Structural polarity and directional growth of microtubules of Chlamydomonas flagella

J. Xol.

Bid.

(1974) 90, 381-402

Structural Polarity and Directional Growth of Microtubules of Chlamydomonas Flagella CAROL~LENAND Departnterd

GARY G.BORISY

of Zoology am? Laboratory of iWoleczllar Biology lJ78iversit2/ of Wisconsirh Madison, Wise., U.S.A.

(Received 11 April

1974, and in revised form 8 July 1974)

A basic question concerning microtubule assembly is the polarit,y of growth, namely, whether subunits can add to either end of a growing microtubule or whether growth proceeds by subunit addition to only one end. To approach this question in an iv, vitro system, experiments were carried out on the addition of microtubule subunits to isolated flagellar axonemes. Flagella were detached from Chlamydomonas by brief treatment with non-ionic detergent, isolated by differential centrifugation, and incubated with crude high-speed extracts of porcine brain tissue or with purified tubulin (obtained by repetitive temperaturedependent assembly and disassembly). Electron microscopy of negatively stained samples showed &8 many as 11 long microtubules added at one end of more than 00% of the axonemes. Colchicine (100 PM), CaClz (2.5 mM), and low temperature (O’C) both prevented and reversed microtubule assembly but had no effect on axonemal length. In crude extracts microtubules formed on both members of the axonemal central pair but on only the A-tubule of the outer doublets. Flagellar fragments, produced by mechanical shearing, were also incubated with microt,ubule subunit. Single tubules formed at only one end of outer doublet fragon one or both members of central ments; the appearance of single tubules pair fragments was predominantly unidirectional. Structural analysis of frayed ;~xonemes and the asymmetry of side-arm attachments permitted the absolute

polarity

of the axonamal fragments to be determined

and revealed that assembly

proceeded by addition tjubules. Using purified

of subunits to the distal ends of the axonemal microbrain tubulin, a limited extent of proximal addition and growth on the B-tubule also occurred. The extent of proximal addition increased with increasing protein concentration and temperature. We conclude that thr microtubules of flagella have an intrinsic polarity reflected in their side-arm attachments and in their directionality of growth.

1. Introduction l!he ordered assembly of microtubules is of importance in many fundamental cellular processes including formation of the mitotic spindle, neuronal outgrowth, flagellar development and the maintenance of cell form (see Tilney, 1971; Bardele, 1973; Olmsted & Borisy, 1973b, for reviews). As originally noted by Porter (1966), in order to influence the shapes of cells, microtubules must be ascribed a directionality in their growth. Polarity of microtubules is also an important feature of some models of chromosome movement (Subirana, 1968; McIntosh et al., 1969) and is suggested by in vitro studies of flagellar motility (Summers & Gibbons, 1971). 381

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In this reportt we describe experiments in which the technique for obtaining functionally active flagellar axonemes (Gibbons & Gibbons, 1972) has been combined with the methods for microtubule assembly in vitro (Weisenberg, 1972 ; Borisy & Olmsted, 1972; Borisy et al., 1974) in order to characterize (1) some aspects of the polarity evident in the axonemal structure, and (2) the directionality of microtubule growth in v&o. Our results demonstrate (1) that the functionally important side-arms are attached to the outer doublets with a structural polarity, and (2) that microtubule growth in vitro occurs in a polar manner but that the directionality is influenced by the source of subunits, protein concentration and temperature.

2. Materials and Methods (a) Cell culture Chlamydomonaa reinhardii strain 21 GR (wild-type) was grown in 160 ml of aerated synthetic medium (Sager t Granick, 1953) in 250-ml Erlenmeyer flasks. Cell synchrony was induced by subjecting cultures to a 14-h light, 10-h dark illumination schedule (Bernstein, 1960). Fluorescent light at an intensity of 1000 foot candles was provided during the light portion of the cell cycle ; throughout the cycle a temperature of 25% 1 deg. C was maintained. Flasks were inoculated with 5 to 6 x lo4 cells and grown for 5 days to a fmal density of 4 to 6 x lo6 cell/ml. (b) Detergent treatment of cm% Cells from 3 or 4 cultures in the 3rd to 5th h of the light cycle were harvested by centrifugation at 230 g (1000 revs/mm, no. 230 swinging bucket head, International Equipment Co.) for 6 min in BO-ml conical tubes. The cells were washed twice with 150 ml of distilled water by sedimentation and resuspension. The resulting pellets were pooled, washed once in 60 ml of buffer I (10 mM-HEPES (N-2-hydroxyethylpiperazine-N’-2-ethane sulfonic acid), 5.0 rnx-MgSO,, 0.5 ma6-mercaptoethanol, 0.6 m-EDTA, adjusted to pH 7.5 at room temperature with NaOH), and resuspended in 1 ml buffer. Flagelle were detached from the cells and flagellar membranes removed by brief extraction in buffer I containing the non-ionic detergent, Non-Idet P40 (Shell Chemicals, Ltd, London). Axonemes thus obtained were then isolated from the cell suspension by differential centrifugation. Flagellar amputation was nearly quantitative and no cell lysis occurred at a final concentration of O*O36e/odetergent, thus this concentration was used routinely for tlagellar isolations. Dense cell suspensions (1 ml) were diluted with 9 ml of buffer I containing 0.04% (w/v) Non-Idet, stirred briefly, and centrifuged at 230 g for 7 to 10 min. The supernatsnt, containing flagellax axonemes, was removed to within 0.5 ml of the cell pellet and the centrifugation repeated. All operations up to this point were carried out at room temperature. The second axonemal supernatant was recovered and centrifuged at 31,000 g at 0°C for 30 min (Sorvall RCBB, SS34 rotor, 16,000 revs/mm) to pellet the axonemes. The loose pellet obtained was resuspended in 10 ml of cold buffer I and the centrifugation repeated. The Snal axonemal pellet was suspended in 0.6 ml of buffer I and stored on ice. Protein concentrations of the axonemal suspensions were determined by the micromethod described by Lowry et al. (1951) and ranged between 0.6 and 1.0 mg/ml expressed in bovine serum albumin equivalents. In some experiments fragments were prepsred from the axonemes by shearing. Samples of an axonemal suspension were forcibly passed through a 4-m, 22-gauge hypodermic needle. A heterogeneous mixture of broken axonemes, outer doublets, and central p8ir microtubules were produced (see Results). In all experiments, the axonemes or fragments were used within a few hours of preparation. aooounta oovering part of the work reported here were presented at the 13th of the Amer. Soo. Cell Biol. (J. Cell. Bill. 59, tie, 1973) and the 67th Annual Meeting of the Fed. Amer. ,300. Exp. Biol. (Fed. PYOC. Fed. Amer. 800. Exp. Bid. 88, 167, 1973). t Preliminary Annual Meeting

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(c) Preparation of porcine brain extracts High-speed supernatants of porcine brain tissue homogenates were used ss the source of microtubule subunits for some experiments (Borisy & Ohnsted, 1972). Brains were obtained within 2 h of slaughter and chilled on ice; all operations were done at 0°C. After removal of superficial blood vessels and meninges, coarsely minced tissue was mixed with buffer II (100 mr+PIPES (piperazine-N,N’-bis (2-ethanesulfonic acid)), 1 mm-EGTA, 2.5 m&r-GTP, adjusted to pH 694 at room temperature with NaOH) in a ratio of 1 g wet weight tissue to 15 ml buffer. The tissue wss homogenized with 6 passes of a tightly fitted motor-driven teflon pestle operated at 2000 revs/min. Cell debris was removed from the homogenate by oentrifugation for 15 min at 31,000 g (Sorvall RC2B, SS34 rotor, 16,000 revs/mm) and the extract then centrifuged at 230,000 g for 90 min (60,000 revs/ min, Spinco type 65 rotor, 9.0.ml polyoarbonate tubes) to provide a high-speed supernatant. Protein concentrations of the high-speed supernatants were between 6 and 8 mg/ml. In some experiments partially purified brain microtubule protein obtained by repetitive cycles of temperature-dependent polymerization and depolymerization (Borisy et aZ., 1974) wae used se the source of microtubule subunit. Homogenates were prepared as described above and centrifuged at 25,000 g for 60 min (Spinco, flxed angle rotor, 20,000 revs/min) to provide a low-speed supernatant. The supernatsnt protein wss polymerized for 20 min at 37°C and a microtubule pellet obtained by centrifugation at 37°C for 30 min at 38,000 g (Sorvall RCBB, SS34 fixed angle rotor, 18,000 revs/mm). The resulting supernatant was decanted and the volume noted. The microtubule pellets were resuspended in cold buffer II to 1/5th vol. of the recovered supernatant. The resuspended pellets were depolymerized on ice for 30 min and the suspension centrifuged as above, but at 2 to 4°C to remove residual aggregates. Microtubule protein from this cold supernatant was again polymerized at 37°C and a second microtubule pellet obtained. These pellets were immediately frozen by immersion in liquid nitrogen and stored at -70°C (Revco freezer). For use in polymerization experiments the pellets were thawed at room temperature, resuspended in buffer II to approximately 5 mg total protein/ml, and depolymerized on ice for 30 min. High-speed supernatants of this partially purified microtubule protein were then prepared by centrifuging at 230,000 g for 90 min (50,000 revs/mm Spinco type 65 rotor, 2.0~ml tubes). As with axonemal suspensions and brain extracts, this material wae used within a few hours of preparation. Dilution of the recovered supernatants to give the desired range of protein concentrations was done immediately before use in polymerization experiments. (d) Polymerization of axonenaes with microtubule subunits Polymerization experiments were done by mixing equal volumes of axonemal suspension and brain extract, or purified microtubule protein, and incubating at 37°C. The course of the polymerization reaction was monitored by electron microscopy. After mixing at 0°C a 5-~1 sample was taken as a zero-time point. Mixtures of axonemal suspensions with buffer II, and of brain tubulin with buffer I were handled in parallel in each experiment,. (e) Electron microscom Carbon-coated 100 and 400 mesh Formvar-covered copper grids were used. For each time point, a 5-~1 sample was placed on the grid and allowed to adsorb for about 20 s. The grid was then treated with 3 drops of each of the following: 1 mg/ml cytochrome c, distilled water, and 1% uranyl acetate. The final drop of stain remained on the grid for 10 to 20 s before excess moisture was removed by touching the edge of the grid to filter paper. Samples were air-dried under cover. In some experiments isolated axonemes or axonemes that had been incubated with microtubule subunits were fixed in solution prior to negative staining. A volume of axonemal suspension was mixed with an equal volume of 5% glutaraldehyde in buffer I ; t)he samples were t,hen fixed for 10 min at room temperature and stained as described above. Grids were examined in a Philips EM300 operated at 80 kV. Because of extreme differences in contrast between the densely stained axonemes and the more lightly stained

384

C. ALLEN

AND

G. G. BORISY

single microtubules, low magnification micrographs were taken without an objective aperture. Higher magnification micrographs were taken with a 36 pm gold foil objective aperture. Plate magnifications were calibrated using a 21,060 lines/mm diffraction grating. The center-to-center spacing of axonemal accessory structures were obtained by measurement of images on pletes using a comparator. Other measurements given were determined either directly on the electron microscope viewing screen (see Results) or from standard enlargements of calibrated plates.

3. Results (a) Structural and functional properties of detergent-extracted Jlagella A method for the preparation of functionally active sperm-tail axonemes has recently been described (Gibbons & Gibbons, 1972). When whole sperm are suspended in low concentrations of detergent the flagellar membrane is removed and the soluble flagellar proteins are free to diffuse. The remaining axonemal complex remains attached to the sperm head and consists of the flagellar microtubules and the associated subsidiary components of the axoneme. This complex can be reactivated by the addition of ATP and will undergo bending motions similar to those observed in viva.

Detergent concentrotlon( per

cent)

FIG. 1. Detergent-extraction of Chlamydomonas flagella. Abscissa, final concentration of Non-Id& P40 in buffer I. Ordinate, percentage of cells in each

category (see Results). ( l ) flagellated and motile cells (F +M + ); (0) flagellated immotile cells (F +M- ); ( 0) deflagellated cells (F- ). 100 cells were scored under phase optics for each detergent concentration.

We have adapted this detergent-extraction technique to provide an isolation scheme for algal flagellar axonemes. Figure 1 shows an experiment in which concentrated suspensions of Chlamydomonas were diluted 1: 10 into buffer I containing Non-Idet P40 and immediately observed by phase contrast microscopy. Three classes of cell types were produced by the treatment: (1) at low detergent concentrations (up to 0405%) 80% or more of the cells remained flagellated and fully motile; (2) as detergent concentration was increased (0*005 to O*O2o/0) immotile cells appeared in the population; (3) at concentrations above 0.04% nearly all of the cells lost both flagella. Flagella on motile cells in the detergent solution appeared as

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385

intact dark fibers under phase optics; in contrast, flagella on immotile cells or free in solution appeared much less refractile than unextracted flagella due to the solubilization of the flagellar membrane. In addition the released, demembranated flagella (axonemes) were seen to be frayed into two or more thinner fibers at one end. We have used this fraying to identify the distal end of the isolated axonemes. When cells were suspended in intermediate concentrations of detergent (see Fig. 1) most flagella were demembranated but remained attached to the cell. The inset in Plate I is a low magnification electron micrograph of a cell from such a population showing that the end of the axoneme distal to the cell is frayed. Since the flagella that were released into solution were also frayed at only one end, we identify the frayed end of isolated flagella to be distal. We have routinely used a final concentration of 0.036% detergent to detach flagella from the cell and remove the flagellar membrane. At this concentration nearly all of the flagella were detached within a few seconds of exposure to detergent, while the few remaining attached flagella were released during the first centrifugation of the suspension. In addition, this concentration of detergent did not cause cell lysis except after several hours of exposure, and cells that were quickly washed free of the detergent regenerated new flagella. Axonemes obtained by detergent-extraction followed by differential centrifugation were examined to determine the structural and functional properties of the isolated complex. When freshly isolated axonemes were suspended in a reactivation solution (10 mM-HEPES, 5 mM-MgSO,, 05 mM-EDTA, 05 mM-mercaptoethanol, 1 mlur-ATP, 2% polyethylene glycol, pH 8.1) the majority of the axonemes displayed some form of bending motion (Allen, unpublished observation). Individual axonemea beat for between 15 and 30 minutes in the reactivating solution. The form of beating was related to the normal beating pattern of living cells. In vivo, Chlumycbmonas are propelled by the ciliary-like lashing of two anteriorly located flagella (Ringo, 1967a), but under some circumstances cells reverse direction and the flagella then form undulatory waves. It had previously been noted that glycerinated flagella of Chlamydomonas moewuaii responded to ATP by swimming through the medium (Brokaw, 1960). We have observed that axonemes free in solution propel themselves through t,he medium by forming undulatory waves, but the majority of the axonemes becamr attached at one end to the cover glass and displayed lashing motions more similar to those of cilia and to the normal motion of flagella on cells. The capacity for bend formation was slowly lost in axonemes stored in cold buffer 1. Approximately 50% of the flagella reactivated after five hours and a small percentage of axonemes (10 to 15%) still reactivated strongly after 24 hours of storage. A detailed account of the reactivation of the isolated axonemal complex will be the subject of a separate paper. In contrast to the capacity for bend formation, the ultrastructural features of the isolated axonemes were very stable upon low temperature storage in buffer I, with no detectable changes observable up to 72 hours after isolation. The following brief review of the current understanding of the architecture in intact flagella will serve as a background for an interpretation of the structural arrangement found in the isolated motile complex. Several excellent descriptions of the components of cilia, flagella and sperm tails as viewed in transverse section are available (Fawcett & Porter, 1954; Afzelius, 1959 ; Gibbons & Grimstone, 1960; Ringo, 1967a; Allen, 1968; Warner, 1970). The 9 + 2 nxoneme consists of a cylindrical array of 9 peripheral doublet microtubules and 2

386

C. ALLEN

AND

G. G. BORISY

single central pair microtubules. Both central pair and outer doublet tubules have several subsidiary components arrayed along their length; the most obvious of these, in sectioned material, are paired outer doublet side-arms which contain the axonemal ATPase, dynein (Gibbons, 1963). The tubule bearing the side-arms has been designated the A-tubule (Gibbons, 1961) and has been shown to be the complete tubule of the doublet (Ringo, 19673; Tilney et al., 1973) consisting of 13 protofilaments. The associated B-tubule is considered to be an incomplete tubule containing 10 protofilaments and sharing three filaments in a common wall with the A-tubule (Ringo, 1967b). In Chlamydomonas flagella the B-tubules of the doublets terminate before the Atubules near the distal end of the axoneme (Ringo, 1967a). The disposition of additional axonemal structures has been clarified by negative staining with uranyl acetate (Chasey, 1969; Hopkins, 1970). In Chlamydomonas axonemes Hopkins observed laterally placed material at periods of 140 A along the A-tubule of the outer doublets and radially projecting spokes and hammerheads occurring in longitudinal pairs at intervals of 1000 A also along the A-tubules. Hopkins also found that the central pair of tubules bear longitudinal rows of projections (rungs) occurring at intervals of 160 A. Examination of axonemes isolated by treatment with non-ionic detergent has confirmed that these preparations retain all of the features described by Hopkins (1970) for Chlamydomonas flagella frayed in situ. In particular, features of the negatively stained material which allow the unambiguous identification of the A and B components of doublet tubules have been examined in detail. Side-arm material appeared in a highly ordered state in these preparations and was attached at an angle to the axis of the A-tubule. This asymmetry is related to the directionality of the axoneme. Several features are evident at the extreme distal tips of frayed axonemes (Plate I). In (a) the outer doublets are lying partially on edge on the grid, and thus provide a clear view of the paired spokes and hammerheads. The spacings of these structures are 320 f 30 A between the members of a pair and 960 & 100 A center-to-center between pairs. Within the range of error of our measurement these figures are identical to those obtained by Hopkins (1970). The micrograph also shows that one member of the outer doublet terminates before the other and that the spokes and hammerheads continue on the longer tubule. Plate I(b) sh ows doublets in a different orientation where both members of the outer doublets can be seen clearly, and again, one member of the doublet terminates first. The structure which continues is a complete tubule and is identified as the A-tubule by the presence of side-arms. Two different orientations of this material are evident in the micrograph. The tubule marked with a single arrow displays scruffy material similar to that identified as the A-tubule side-arms in negative staining (Hopkins, 1970). The tubule marked with triple arrows has a more distinct array of material along one side. The side-arm spacing measured from images of this type was 241 & 10 A, a value significantly greater than the 140 A repeat reported previously (Hopkins, 1970). It seems reasonable to assume that the 240 A repeat we have observed arises from the rows of paired side-arms which have previously been identified as A-tubule attachments from transverse sections (Gibbons, 1961; see Warner, 1970, for review). The side-arms appear as ellipses with the major axis of the ellipse marking an angle of approximately 45’ to the axis of the A-tubule. An arrow drawn parallel to the major axis and originating in the

PLATE T. Frayed distal ends of isolated axonemea. (*I) Out,er doublet,s showing spokes and hammerheads and B-tubule tet’minatilm (arro\\h). Sots that, the at,tachod structures continue on the longer tubule. (b) Outer doubletti showing orirnted side-arms (triple arrows) and less ordered material (single arrow). x 80,000. (c) Insf,t, sho\vs t,hat fraying of extracted flagella occurs at end distal to the cell (details in Results). x 20Ol1. In this and all following plates the distal end of the axoneme or axrmemal fragment, appears :~i. t htt t,op of the Platv.

D

RS

-SA

(a)

PLATE II. Structural detail of isolated flagellar axonemes. (a) Perspective diagram (modified from Hopkins, 1970) of axonemal components revealed by negative staining. Side-arms appear in anti-clockwise orientation that would be seen if the axoneme were viewed from the distal end toward the cell body (Gibbons, 1961). A, A-tubule; B, B-tubule; RS, radially placed spokes and hammerheads; SA, side-arms; CP, central pair microtubules. (b) to (i) Electron micrographs and diagrammatic int,erpretation of outer doublet images. (b) and (c) Doublet on edge on grid with spokes and hammerheads visible as lateral projections; (d) and (e) face view of doublet with hammerheads appearing as projections above side-arms on the left, and (h) and (i) on the right the doublet oommon wall; (f) and (g) oriented of the doublet. x 165,000.

PLATE III. Addition of microtubule subunit to isolated axonemes. (a) ‘I’hin fibers emerge from the frayed (distal) ends of’ axonemes after 10 min inoubatioll at Xi (’ with brain extract supernatant. (b) Inset is axoneme from parallel experiment in which 100 p&r-colchicine was added to the incubation mixture at zero time. I’, proximal; T1, distal. ‘L’hls axoneme remained intact upon incubation at 3792, but no microtubules have formed. x 2000.

.

Axonemes the axonemal

^

PLATE IV. Continuity of added microtubules were incubated as described in the legend outer doublet miorotubulcs. x 10,000.

-

and flagellar axoncme. to Plate III. Arrows mark

the

ends

of

PLATE VI. Unidirectiorml addition uf microtubules t,u oentral pair frttgmerlts. Fragments were incubated for 20 min at 37°C with brain extract supernatant. (a) Fragment of approximately 5 @n showing the addition of 20.pm polymer. x 11,500. (b) Higher magnification of image in (a) showing transition between added tubules and central pair. x 40,000. (c) Nearly full length central pair fragment (8 pm) with 10 pm added polymer visible on Plate (total length of addad polymer was 20 wm). x 26,000. Note that the. fragments remain ass&&cd as pairs and display a characteristic curvature.

_.__.-

- -.

..--

-.-

--

hATE VII. Unidirectional addition of microtubules to outer doublet fragment,s. ( ‘onditions uf incubation were as given in the legend to Plate VI. (a) and (b) paired, ant1 (c). (d) and (e) single, outer doublet fragments in continuity with single added microtubules. .n: 11,500. (f) Highor magnification of image in (e) showing tran&ion from doublet to single microt,ltbull~. K1.1)00.

.--.“.-- ___._ _-_-..PLATE VIII. Bi-directional addition of purified tubulin to axonemes. Axonemes were incubated for 10 min at 37°C at a final concentration of 0.4 mg of purified tubulin/mi. Proximally and distally added microtubules are evident. Tubule marked with arrow would not be scored under our selection criteria (see text). Note that the distally added tubules are considerably longer (about 10 pm) than thaw added at the proximal end of t,he axoneme (2 to 3 pm). x 10,000.

PLATE IX. Bidirectional addition of purifictl tubulin tjo uuter doublet fragmrnts. I~y‘ragmcnts were incubated at 37°C for 10 min with a final concentration of 0.4 mg tuhulinjml. (a) Added single microtubules are evident at both ends of the fragment bundlr~. ’ ~o.OO~I. (h) Higher magnification of proximal end of bundle *hewn in (a). Iknthlr~t~ tnark~~~l with ar’rr~u pi is in rr)rltinllity with twvo complrtc acltlctl microtubulw. .’ 1f10.000. 18

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common wall would then point toward the distal end of the axoneme (see also Plate II (f) to (i)). Although well-preserved side-arms have not been observed on all nine outer doublets of a single flagellum, they have been observed on as many as six doublets, and of hundreds of individual doublets examined, when the polarity of the side-arms could be determined, it was invariably distal. Thus in answer to one of our original questions, the axoneme does display structural polarity with respect, to this functionally significant tubule attachment. The two micrographs in Plate I are extreme examples of distal tip images in that one or the other of the two common views predominates in each figure. More commonly a frayed axoneme will display outer doublets in a variety of orientations on the grid surface, and doublets displaying both side-arms and spokes and hammerheads may be recognized. Plate II illustrates this point in greater detail. The upper portion of the Plate is a perspective diagram, modified from Hopkins (1970), which shows our present interpretation of the tubular and subsidiary components of thr Chbmydomonas motile axoneme. The major differences between our reconstruction of outer doublet arrangement and that presented previously (Hopkins, 1970) are the inclusion of distally oriented side-arms and the placement of the radial spokes and hammerheads on the common wall of the outer doublets. Central pair projections have been observed in the detergent extracted preparations but have been omitted from the diagram for clarity. Since in transverse sections, side-arms extend in the same direction from the A-tubule of all the doublets of a given flagellum, the axoneme is characterized by a configuration which could exist, theoretically, in either of two enantiomorphic forms. Gibbons (1961) has shown that the clockwise form (defined as that in which, to an observer looking outwards along the cilium from base to tip, the arms all point in a clockwise direction) is the configuration observed in cilia, flagella and sperm tails from widely separate groups of protozoa, insects and other animals. The lower portion of Plate II shows individual outer doublet images together with a diagrammatic interpretation of the attached structures. The first micrograph shows a doublet lying on edge thus displaying a clear image of the spokes and hammerheads as a set of lateral projections. In this configuration it is impossible to determine whether the projections originate from the A or the B component of the doublet. However, doublets occasionally lie twisted on the grid surface in such a way as to show this image alternating with the complete doublet profile, and in such cases it was clear that the hammerheads emerged from the same tubule which had the sidearms. Plate II(c) shows a doublet lying flat on the grid surface. In regions of dense stain the paired hammerhead projections are seen as bright dots superimposed on the common wall of the doublet, leading us to suggest that these structures origina,te from the common wall. Plate II(f) to (i) shows that side-arm images are obtained on both the left and t)he right side of a doublet, as viewed distally. Based on the clockwise enantiomorphic configuration of the axoneme, these views result when a doublet lies with either its outer or its inner face, respectively, in contact with the grid surface. Because the projection of hammerheads over the doublet image is only observed in regions of very deep stain, which obscures side-arm identification, we have only rarely seen doublets with both classes of attachment visible in the same region of the tubule. However, in a few cases we have seen hammerhead projections superimposed on the common wall of a distally oriented tubule which had side-arms at the left of the A-t,ubuIe. We interpret this image as indicating that the out)er surface of the doublet 25

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is in contact with the grid and the hammerheads are projecting toward the viewer. The hammerhead projections were never seen when the arms were displayed on the right of the doublet. The salient point for this paper is that regardless of which doublet surface is exposed to view, the side-arms are attached at an angle to the axis of the A-tubule and thus reveal the polarity of the doublet. (b) Addition of microtubule subunit to isolated axonemes Having obtained preparations of axonemes displaying good preservation of both structural polarity and in vitro function, we next asked if the isolated axonemal tubules were capable of supporting the addition of microtubule subunits in vitro. Since axonemes are difficult to solubilize under mild conditions and the solubilized subunits rapidly lose the colchicine-binding activity thought to be indicative of the native configuration (Meza & Wilson, 1973), initial experiments to test the growth competence of axonemal tubules were done using extracts of porcine brain tissue as a source of microtubule protein subunits. It has been shown that low-speed supernatants (25,000 g) of extracts will form microtubules when the solution is raised t’o 37°C (Borisy & Olmsted, 1972). The specificity of the polymerization has been characterized by viscometry (Olmsted & Borisy, 1973a) and electrophoretic analysis (Shelanski et al., 1973; Borisy et al., 1974). Axonemes and low-speed supernatants from brain homogenates were mixed in a volume ratio of 1: 1 and incubated at 37°C. Axonemes were also incubated with either one volume of buffer II or one volume of buffer I. Samples were taken for electron microscopy at zero-time and at five-minute intervals thereafter. Axonemes incubated with either of the buffers at 37°C remain structurally intact for up to 30 minutes with some increase in fraying occurring after longer periods of incubation. Axonemal length in these incubated samples remained constant. Axonemes incubated with brain supernatant also remained intact, but frequently had from one to several long single microtubules emerging from the frayed distal end. Examination of these single microtubules at high magnification revealed that they were continuous with the microtubules of the distal end of the axoneme. In addition, numerous single microtubules unattached to axonemes were observed on the grids, thus making further analysis of this system complex. It seemed desirable to attempt to add microtubule subunits to the axoneme under conditions where tubule formation in the extract would normally not be observed. Previous experiments had indicated that the assembly of microtubules in supernatants of brain tissue required the presence of a particulate fraction which could be removed by high-speed (230,000 g) centrifugation of the extract (Borisy & Olmsted, 1972). Therefore, axonemes were incubated with high-speed supernatants of brain extract as the source of microtubule subunit. Under these solution conditions extensive addition of subunit occurred on the axonemal microtubules, but free microtubules were not observed (Plate III(a) and Plate IV). The tubule addition was inhibited by colchicine (Plate III(b)). The extent of addition to axonemal tubules was quantitated with respect to number of microtubules added per axoneme, their approximate length, and their position relative to the proximal or distal ends of the axonemes. For each determination 100 axonemes were examined per grid. Each grid was traversed completely and axonemes were selected for detailed examination when the following criteria were met : (1) both proximal and distal ends of the axoneme were visible ; (2) preservation

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and staining were adequate to distinguish axonemal features; and (3) distal fraying of the axoneme was sufficient to determine the origin of the added tubules. Scoring was carried out as follows : a grid was scanned at low magnification ( x 4000) ; axonemes meeting the selection rules were then examined at higher magnification ( x 22,000) and the transition between flagella and single microtubules observed. Only tubules seen to be in direct continuity with axonemal structures were counted. TABLE

1

Quarditative analpsis of microtubule polymerization cm axonemes Experiment number

(1) (‘1 (W (4) (5) (6) (9) (10) (14) (18)

Number of axonemes with distal tubules 98 99 99 100 97 97 99 97 100 98

Modal number

5 7 6 5 6 6 7 6 6 5

Average length (d 12 20 28 24 10 53 34 23

Number of axonemes with proximal tubules 0 0 0 0 9 0 27 3 0 0

-Modal number -

Average length (cLm) --

1

-11 --

2

r_-1 ---

-

Scoring was clone on negatively stained samples of asonemes after incubation eskact for 10 min at 37°C. Selection criteria for scoring are described in t.he test.

with

brain

Table 1 shows that in ten separate experiments 97$/6 or more of the axoneme.3 had one or more added tubules with the modal number ranging between fire and seven per axoneme. Data given here were obtained from analysis of axonemes in. cubabed for ten minutes with porcine extract, at which time the single tubules had attained a length equal to or greater than the original axonemal length (details in section (c), below). Similar determinations made at earlier time points (from 2 min) or lat,er incubation times (up to 30 min) showed essentially identical tubule diskibutions, differing only in the length of added tubules. As can be seen in Table 1, t,he great majority of added tubules were observed at the distal end, and their length often exceeded the original length of the axoneme. In a few of the experiments a small percentage of axonemes had both long distally added tubules and a lesser number of short proximal tubules visible. Differences in the behavior of these two types of tubules are discussed more fully in the following section. Length determinations of added tubules (Table 1, column 3) were made at low magnification ( x 4000) in the electron microscope using the diameter of an inscribed circle (3 cm) on the electron microscope viewing screen as a reference rule. Thus an added tubule was traversed t,hrough the circle and assigned a length accurate to &loo//0 of the tubule length. Measurements were made on each tubule from ever.v tenth axoneme chosen for scoring; as the modal number of tubules per flagellum was five to seven, this means that an average of 60 tubules were measured on each grid scored. The lengths of the added tubules varied widely, with tubules of lengths five

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to ten times the axonemal length observed at later time points (30 min). At later times, added tubules usually crossed more than one grid square and their lengths could not be determined regularly. In contrast, proximal tubules never exceeded a length that was more than a fraction of the axonemal length (5 1 pm). The presence of single microtubules projecting from isolated flagellar axonemes after incubation with porcine extract could have been the result of either (1) partial breakdown of axonemal outer doublets, or (2) the addition of subunits to the axoneme. The B-tubule of isolated outer doublets is unstable at elevated temperature (41°C) (Stephens, 1970) and the first possibility could conceivably have arisen if the B-tubule were breaking down from the distal end, resulting in images of doublets with continuous single tubules at one end. This interpretation was excluded by the following observations: (1) single tubules projecting from doublets occurred only upon incubation with extract, whereas if breakdown were occurring at 37°C a similar image would have been expected in material incubated with buffer and this was not observed ; and (2) single tubules in direct continuity with axonemal tubules attained lengths that were up to five times the original length of the axoneme. However, these observations alone are not sufficient evidence that porcine microtubule subunits had polymerized onto the axonemes. It could be argued that at elevated temperatures an equilibrium exists such that a fraction of the axonemes are depolymerizing, and solubilized axonemal subunits are then available to add on to other axonemes. The latter interpretat,ion is not consistent with the following findings : (1) t’he number of axonemes per grid during the course of the incubation remained roughly constant, while the amount of added tubules observed even at early time points (i.e. 10 min) would require that about one third of the axonemes had been solubilized ; (2) the phenomenon was only observed in the presence of extract, not in buffer; (3) only complete axonemes were seen, whereas partially dissolved axonemes should have been observed if breakdown were occurring; and (4) the added microtubules were sensitive to agents which inhibited polymerization of brain tubules but which did not affect axonemes in vitro (see below). It is well known that the stability of microtubules from different cellular sources varies greatly with respect to colchicine concentration and temperature (see Margulis, 1973; Olmsted t Borisy, 1973b; and Wilson Q Bryan, 1973, for reviews). Flagellar tubules are resistant to several agents or treatments which can cause depolymerizat,ion of cytoplasmic tubules. Under the conditions described in this paper detergentextracted Chlamydomonas flagella were structurally stable at 0°C for several days in buffer I, and exposure to 100 PM-colchicine for several hours at 37°C did not lead to any obvious disruption of axonemal microtubules or their subsidiary attachments. In contrast, the in vitro assembly of microtubules in brain extracts was both inhibited and reversed by low temperature and colchicine (Weisenberg, 1972; Borisy & Olmsted, 1972). Further, calcium ion at low concentrations (1 mM) inhibits tubule assembly, and causes the rapid depolymerization of microtubules formed in vitro. Table 2 summarizes data from an experiment where disruptive agents were used as inhibitors of tubule assembly. It is seen that no distal tubules form. For comparison to Table 1, data are given for a ten-minute incubation; however, observations up to 30 minutes failed to reveal any distal tubule addition. In contrast, the infrequent short proximal tubules noted previously did appear in the presence of colchicine and CaCl,, but were not present in material incubated at 0°C.

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TABLE 2 Inhibition

of microtubule polymerization in intact axo1teme.s

Number of axonemes with distal tubules

Inhibitory agent

(a) None (b) Low temperature (OV (c) 100 PM-colchicine (d) 2.6 mM-CaCl,

Modal number

Average length (pm)

6

Number of axonemes with proximal tubules

98 0

-

22 -

8 0

0 0

-

-

8 8

Modal number

Average length bm)

2

51
2 2

51
-

Axonemes were mixed with brain extract and incubated at 37°C (a) or 0°C (b) for 10 min, or made 100 pM in colchicine (c) or 2.6 mM in CaCl, (d), and incubated at 37°C for 10 min.

Depolymerization experiments with these agents were also carried out and Table 3 shows the time-course of tubule disappearance in one such experiment. Axonemes and porcine extract were incubated at 37°C for ten minutes and then samples were either transferred to 0°C or made 100 pM in colchicine or 2.5 mM in CaCl,. Low temperature led to complete depolymerization of tubules by progressive shortening over a course of fifteen to twenty minutes. Colchicine treatment resulted in tubule shortening at a rate similar to the value for low-temperature treatment, but short TABLE 3

Reversal of microtubule polymerization in intact axonemes

Agent appliecl

(a) None (37°C)

(b) Low temperature (0°C) (c) 100 @i-colchicine

(d) 2.5 mM-CaCl,

Time (min)

Number of axonemes with distal tubules

5 15 30

96 97 96

6 6 6

22 23 26

-

6 15 30

96 97 0

6 6 0

11 2 0

-

6 15 30

97 96 96

6 6 6

11 2 1

-

5 15 30

0 0 0

0 0 0

0 0 0

-

Modal number

Averagt 3 ‘alms

Number of axonemes with, nroximal

MO&l number

7

2 -

8

1 -

2

7

1

2 -

8

2

7

1 -

2 -

7

2

6

1 -

2 -

7

Average length

2

1 -

Axonemes were incubated with brain extract at 37°C. After 10 min samples were allowed to continue polymerization (a), removed to 0°C (b), or made 100 q in colohicine (c), or 25 rnM in CaCl, (d) and maintained at 37°C. Times indicated for sooring were times after transfer to depolym&zing conditions.

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lengths of single tubules (approx. 0.5 pm) often persisted even after prolonged incubation (60 min). Loss of added tubules was rapid and complete in the presence of CaCl, after five minutes. In contrast, the distribution and length of proximal tubules was not affected in these reversal experiments. The results of the inhibition and reversal experiments indicate that the added tubules showed the response expected for cytoplasmic tubules. From this and the previous data we conclude that porcine microtubule subunits can polymerize onto the distal ends of flagellar axonemes. The observation of infrequent, short proximal tubules which are not sensitive to agents which disrupt cytoplasmic tubules suggests that the tubules seen at the proximal end in these experiments are probably of flagellar origin and perhaps arise by slippage or sliding of tubules in the shaft of the axoneme. Repetition of these experiments in which samples were fixed with glutaraldehyde prior to negative staining failed to show any significant difference in the distalproximal location, numbers, or length of the added microtubules. At later time points, when microtubule elongation was extensive, the inclusion of the fixation step did prevent the breakage of long microtubules (>20 pm). However, we did not routinely use fixed material in our examinations because, in this system, glutaraldehyde obscured the fine detail of axonemal attachments, presumably by cross-linking free proteins in the brain extracts to the axonemes. (c) Ident$mtion

of growth zones of isolated uxonenaes

Microtubulee added to flagellar axonemes were observed in continuity with both central pair and outer doublet tubules. Axonemes with tubules added to either one or both central pair members were seen, but individual outer doublets gave rise to

ILL IO

8

Number of added mtcrotubules

per

12

flOgellUm

FIGI. 2. Distribution of numbers of added microtubules per exoneme. Axonemes were incub&ed for 10 min at 37°C with brain extract supernatant. The histogram was derived by counting the number text for selection criteria). N = 104.

of microtubules

in continuity

with

each exoneme

(see

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only a single added tubule. As shown in the previous section, the modal number of microtubules added to a population of axonemes was between five and seven (Tables 1, 2 and 3). However, more extensive addition to individual axonemes was also observed. The histogram in Figure 2 demonstrates the distribution of numbers of tubules added per axoneme in a typical polymerization experiment. Since the maximum number of tubules per axoneme observed was eleven we next asked if only the complete microtubules of the axoneme (9 A-tubules + 2 central pair tubules) were capable of in. vitro growth. Axonemes were chosen for examination by the selection rules stated previously and the added tubules were scored as to origin in either an outer doublet or central pair microtubule. Examples of the type of image used for this analysis are given in Plate V. From the micrographs, the central pair is distinguishable from the outer doublets by the 160 a periodicity along their length and by t,heir tendency to bend into an arc of regular curvature (Plates VII and IX). The results from scoring 100 added microtubules in each of three experiments are given in Table 4. The percentage of microtubules originating from the central pair tubules, 16 f 40/, was in good agreement with the predicted value of 18.1%. The predicted value assumed that only complete tubules served as growth sites, and that all complete tubules initiated microtubule assembly with equal frequency. TABLE 4 Distribution

Experiment number

of added tubules originating from central pair or outer doublets in axonemes Number of tubules examined

Central pair origin

100 100 100

(5) Average

Outer doublet origin

16 20 12 16*4’$&& (18.1)

Values in parentheses are expected percentages doublets if only complete axonemal microtubules

84 80 88 8454%

(81.9)

of tubules originating in central pair and outer have initiated growt#h.

To test this assumption further another type of analysis was done. Although it had been found that an individual outer doublet would initiate the growth of only a single microtubule, this finding could have resulted from two different, phenomena: (1) only the complete A-tubule was able to form extensions in vitro; or (2) either the A or the B-tubule could act as a growth zone, but not both in the same doublet. This point, was clarified by examination of the transition zones between outer doublet tubules and the added polymer. As noted in section (a), above, outer doublet microtubules may be displayed in several configurations on the grid surface and it is not always possible to distinguish the A from the B-tubule (Plate II(c) and (e)). Thus to the

selection

rules

already

established

the

following

criteria

for

A-tubule

identi-

fication were added: (1) the B-tubule termination must be visible; and (2) the remaining tubule must bear side-arms. Plate V(a) shows two outer doublets in continuity with added polymer. By our conventions only the doublet on the left is adequately displayed to identify the A-tubule unambiguously. The doublet, on the right would

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have been scored as an example of single tubule addition to a doublet, but would have been listed as “not identified”. Examination of 100 microtubules added to outer doublets in each of three experiments showed that only about 50% of such images allowed a determination of the precise origin of the new polymer. However, in every case where identification was possible (152) the added tubule was in continuity with the complete A-tubule of the doublet. (d) Unidirectionul addition to axonenud fragments The experiments just described indicated that the polarity of axonemal structure exhibited by the side-arm placement was also reflected in an intrinsic polarity of microtubule growth. However, the previous results with intact axonemea could not preclude the possibility that a specialized structure at the proximal end of the isolated axonemes interfered with the addition of subunit at that end. Therefore, experiments similar to those described above were carried out using fragments of axonemes to provide nascent proximal and distal ends. Fragments of axonemes, prepared by shearing, were a heterogeneous mixture of types: (1) central pair fragments of a variety of lengths; (2) outer doublet fragments occurring either singly or in lateral groups of two or more; and (3) transversely broken axonemes containing the entire 9 + 2 complex. Although previous work had shown that the intact isolated axonemes were stable at elevated temperature, it was not clear that this would be the case for the fragmented axonemal tubules. Accordingly, fragments were mixed with an equal volume of either buffer I or buffer II and incubated at 3’7°C. Examination of these samples revealed that outer doublet fragments remained intact upon incubation. At both ends of these fragments the outer doublet profile was discernable and in no case were single tubule images observed which might have resulted if t,he B component of the fragmented doublets were labile in this situation. However, a minor alteration of central pair morphology was noted. Although the tubules in most central pair fragments remained associated along their entire length, in a few cases one end of the pair was dissociated and two single straight tubules projected from the pair. Thus a sufficiently long central pair fragment, if extensively separated, might appear to be a short fragment bearing two added microtubules. To preclude the possibility of scoring such an image as a case of microtubule growth, we considered addition to have occurred only whon the single tubules projecting from axonemal fragments were twice the length of the original axoneme. Thus even if the portion of a single tubule image immediately contiguous to a fragment were of axonemal origin, the extent of addition would still represent an axonemal length equivalent (10 pm). Examples of central pair tubules after incubation with brain supernatant are shown in Plate VI. Plate VI(a) shows a fragment having two added tubules in excess of 20 pm. Addition is, as in the case of the intact axoneme, unidirectional. Plate VI(b) shows a central pair which is of almost native length (8 pm), and again extensive unidirectional addition has occurred. Plate VII gives several examples of unidirectional addition to individual and paired outer doublet fragments. Table 5 shows that whenever polymer addition could be scored on central pair and outer doublet fragments, the addition was unidirectional. We have also determined that the addition of subunit to tubule fragments was inhibited at 0°C and by 100 ELMcolchicine and 2.5 IUM-CdCiUUI.

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TABLE 5 Unidirectional Experiment number

(7) (11)

(12)

addition

of tubules to axonemal fragments

Central pair Number observed

37 42 48

Unidireotional

37 42 48

Bidireotional

0 0 0

Number observed

Outer doublet Unidirectional

Bidirectional

51

51

0

63 49

63 49

0 0

Since microtubule addition to axonemes was polar and addition to fragments was unidirectional, it would be reasonable to assume that growth onto fragments also occurred on their distal ends. Examination of a category of outer doublet fragments tested this assumption. When outer doublet fragments were found together in bundles of three or more, stain trapped between the tubules sometimes allowed the detection of the A-tubule side-arms. These fragments were assigned a directionality by observation of the angle that the side-arms made to the axis of the tubules (refer to section (a), above). Of 104 fragment bundles examined, the directionality of the fragments could be identified in 42 instances, and of these, the added tubules were always observed on the distal end. (e) Parameters affecting the directionality of microtubule growth In the experiments described above, microtubule subunits were obtained from high-speed supernatants of brain homogenates. Total protein concentration in the preparations ranged from 6 to 8 mg/ml which represented 1.5 to 2.0 mg/ml microtubule protein as determined by planimetry of densitometer tracings from sodium dodecyl sulfate-acrylamide gels (Borisy et al., 1974). The preparation of homogenates in smaller buffer volumes resulted in only a small increase in recoverable tubule protein. Thus it was difficult to assess the effect of higher protein concentrations on the directionality of microtubule growth in vitro and we could not exclude the possibility that some component of the extract other than the microtubule subunit was affecting the pattern of microtubule assembly in this system. The development of a method for the purification of the tubule subunit contained in brain extracts (Shelanski et al., 1973; Borisy et al., 1974) allowed the exploration of these questions. Figure 3 shows the results of an experiment in which intact axonemes were incubated at 37°C at several concentrations of purified microtubule protein. Substantial distal addition of tubules was observed over the entire range of protein concentration examined. However, as can be seen from the histograms, proximal addition of subunit also occurred as protein concentration increased. Moreover, in contrast to the rare proximal images noted in previous experiments, the proximal tubules added under these conditions were both inhibited and depolymerized by low temperature (O”C), 100 PM-colchicine, and 2.5 mu-CaCl,. Plate VIII shows that the length of polymer added at the distal end of an axoneme was significantly greater than the length at the proximal end. This difference was maintained even after prolonged incubation, with proximal lengths rarely exceeding 5 pm. The extent of proximal and distal addition also differed in their temperature

398

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95

mg/n

30 20

I

0.70 mg/m

I

I.4 mg/ml

0

2

4

6

8

Number of added microtubules

IO

12

14

per flagellum

FIG. 3. Distribution of proximal and distal addition of microtubules to flagellar axonemes as a function of protein ooncentration. Axonemes were incubated for 10 min at 37°C with purified brain microtubule protein at final concentrations indicated. Open bars represent numbers of distally added microtubules and shaded bars represent proximal addition. For each histogram 100 axonemes were scored (selection criteria described in text).

dependence. At 25”C, extensive addition occurred on the distal end of the axoneme; however, no proximal tubules were evident, nor did proximal tubules form after longer periods of incubation (up to 60 min). Figure 4 presents the combined data from an experiment in which axonemes were incubated in parallel at three temperatures with purified brain tubulin. In the graph the total extent of polymer added to each end of the axoneme is given as a function of time of incubation. The value for total polymer added was obtained by multiplying the mean number of tubules by the average length of the tubules at each end of the axoneme. The data show that over the range 25 to 37°C both the rate and extent of polymerization were greater at the distal end of the axoneme. The absence of fraying at the proximal end of the axoneme made t,he identification of the microtubule growth zones in this region impossible. For this reason, further observations were made using fragments of axonemes incubated with purified tubulin &s described previously. Examination of fragments after incubation at 37°C with purified tubulin showed that both central pair and outer doublet microtubules supported b&directional addition of subunits. Plate IX shows bi-directional growth onto a bundle of outer doublet fragments.

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r

FIG. 4. Time-course of addition of purified brain tubulin to isolated axonemes. Axonemas were incubated with a final concentration of tubulin of 0.4 mg/ml at 3 different temperatures. Solid data points represent distal addition, open points indicate proximal addition (~,o) 25°C; ( n ,lJ) 30°C; ( l ,O) 37°C. The value plotted on the ordinate represents the total amount of polymer added per axoneme (given in pm) and was obtained by multiplying the mean number of tubules formed at each end of the axoneme by the average length of the added microtubules.

The simple& interpret.ation of the protein concentration and temperature dependence of proximal addition is that the tubule subunit has a different affinity for the two ends of the formed microtubules. However, further observations indicate that the preferential addition of subunit to the distal end of the axoneme may be regulated in a more complex manner. It was noted earlier that the high-speed supernatants of brain extract contained between 1.5 and 2.0 mg/ml tubule protein, yet Figure 3 shows that proximal addition occurred at much lower concentrations when purified material was used as the source of microtubule subunit. This suggests that some factor in the brain extract is affecting the directionality of tubule growth. In order to test this possibility, microtubule protein solutions were prepared by diluting purified brain tubulin with (1) buffer II, (2) 10 mg bovine serum albumin/ml in buffer II, and (3) the supernatant obtained by centrifugation of a polymerized brain extract (see Materials and Methods). Examination of axonemes incubated at

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37°C with these solutions showed that both dilution of tubulin with buffer II and dilution with bovine serum albumin allowed the formation of both proximal and distal microtubules. However, incubation with purified protein diluted to the same ooncentration with the supernatant from a polymerized extract yielded only distal microtubules. These observations would seem to preclude the possibility that the non-specific interaction of proteins other than tubulin influences the pattern of microtubule addition to formed tubules. Our data is insufficient to determine whether the suppression of proximal addition by the extract supernatant represents the presence of a physiologically significant regulatory moiety in such preparations. We note this phenomenon merely as evidence that, like protein concentration and temperature, the exact solution conditions under which tubule subunit is added to polymer can influence the directionality of growth. (f) Growth on B-tubules One further difference was noted when axonemes were incubated with purified brain tubulin. At high protein ooncentrations (2 to 4 mg/ml), the number of microtubules added at the distal end of the axoneme occasionally exceeded eleven. In addition, doublets at both proximal and distal ends of the axoneme were occasionally observed in continuity with flattened sheets of protofilaments. Since these images also appeared in material that had been prefixed with glutaraldehyde before negative staining it was unlikely that they had arisen from the breakdown of intact microtubules. These observations suggested that when purified protein was the source of microtubule subunits, the B-tubule could also initiate polymerization; however, we have not quantitated the rate of initiation by the B-tubules. Plate IX shows the B-tubule of a doublet which has initiated the formation of a complete microtubule. Flattened sheets of protofilaments originating from the B-tubule were also observed. B-tubule addition was observed at both the proximal and distal ends of the fragments. However, the B-tubule extensions were always independent of the A-tubule extensions and we have not yet observed a true doublet formed at either end of the axoneme.

4. Discussion (a) Structural polarity and directional growth of axonemal microtuhh The data presented in this paper demonstrate that detergent-extracted axonemes from Chlamydom0na.s maintain two significant properties in vitro: (1) generation of bending movements in the presence of ATP; and (2) growth of the tubules in the presence of free microtubule subunit. Examination of the isolated structure revealed that the flagellar axoneme has polarity at two levels: (1) the ATPase containing side-arms (Gibbons, 1963) have a native structural polarity; and (2) microtubule subunits are added predominantly unidirectionally to the distal tip of the axonemal tubules. The observation of asymmetrically oriented side-arms in both fixed and unfixed material suggests that this asymmetry may reflect a biologically significant property of side-arm attachment to the axoneme. As has been discussed by others (Summers & Gibbons, 1971) the side-arms most probably function in vivo to slide outer doublet microtubules past one another. These authors’ analysis of outer doublet sliding in vitro suggested that some portion of the axonemal structure would be polar and

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(a)

Bipolar

(b)

Polar

(c)

Biased

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assembly

assembly

polar

assembly

FIG. 6. Schematio diagram of subunit addition to microtubules. (a) Bipolar assembly would OOOUP if the subunit had an equal affinity for both ends of the elongating polymer. (b) Polar assembly would ooour if subunit-polymer interaotion was limited to one end of the growing microtubule. (c) Biased polar assembly has been observed in the experiments described in this report. This could result from a greater affinity of subunit for the distal (Kd) than the proximal (Kp) end of the microtubule.

they noted that an individual doublet tubule would have the same polarity along length. Our observations of side-arm orientation suggest that this is the case, and that all of the doublet tubules have the same polarity. The second level of polarity observed is that of the directional addition and subtraction of microtubule subunits at the end of the flagellar tubules. A priori one might have expected that subunit addition would be strictly limited to one end of a growing tubule, be equally permissible at either end, or that the affinity of the subunit for the two ends of the tubule would be different so that a biased polar growth would result (Fig. 5). Our data show that when crude brain tissue extract was used as a source of microtubule subunit or when purified tubulin was used at low temperature (25°C) and protein concentration, microtubule growth occurred directionally from the distal end. However, when purified tubulin was used at high temperature (37°C) and protein concentration, tubule growth was bi-directional, although growth was still favored at one end. Therefore, we conclude that microtubule assembly in this heterologous system has an intrinsic directionality which under certain conditions may be expressed as biased polar growth rather than absolutely polar growth. The question of polarity of assembly has also been examined using a homologous system in which fragments of polymerized tubules from brain were morphologically labeled with DEAE-dextran and used as seeds. Experiments carried out in parallel with ones using flagellar seeds showed that the homologous system was also characterized by a similar biased directionality of tubule assembly (data not included; also, see Olmsted et al., 1974). Directional growth of microtubules hamsalso been reported using isotopically labeled chick brain tubules as seeds (Dentler et al., 1974). its

400

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In vivo the polarity of microtubule growth is expected to be closely controlled. In some cases, such as axopodial extension, neuronal outgrowth, and flagellar elongation, the asymmetric cell process grows away from the cell body. Such directionality could result from the addition of subunit to either the proximal or the distal end of the elongating microtubules. Observations of flagellar elongation in vivo using natural markers (Tamm, 1967) or autoradiographic studies (Rosenbaum & Child, 1967; Witman, 1973) suggested that growth occurred by addition of precursor to the distal end of the flagella. The present study confirms these observations and further suggests that the information required to specify the directionalit,y of flagellar growth is intrinsic to the microtubule component of the axoneme.

(b) Co-polymerization and classes of microtubules In addition to indicating that microtubule growth is an intrinsically polar process, the experiments described touch on another question: the possible variety of species of microtubule subunits in cells and the significance of this variety. Early cytological observations demonstrated that microtubules associated with different cellular structures varied markedly in their response to a variety of physical and chemical agents. Behnke & Forer (1967) suggested, on the basis of temperature stability and sensitivity to proteolytic digestion, that cells contain four classes of microtubules: the labile cytoplasmic microtubules, the central pair, B-tubules, and A-tubules of flagellar axonemes, which are of increasing stability in the order given. More recently it has been suggested that microtubules be classified as simply labile or stable (Bryan & Wilson, 1971). Despite these differences, however, an increasing literature on the chemistry of the microtubule subunit isolated from a wide variety of cellular sources indicates that the size, charge, amino acid composition and drugbinding affinity of tubulin are highly conserved (reviewed in Olmsted & Borisy, 1973b; Wilson t Bryan, 1974). Recently two further reports concerning the similarity of microtubules from various sources have appeared. Tilney et al. (1973) have analyzed microtubules from heliozoan axonemes, the mitotic apparatus, contractile axostyles, repolymerized microtubules from chick brain, the flagellar central pair, and the flagellar and basal body A-tubules, and have shown that these microtubules are all composed of 13 protofilaments. In addition, amino acid sequence analysis of tubulin polypeptides from sea urchin sperm tail and chick brain microtubule protein has shown that these proteins differ at only two positions in the first 25 residues sequenced from the N-terminus (Luduena & Woodward, 1973). These authors have calculated a mutation rate of 0.45 point mutations per hundred residues per hundred million years, which makes tubulin one of the most highly conserved protein classes examined. The polymerization of porcine brain tubulin onto the axonemes of algal flagella described in this report provides evidence for the functional conservatism of tubulin as well. Recently, similar results were also obtained using chick brain tubulin and sea urchin sperm tails (Binder et al., 1973). Brain tubulin has now also been demonstrated to augment the birefringence (microtubule structure) of the mitotic spindles of Chaetopterus oocytes (Ino& et al., 1974), Spisula oocytes (Rebhun et al., 1974) and mammalian tissue culture cells (Cande et aE., 1974). The copolymerization of tubulin from diverse cell types suggests that t,he characterization of directional assembly described here with the brain-fla,gellar syst,em will have general applicability in other systems also.

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These considerations suggest that the differential stability observed in vivo among classes of microtubules may result not from differences in subunit chemistry but from variation in subsidiary attachments to microtubules in different cellular structures. Alternatively, it is also possible that cells which produce more than one tub&n structure contain more than one tubulin gene. Luduena & Woodward (1973) have suggested that u and /l tubulin arose by duplication of a single tubulin gene. Such gene duplication could have occurred frequently during species divergence and given rise to tubulins which are specific to different structures such as the mitotic apparatus or the flagellar axoneme. The resolution of these possibilities will require peptide, analysis of tubulins derived from the different structures. (c) Microtubule

assembly in vivo and in vitro

The literat’ure concerning the temporal and spatial regulation of microtubule formation in viva is large (Porter, 1966; see Tilney, 1971; Bardele, 1973; Olmsted & Borisy, 19736, for reviews). Two observations have recurred in many investigations: (1) cells are able to maintain a pool of microtubule protein which can at some later time be mobilized for polymer formation; and (2) the production of orderly arrays of microtubules has often been thought to be associated with discrete cellular structures designated as microtubule organizing centers (Pickett-Heaps, 1969). It is possible that different initiat,ion sit,es for microtubule assembly can specif) the form of the tubule achieved. For example, although the axonemal outer doublet structure appears to arise by continuity and simplification of the triplet microtubules of t,he basal body, the central pair microtubules arise in the vicinity of a non-tubular basal plate. In vitro assembly of tubulin isolated from brain tissue has been shown to require the presence of a disk (or ring) structure (Borisy t Olmsted, 1972; Olmsted et ccl.> 1974; Kirschner et al., 1974). However, the relation of this structure to the microtubule organizing centers postulated to exist in cells is not known. The analysis of pattern formation in microtubule structures would be assisted by presumptive organizing centers. The conservatism an in. vitro system for investigating of tubulin and its ability to copolymerizc with subunits derived from diverse sources suggests that purified brain tubulin might be used as a reagent to test for the existence in various cell extracts of structures competent to initiate microtubule growth. In summary, we have shown that the microtubules of flagella have an intrinsic polarity reflected in their side-arm attachments and in their directionality of growth. The ilz vitro assembly system described here may be of use for further studies on flagellar development and patterned microtubule formation. The collaboration of Dr J. B. Olmsted in the initial experiments fully acknowledged. We also thank Dr Olmsted for her continued ment. The cheerful assistance of MS E. Larison in all phases of deeply appreciated. This research was supported by National Science Foundation one author (G. G. B.). The other author (C. A.) was a National prodoctoral trainer. REFERENCES Afzelius, B. (1959). J. Biophy.~. Biochem. Cytol. 5, 269-281. Allen, R. (1968). J. Cell Biol. 37, 825431.

reported

here is grate-

advice and encourageelectron

microscopy

ix

grant no. GB36454 to Institutes of Hea,lth

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