see table 2, com-ponents Ee 21, 22 and 23, of Linck, 1976). We can rule out the possibility thatthese unique proteins are in vitro artifacts (proteolytic breakdown products of a- or/?-tubulin) since, for any of the animal species studied, the physical/chemical proper-ties of these proteins and their relative stoichiometric ratios to a- and ^-tubulin areconstant from preparation to preparation under a variety of laboratory conditions.Furthermore, one stable filament protein (S. purpuratus polypeptide 1, Fig. 7), whichmigrates closely with /?-tubulin on SDS-PAGE gels, is very different from a- or /?-tubulin on the basis of peptide mapping analysis; thus 5. purpuratus polypeptide 1 isnot a large proteolytic derivative of a- or/9-tubulin. Likewise, S. purpuratus polypep-tides 2 and 3, although they are partially related in their peptide maps to a- and

Fig. 7. Comparative peptide mapping of three different microtubule filament proteinswith tubulin and desmin. a. Two stable filament fractions were first prepared by theextrac; ion of S. purpuratus (S.p.) flagellar doublet microtubules with 3 M and 5 M-urea;these two fractions were analysed on SDS-PAGE gels as shown: polypeptides marked1, 2 and 3 have apparent molecular weights (Afr) of approximately 55000, 51000 and47000, respectively. The relative positions of a- and /9-tubulin are indicated in the3 M-urea fraction; they are absent in the 5 M-urea fraction. For subsequent peptidemapping, unstained bands 1 and 2 were excised from slab gels of the 5 M-ureapreparation, and band 3 from the 3 M-urea preparation (see Materials and Methods).Similarly, previously purified flagellar B <x- and /?-tubulin and chicken gizzard desminwere electrophoretically purified, b. Limited proteolytic digests (using 5. aureusprotease (Cleveland et al. 1977) of S.p. flagellar B a- and /9-tubulin (a, P), threestable filament proteins from S.p. {S.p. 1, 2 and 3) from a, and desmin (D). Approxi-mately equal amounts of the proteins were compared. All proteins were cleaved tosome extent (there is no staining above the top stained band of undigested protease,27700 Mr). In comparing the peptide maps we have considered similarities in thenumber of proteolytic fragments, their relative mobilities (i.e., M, values) and theirrelative densities (i.e., relative number of fragments per molecule) and the fact that atotally unrelated protein (bovine serum albumin) shows a very different pattern ofdegradation (see Fig. 8); four separate peptide mapping analyses were performed andthe data were reproducible. Horizontal dashes indicate some of the peptide fragmentsimilarities between adjacent samples; open circles indicate fragments common totubulin and desmin. Results of the comparisons are as follows: (i) except for slightdifferences, a- and /?-tubulin are indistinguishable under the conditions used, (ii) Thepeptide maps of S.p. proteins 2 and 3 (51000 and 47000 Mr, respectively) are nearlyidentical, with the major difference being that S.p. 3 has some higher M, fragments,(iii) S.p. 2 and 3 are both quite different from S.p. 1 in the M, values and relativeamounts of most fragments, although there are some similarities in the lower M,fragments, (iv) S.p. 1, which comigrates closely with /?-tubulin (see a), is quitedissimilar from /^-tubulin, although certain fragments are related in MT values butnot relative amounts, (v) The M, values and relative amounts of five fragments ofS.p. 2 and 3 are similar to those of ct- and /?-tubulin, and the Mr values (but notrelative amounts) of most fragments of S.p. 2 and 3 are similar to desmin. (vi) Finally,a- and y?-tubulin and desmin show similarities in the Mr values and (to some extent)the relative amounts of their fragments (open circles). See also Fig. 8.

R. W. Linck and G. L. Langcvin

SA I A I )3 I OL I D

I I I I

I I I IFig. 8. Peptide maps as described in Fig. 7 comparing bovine serum albumin (SA),rabbit skeletal actin (A), S. purpuratus flagellar B-tubulins (a and ff) and chickengizzard desmin (D). Serum albumin bears little similarity to the other proteins,whereas there are considerable homologies between actin and ̂ -tubulin, and betweena-tubulin and desmin, under the conditions used.

/ff-tubulin, do not appear to be simply shorter lengths of tubulin; this conclusion issupported by the fact that polypeptide 2 is a major component forming the 2 nmfilamentous material (Figs. 4, 9B) of high a-helical content (Fig. 10).

Regardless of the actual identity of the proteins forming the unique filaments, ourelectron microscopic studies indicate that both the 08 M-NaSCN- and the 4-5 M-urea-insoluble filaments have a 'filamentous* rather than a 'globular' substructure(Figs. 2c, 3, 4). In the splayed-out sheets the centre-to-centre spacing betweensubfibrils is approximately 29 nm; given that some lateral separation of these elementsmay take place during the dissolution process, the diameter of an individual subfibrilis probably less than 2-9 nm. The fibrous substructure (Fig. 4) and the relatively higha-helical content (Fig. 10) of the insoluble fraction suggest that each 2-2-5 mn

subfibril corresponds to longitudinally associated (possibly paired) a-helices (Pauling,Corey & Branson, 1951; Cohen, 1966).

Origins and functions of the insoluble filaments in flagellar microtubults

Our work here suggests the sequential chemical breakdown of flagellar microtubulesto yield ribbons of two or three protofilaments, which subsequently decompose toform 3 nm filaments and sheets of subfibrils of unique chemical composition. Theorigins and functions of these filaments in flagellar microtubules have not been deter-mined, but there are several possibilities. The filaments or subfibrils could stabilize


b 1

\ &T F

f\

rt

Fig. 9. Subfractionation of 5. purpuratui flagellar doublet microtubules into subfibrilsfollowing extraction with Sarkosyl/urea. a. SDS—PAGE gels of doublet micro-tubules (T) and the isolated filament fraction (F) showing a-tubulin, /?-tubulinand polypeptides 1, 2 and 3, as in Fig. ya. b. EM image of the negatively stainedfilament fraction, showing individual 2 run fibrils (small arrows) and bundles ofsame (large arrow). Note that the filaments appear to form a branching and anastomos-ing network. Bar, 100 nm.

the two to three protofilament ribbons by lying alongside or between tubulin proto-filaments in the same way as tropomyosin stabilizes an actin filament. These stablefilaments and fibrous sheets could also arise from the 'adluminal components' of theA-tubules (see Fig. 1), or they could be artifacts formed by aggregation, althoughthis latter possibility seems unlikely.

Another possible interpretation, however, is shown in Fig. n . Certain imagessuggest that the fibrous sheets may have originally been rolled up or twisted to form5-7 nm diameter filaments. Our data are consistent with the hypothesis that these5-7 nm filaments may represent one or more unique protofilaments in the A-micro-tubule. In particular, two of the urea-insoluble filament polypeptides (S. purpuratus2 and 3 of Fig. yd) are similar to each other and bear some peptide homologies totubulin. Thus, they may contain certain molecular domains in common with tubulin

i8 R. W. Linck and G. L. Langevin

o

24

20

16

12

8

4

0

A

—4

- 8

-12

\

\

\

• \

\

2O0 220

X (nm)

240

Fig. io. Preliminary analysis by circular dichroism spectroscopy indicates that thelimiting filament fraction from 5. purpuratus (see Fig. 8) has an a-helical content ofapproximately 71 %. {0), mean residue ellipticity in (deg cm»)/dmol. ( )measured spectra; ( ) calculated spectra.

and may be capable of forming fibrous protofilaments and associating laterally withtubulin protofilaments in the microtubule wall. Also in agreement with the possibilityof protofilament heterogeneity are certain quantitative data available on the insoluble-filament polypeptides and tubulin. The o-8 M-NaSCN-insoluble filament fractionshows only extended filaments and fibrous sheets, yet is composed of five or morepolypeptides (Fig. 56, o-8) in nearly equimolar amounts. By quantitative gel densito-metry each of these five polypeptides is present in doublet microtubules in a ratio ofapproximately 1 :20 relative to the total a- and /?-tubulin content (refer to table 2 ofLinck, 1976); these polypeptides are thus present in sufficient quantities to accountfor approximately three to six protofilaments in a doublet microtubule composed of 23protofilaments (see Fig. 12), given the observed two-fold variations in protein:dyebinding ratios (Potter, 1974).

These possibilities are summarized in the model shown in Fig. 12, which illustrateshow structurally or chemically distinct protofilaments might be arranged in thedoublet microtubule, and thereby how the organization of the various axonemecomponents might be determined. This model is also supported by the followingobservations.

(1) Previous work indicates that there are two or more stable protofilament ribbonmoieties per A-tubule (Linck, 1976). Given the asymmetric organization of the


Fig. 11. Simplified schematic model showing the pattern of breakdown of spermflagellar microtubules deduced from this study. Following the solubilization of thetubulin dimers (white), the microtubule decomposes first into protofilament ribbonsof decreasing width, then into chemically stable filaments (black or shaded), andultimately into 2 nm subfibrils of high a-helical content. The precise origin of the2 nm subfibrils is not certain. Our data suggest that six or more of these fibrils may becoiled together (as shown here) or organized in parallel to form special protofilamentsin doublet and singlet microtubules; alternatively, these fibrils may be accessoryfilaments laterally associated with certain protofilaments.

2O R. W. Linck and G. L. Langevin

Fig. 12. A cross-sectional diagram of a flagellar or ciliary doublet microtubuleshowing the A- and B-tubules, the protofilaments (numbered) forming the walls, andthe approximate positions of various axoneme components, e.g., the outer dyneinarm (OA), the inner dynein arm (IA), the radial spoke (S), and the 'adluminalcomponent' (shaded). This model suggests that there may be several classes of proto-filaments (black; arranged hypothetically here), which are structurally and chemicallydistinct and may act to specify the organization of the associated components.

axoneme components on the A-tubule (see Fig. i), there is reason to suppose thatthese two protofilament ribbon moieties are distinct, differing possibly in theirprotofilament structure or chemistry.

(2) Given that the central-singlet microtubules shown here (Fig. 1) do not have anyapparent extrinsic filaments associated with their walls and yet break down to yieldribbons of four protofilaments, it is possible that certain of the central-singlet microtu-bule protofilaments are also structurally or chemically heterogeneous.

(3) The 5 M-urea-insoluble filament polypeptides present in doublet microtubulesare quantitatively retained in the two- and three-protofilament ribbon fractions,following the solubilization of most of the tubulin-containing protofilaments. Thus,these 'fibrous' polypeptides are associated with or possibly form a subset of themicrotubule protofilaments. In fact, certain microtubule protofilaments appear tohave a helical substructure (e.g. Fig. 3 A), as would be expected if these protofilamentswere composed of coiled coils of a-helices.

(4) Similarly, a constant amount (~ 10%) of the polypeptides in the dynein regionon SDS-PAGE gels remains associated with the insoluble filament fraction followingthe fractionation of the axoneme with NaSCN (Fig. 5), indicating that these highmolecular weight components may be associated with specific protofilaments.

Possible implications of this work

This report has centred on the characterization of these unique filaments isolatedfrom flagellar microtubules. It is not unreasonable, however, to suppose that non-flagellar, cytoplasmic microtubules might also contain chemically resistant protofila-


ments or filamentous components. Earlier studies in fact suggested that microtubuleswere to a certain extent 'interconvertible' with the class of cytoplasmic filamentstermed ' 10-nm filaments' or 'intermediate filaments' (Ishikawa, Bischoff & Holtzer,1968; Moellman & McGuire, 1975; Peters & Vaughn, 1967), which are known to berelatively chemically resistant to solubilization (see Davison, 1981; Lazarides, 1980).We have noted earlier (Linck et al. 1981; Linck, 1982) and we observe here in greaterdetail that the insoluble filamentous components from sperm flagellar microtubulesare strikingly similar to intermediate filaments (IFs). Specifically, the insolubleflagellar filaments and constituent proteins described here are similar to certain IFsand IF proteins in terms of their solubility properties, isoelectric points, apparentmolecular weights (i.e., electrophoretic mobilities on SDS-PAGE gels), their fila-mentous substructure, their high a-helical content and certain apparent similarities intheir peptide maps. In the light of our findings and the above discussion, it is possiblethat the microtubule model in Fig. 11, although preliminary, may explain the apparent' interconvertibility' of microtubules with certain intermediate filaments and othercytoplasmic filaments, and it may offer suggestions and explanations for the mechan-ism of microtubule assembly and the mechanisms of various forms of cell motility.

We are deeply indebted to Drs Linda and W. B. Amos for their constructive criticisms ofthis paper and to W. B. Amos for completing Figure 11. We are also grateful to Drs DavidAlbertini and Raymond Stephens for invaluable discussions during the course of this work.

This work was supported by grants from the U.S. National Institutes of Health (GM 21527)and S07-RR-05381-20) and the National Science Foundation (PCM 78-16158).

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{Received 1 July 1982)

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STRUCTURE AND CHEMICAL COMPOSITION OF INSOLUBLE ... · components are only approximate, c. Enlargement of another decomposed central pair of microtubules, showing the remaining stable