The ultrastructure of the Chlamydomonas reinhardtii basal apparatus: identification of an early marker of radial asymmetry inherent in the basal body

Basal bodies (present at the base of cilia and flagella) and centrioles (which form the core of the centrosome in mammalian cells) are two manifestations of the same conserved eukaryotic organelle. They consist of nine triplets of microtubules arranged in a ninefold radially symmetric array. Basal bodies and centrioles display structural and functional polarities that play an important role in the spatial arrangement of the cytoskeleton and hence the polarity of the cell and, in the case of basal bodies, the direction of ciliary/flagellar beat (reviewed in Beisson and Jerka-Dziadosz, 1999; Preble et al., 2000).

The basal apparatus of the biflagellate green alga Chlamydomonas reinhardtii is a complex organelle located at the anterior end of the cell consisting of two basal bodies (often referred to as mature basal bodies), two probasal bodies (nascent basal bodies) and an array of fibers and microtubules appended to each basal body in an asymmetric manner. The basal bodies are anchored to the cell membrane, organize the formation of the flagella and serve as an organizing center for the microtubular cytoskeleton in interphase and mitosis (reviewed in Dutcher, 2003).

Green flagellates are excellent model organisms for the analysis of the function and protein composition of basal bodies (reviewed in Marshall and Rosenbaum, 2000). The ubiquitous centrosomal protein centrin (also known as caltractin) was first discovered as the major component of a basal-body-associated fiber in a green alga (reviewed in Salisbury, 1995). The biochemical analysis of isolated basal apparatuses from the green flagellate Spermatozopsis similis led to the identification of several novel basal apparatus proteins (SF-assemblin, BAp95, BAp90 and p210) that are localized to different fibers associated with specific sites of the basal bodies (Lechtreck and Melkonian, 1991; Geimer et al., 1998a; Geimer et al., 1998b; Lechtreck et al., 1999). In Chlamydomonas, mutations affecting basal body function can be identified by the absence of flagella or their presence in an aberrant number (bld mutants are bald, having no flagella; uni mutants are uniflagellate; vfl mutants have a variable number of flagella). The UNI3 gene has been shown to encode an entirely new class of tubulin, δ-tubulin (Dutcher and Trabuco, 1998), and the Vfl1 and Vfl3 proteins are thought to play a role in the rotational orientation of the basal bodies (Silflow et al., 2001; Silflow and Iyadurai, 2002). Within the past few years, new molecular tools have been established, such as Chlamydomonas-adapted green fluorescent protein (GFP) (Fuhrmann et al., 1999) and RNA interference methods (Fuhrmann et al., 2001), which can be used for the in vivo analysis of basal apparatus proteins (Ruiz-Binder et al., 2002; Lechtreck et al., 2002; Koblenz et al., 2003). These tools, together with the availability of large expressed sequence tag databases (Asamizu et al., 1999; Asamizu et al., 2000; Shrager et al., 2003), the complete nuclear genome sequence (http://www.biology.duke.edu/chlamy_genome/cgp.html) and progress in proteome research will greatly facilitate the molecular analysis of the basal apparatus.

The overall structure of the Chlamydomonas flagellar apparatus (the flagella and the basal apparatus) and the major ultrastructural components of the basal apparatus have been described in two classic studies (Ringo, 1967; Cavalier-Smith, 1974), but these data are not sufficiently detailed for the new tasks. This is illustrated by a recent study in which minor defects in the basal bodies of a Chlamydomonas δ-tubulin deletion mutant were detected using dual-axis electron tomography (O'Toole et al., 2003). We felt that a detailed high-resolution map integrating all structural components of the Chlamydomonas basal apparatus was needed and decided to reinvestigate its ultrastructure using serial ultrathin sections of isolated cytoskeletons. We describe several novel aspects of the basal apparatus architecture (including new information about the attachment of the microtubular flagellar roots to specific basal body triplets), a numbering system for the triplets of the probasal bodies and a detailed diagram illustrating how all components of the basal apparatus together form the highly ordered architecture of this complex proteinaceous machinery. Most importantly, a structural marker for the rotational asymmetry inherent in the basal bodies and probasal bodies has been discovered. This structure (termed the `acorn') is attached to the inner wall of the microtubular cylinder at its distal end, representing the counterpart of the well known cartwheel usually present at the proximal end of basal bodies and centrioles. Whereas the cartwheel is thought to nucleate the ninefold rotational symmetry of the microtubular triplets (reviewed in Beisson and Wright, 2003), the acorn might play an equally important role imposing rotational asymmetry on the microtubular triplets, perhaps leading to the asymmetric assembly of basal-body-associated fibers and hence cellular asymmetry in general.

C. reinhardtii cw 15⁺ Dangeard (SAG 83.81) (Schlösser, 1994) was cultured in aerated 10 l flasks (∼1 l minute^–1) at 15°C with a light:dark cycle of 14:10 hours and a photon flux density of 20 μE m^–2 second^–1 (Osram 40W/25 universal white) in WarisH culture medium (McFadden and Melkonian, 1986).

Cytoskeletons were isolated as described in Wright et al. (Wright et al., 1985).

The isolated cytoskeletons were resuspended in MT buffer (30 mM HEPES, 5 mM Na-EGTA, 15 mM KCl, pH 7.0), fixed by adding an equal volume of 5% glutaraldehyde in MT buffer for 15 minutes at 15°C, sedimented (1000 g for 10 minutes) and resuspended in MT buffer containing 1.25% glutaraldehyde and 0.5% tannic acid (TAAB, Aldermaston, UK). After 25 minutes at 15°C, the cytoskeletons were sedimented, washed twice in MT buffer and osmicated for 20 minutes (1% OsO₄ in MT buffer) on ice. Pellets were washed twice in MT buffer, transferred to agar, dehydrated and embedded in Epon 812 (Serva, Heidelberg, Germany) as described by McFadden and Melkonian (McFadden and Melkonian, 1986).

Ultrathin serial sections (50-70 nm) were cut with a diamond knife (Delaware Diamond Knives, Wilmington, DE, USA) on a RMC MT 6000-XL microtome (RMC, Tucson, AZ, USA) and mounted on Pioloform-coated copper grids (Plano, Wetzlar, Germany). The sections were stained with lead citrate and uranyl acetate (Reynolds, 1963). Micrographs were taken with a Philips CM10 transmission electron microscope using Scientia EM Film (Agfa, Leverkusen, Germany). In cross-sectioned basal bodies or axonemes, the view is from the distal to the proximal end.

For a general terminology of the components of the flagellar apparatus, see Andersen et al. (Andersen et al., 1991). All structures associated with the basal bodies (bbs), including the X-shaped 4-2-4-2 microtubular flagellar root system, were labeled as previously described, so 1 indicates an association with the older bb and 2 an association with the younger bb (Heimann et al., 1989; Moestrup and Hori, 1989), and s indicates sinister (left) and d dexter (right) of the bb viewed from its distal end with triplet 1 in the 12 o'clock position (Melkonian, 1984) (see Fig. 7G-I). The numbering of individual flagellar root microtubules follows Melkonian (Melkonian, 1978) (see Fig. 7G).

A numbering system of the bb triplets can be established by tracing axonemal doublets in serial sections from the axoneme down into the bb. In the axoneme of Chlamydomonas, doublet 1 is recognized by the absence of the outer dynein arm and a (two-part) bridge extending from its A-tubule to the B-tubule of doublet 2 (Fig. 1A,B, arrowheads) (Hoops and Witman, 1983). Triplet 1 of the bb is the middle of the three triplets to which the distal connecting fiber (dcf) is attached (Fig. 1A-J, arrowheads) (Hoops and Witman, 1983). A numbering system for the triplets of the probasal body (pbb) was established in this study (see below).

At the distal end of the bb, exactly at the transition between the microtubular triplets of the bb and the doublets of the transitional region, a filament of about 10 nm diameter is attached to the microtubular doublets (rarely triplets) in a rotationally asymmetric pattern linking A-tubules of doublets 7, 8, 9, 1 and 2 (Fig. 1G, Fig. 2D,E). From doublet 2, the filament arches back to doublet 7, passing through the lumen of the bb. The overall outline of this closed structure resembles that of an (capless) acorn with its broad base located between doublets 1 and 2, and its pointed tip at doublet 7 (see schematic drawing Fig. 7D). At doublets 2 and 7, the acorn has a prominent knob through which linkage with the A-tubule is established. The side of the acorn that is not attached to bb doublets/triplets will, in the following, be called the `lumenal' side of the acorn.

A second, V-shaped, system of filaments of about 8 nm diameter is present in this region of the bb. Two filaments extend from triplets (rarely doublets) 4 and 5 into the bb lumen, bisect the lumenal side of the acorn (Fig. 1G, Fig. 2D,E) and, in the center of the bb lumen, connect to another filament that runs perpendicular to the two filaments, extending down from the proximal end of the stellate structure in the transitional region (Fig. 1G,K, small arrowheads). The positional relationships of the acorn to these filaments is best seen in longitudinal sections through bbs (Fig. 2G). In such sections, the acorn is seen as two asymmetrically positioned electrondense dots (Fig. 2G, arrowheads). The filaments extend from triplets 4 and 5 at the level of the acorn and bend downwards with an angle of about 35° after reaching the lumenal side of the acorn. They continue to the other side of the bb, making contact with triplets at the level of the dcf attachment with the bb (Fig. 2G, small arrows). In cross sections through bbs, depending on the sectional plane, these filaments can be seen to contact triplets 8, 9 and 1 (Fig. 2E, arrowhead) (S. Geimer and M. Melkonian, unpublished).

Most interestingly, both systems of filaments were already present at the distal end of the probasal bodies (cross section in Fig. 2A, compare with Fig. 2D,E in the bb; longitudinal section in Fig. 2F, compare with Fig. 2G in the bb).

The flagella-bearing bbs are composed of nine microtubular triplets with connections between the A- and C-tubules of adjacent triplets (Fig. 3F). They have an average length of 320 nm (n=10) based on the length of the microtubular triplets and their diameter is about 210 nm (n=10). Most of the C-tubules in the bb triplets are filled with electron-dense material (Fig. 1H-J, Fig. 3F, Fig. 4A-E). At the proximal end of each bb, the hub and spoke complex of the cartwheel is present (Fig. 1K, cw; Fig. 3F) (Ringo, 1967). The cartwheel consists of a central hub (diameter about 25 nm, with a central dot; Fig. 3F, Fig. 4E) and nine spokes that are connected to the A-tubules by nine triangular electron-dense knobs (Fig. 3F, Fig. 4E) (Cavalier-Smith, 1974; O'Toole et al., 2003). The dot in the central hub might represent a fine filament in cross section because, in some median longitudinal sections of bbs, a fine filament was seen emanating from the central hub at the proximal end of the cartwheel (not shown).

In the present study, the developmental status of the bbs was deduced based on the number of cartwheel tiers. It has been shown previously in other flagellate green algae that the cartwheel of the developmentally oldest bb (bb 1) shows fewer tiers than the cartwheel of the younger bb(s) (Beech et al., 1991; Beech and Melkonian, 1993). Because serial sectioning through both bbs (n=10) consistently revealed that there are differences in the number of cartwheel tiers in the two bbs we assume that the same is true for C. reinhardtii and thus label as 1 the bb that has the fewer cartwheel tiers (Fig. 3A-D,E). A statistical analysis of randomly sectioned bbs also revealed that cartwheels consist of two to seven tiers. The number of cartwheel tiers was analysed in 236 longitudinal sections of bbs; 99 bbs (41.9%) exhibited a cartwheel consisting of three tiers and 99 bbs (41.9%) a cartwheel consisting of four tiers. Only 11 bbs (4.7%) showed a cartwheel with only two tiers, 22 bbs (9.3%) a cartwheel with five tiers, three bbs (1.3%) a cartwheel with six tiers and two bbs (0.9%) a cartwheel with seven tiers (Fig. 3A-D,E).

In median longitudinal sections of the bb, a series of projections extend about 25 nm into the lumen of the bb (Fig. 1K, between two pairs of dashes) (Cavalier-Smith, 1974). They start about 25 nm (n=5) distal to the cartwheel and extend for about 215 nm (n=5) along the microtubular triplets. These structures were initially described as projections attached to the A-tubules and named `A-tubule feet' (Cavalier-Smith, 1974). Our data show that these projections are associated with both the A- and B-tubules of each triplet (Fig. 4A-C, small arrowheads) and are interconnected by a very fine filament (Fig. 4A-C, small arrow). However, in preparations of isolated cytoskeletons fixed in the absence of tannic acid, the fine structure of the A-tubule feet is more complex (S. Geimer and M. Melkonian, unpublished). Similar projections attached to the microtubular triplets are also present in mammalian centrioles (Stubblefield and Brinkley, 1967; Ross, 1968; Paintrand et al., 1992).

The lumen of the bb is filled with filaments (Fig. 3A-C,E) of which some are attached to the A-tubule feet (Fig. 1H). In some micrographs the bb lumen appears to be empty, presumably because the filamentous material was extracted during isolation of the cytoskeletons (Fig. 1I-K, Fig. 4A-C).

The probasal bodies (pbbs) are – like the flagella-bearing bbs – composed of nine microtubular triplets with connections between the A- and C-tubules of adjacent triplets (Fig. 2B) (Ringo, 1967; O'Toole et al., 2003). The microtubular triplets were complete in all observed pbbs (n=20), the B- and C-tubules are filled with electron-dense material (Fig. 2B). The pbbs have an average length of 95 nm (n=10), based on the length of the microtubular triplets, and their diameter is the same as that of the bbs (about 210 nm, n=10).

It has previously been shown for other flagellate green algae that the number of cartwheel tiers in the pbbs is higher than in the bbs (Beech et al., 1991). This is also true for Chlamydomonas – a statistical analysis of 53 longitudinal sections of pbbs revealed that the cartwheel of the pbb consists of four to seven tiers. Of the 53 pbbs analysed, 23 (43.4%) exhibited a cartwheel consisting of five tiers, 17 (32.1%) a cartwheel with four tiers, 12 (22.6%) a cartwheel with six tiers and only one pbb (1.9%) a cartwheel with seven tiers. Only three or four tiers are embedded in the microtubular cylinder (Fig. 2F, Fig. 5E) and the cartwheel protrudes proximally from the microtubular triplets in a conical shape; that is, the diameter of the tiers decreases gradually (Fig. 2F, open arrow).

The position of the acorn (see above) in the pbb allows the introduction of a tentative numbering system for the pbb triplets (Fig. 2B, see also Fig. 7G,H). Based on this observation, we estimate that pbbs are rotated by 140° with respect to the flagella-bearing bbs with which they are distributed to progeny during cell division (Fig. 7G,H).

About 50 nm (n=5) distal to the end of the C-microtubules of the bb, there is a transitional region (tr) characteristic for green flagellates. The tr contains two distinct stellate structures that are separated by the transitional plate (tp) (Fig. 1C-F,K, tr) (Ringo, 1967; O'Toole et al., 2003). The stellate structure is formed by filaments that connect the A-tubules of every third axonemal doublet with each other, constituting (as seen in transverse sections) a nine-pointed star with a central ring-like hub (Fig. 1D,F) In the distal stellate structure, the hub is lined with electron-dense material that is not present in the proximal stellate structure (compare Fig. 1D with 1F). In longitudinal sections, the two stellate structures form an H-piece made up of the distal (about 95 nm long) and the shorter proximal (about 55 nm long) stellate structure, and separated by the tp (Fig. 1K, tr). As typical for chlorophycean algae (Melkonian, 1984), the proximal end of the distal stellate structure is attached to and closed by the tp, which extends to the microtubular doublets (Fig. 1K, Fig. 2G, Fig. 3C,E). The distal end of the proximal stellate structure is not attached to the tp but is closed by a second plate, which, unlike the tp, does not extend to the microtubular doublets (Fig. 2G, Fig. 3E, bb1). This plate has not been observed in previous studies, presumably because it is sometimes closely appressed to the tp and thus not discernible (Fig. 1K, Fig. 3C). Cross sections of the tp have previously not been documented in conventional thin sections, but they were shown in a recent study using dual-axis electron tomography to be a central, amorphous disk (O'Toole et al., 2003). In our preparations, the central, amorphous disk is also revealed (Fig. 1E). However, the tp clearly extends between the microtubular doublets into the doublet outer projections (see below), which, at this level, form saw tooth-like structures projecting from the microtubular cylinder (Fig. 1E, compare with Fig. 1K showing the tp in longitudinal section).

In the area of the tr, each of the nine doublets bears a short (about 40-50 nm) T-shaped fiber termed the `doublet outer projection' (Cavalier-Smith, 1974), which is attached to the A/B-tubule junction (Fig. 1C,D,F, small arrows). In longitudinal sections, the doublet outer projections form two discrete zones of wedge-shaped, dense material (Fig. 1K, small arrows). In cross sections through the proximal stellate structure, the doublet outer projections are sometimes interconnected, forming a ring (Fig. 1F, small arrows). Membrane fragments attached to the doublet outer projections can be seen making contact with the distal end of the transitional fibers (Fig. 3B,C, double arrowheads).

The transitional fibers (tfs) were first described by Ringo (Ringo, 1967). In cross sections of the bb, they project radially counterclockwise from each triplet. The tfs are about 170 nm in length and have a swollen distal end (Fig. 1G, Fig. 2D,E, large arrows). They are attached to the distal third of the bb over a region of about 120 nm (Fig. 1G-I,K, large arrows). The tfs show a faint striation pattern (Fig. 1K, Fig. 3C) but, as in cryofixed cells (O'Toole et al., 2003), the exact number and spacing of the cross-striations are difficult to determine. The swollen distal ends of the transitional fibers are interconnected by a fine filament (not shown) and a fine filament runs from the distal end of at least the transitional fiber attached to triplet 9 to a triangular structure located above the center of the dcf (Fig. 3B, open triangle and asterisk).

An array of morphologically distinct fibers link specific bb triplets to the microtubular flagellar roots. Two sets of fibers extend laterally from each bb: left fibers (sf¹ and sf²) connecting the bb to the four-stranded microtubular root (s-root), and the right fiber (df) connecting the bb to the two-stranded microtubular root (d-root). The spatial arrangement of these fibers within the basal apparatus is illustrated in Fig. 7.

The sf¹ is attached to the mid-region, the sf² to the more proximal region of the bb (Fig. 5D, sf¹ and sf²). The fan-shaped sf¹ arises from the C-tubule of triplet 4 and traverses about 90 nm (Fig. 4C, sf¹) to contact the upper surface of the corresponding s-root (root microtubules 2 and 3; Fig. 5D, see also Fig. 7G, sf¹). The sf² is mainly attached to triplet 3 but projections are also in contact with triplets 2 and 4. It traverses about 45 nm (Fig. 4D,E, sf²) and contacts the lower surface of the corresponding s-root (root microtubules 1, 2 and 3; Fig. 5D,E, Fig. 7H, sf²). The sf² extends to the most proximal end of the bb and underlies at least triplets 2 and 3 (Fig. 4F, Fig. 5B,C, Fig. 7I, sf²).

The df is attached to triplets 8 and 9 (Fig. 4C, triplets 8 and 9 numbered, see also Fig. 7G, df) and extends about 30 nm to contact the lower surface of the corresponding d-root (with root microtubule 1; Fig. 6D and inset, small arrows).

The pbbs are connected to the bbs, the d-roots and their corresponding striated microtubule-associated fibers (SMAFs), and the s-roots by different fibers, and we describe the attachment of these fibers to particular triplets of the pbb (Fig. 7H). In cross sections through the basal apparatus, a striated fiber originates at a d-root mid-way between the pbb and a bb, extends about 280 nm towards the pbb, and then passes between the pbb and the opposite bb (Fig. 2B,C,H, large arrowheads; Fig. 7H, arrows). This fiber was first observed and termed `lateral striated fiber' (lsf) by Weiss (Weiss, 1984). The lsf shows a cross-striation pattern consisting of electron-dense bands of about 30-45 nm that are separated by electron-translucent areas of about 10-20 nm and is attached to pbb triplets 7, 8 and 9 (Fig. 2B,C,H). In cross sections of the d-root, the lsf (Fig. 6E,F, small arrowheads) attaches to an electron-dense plate underlying both root microtubules (Fig. 6E,F, open triangles). Filaments of the lsf (Fig. 5D-F, Fig. 6E, small arrowheads) continue to the sf² (Fig. 5D,E, sf²) and the s-root (Fig. 6E, s). The pbb is linked to the d-root at a second site by thin filaments extending from the SMAF overlying the d-root to triplets 1 and 2 of the pbb (Fig. 2B, Fig. 6H, double arrowheads). A direct connection between a flagella-bearing bb and a pbb is made by material of the proximal connecting fiber (Fig. 4D,E) contacting pbb triplets 8 and 9 (Fig. 7H).

Previously, only two cross-striated proximal connecting fibers (pcfs) had been described attached to the bbs (Ringo, 1967). The two pcfs are linked to triplets 8 and 9 (Fig. 2H, pcf, Fig. 3F, pcf, Fig. 4D,E, arrows). The connection of the pcf to triplets 2 and 3 of the opposing bb is made through the sf² (Fig. 2H, Fig. 4E). In this study an additional pcf was detected. A similar fiber had been previously described in zoospores of the green alga Chlorosarcinopsis and termed a `median pcf' (mpcf) (Melkonian, 1978) (Fig. 3A-D, Fig. 4F, Fig. 6J, large arrowheads). The mpcf is not cross-striated and, at its distal end, is attached to triplets 5 and 6 (Fig. 4D, large arrowhead), whereas, more proximally, it is linked to triplets 4, 5, 6, 7 and 8 (Fig. 4E,F, large arrowheads). At triplet 8, the mpcf is attached to a hook-like structure (Fig. 6J, small arrow). Material of the mpcf underlies the proximal ends of the bb (Fig. 3A-D,E, Fig. 6J, large arrowheads) and forms, together with parts of the sf², an electron-dense, open ring (Fig. 4F).

The ultrastructure of the basal apparatus of C. reinhardtii has been analysed in detail by serial ultrathin section electron microscopy of isolated cytoskeletons. New information has been presented on the structure of the pcfs, the fibers connecting the bb with the microtubular flagellar roots, the linkage of the pbbs to other elements of the basal apparatus and the bbs/pbbs themselves. These data will facilitate the precise mapping of previously identified as well as novel basal apparatus proteins, the prediction of possible protein-protein interactions and the analysis of mutants and RNA interference cells with only subtle defects in structural components of the basal apparatus.

The high density of basal apparatuses in our preparations made it possible to statistically analyse a large number of basal apparatuses. The diagram illustrating the basal apparatus ultrastructure (Fig. 7) is based on data derived from the analysis of 60 complete series and several hundred non-serial sections through basal apparatuses. An important issue when studying subcellular structures is whether or not the isolation procedure has led to structural modifications. Information about the ultrastructure of the basal apparatus of C. reinhardtii in situ has been provided previously (Ringo, 1967; Johnson and Porter, 1968; Cavalier-Smith, 1974; Goodenough and Weiss, 1978; Hoops and Witman, 1983; Weiss, 1984; Gaffal, 1988; O'Toole et al., 2003). Careful comparison of these data with our own findings leads us to conclude that the basal apparatuses that we have analysed were structurally intact.

Using this approach, several novel basal apparatus structures were identified during this study. Three types of specialized fibers were identified that link the microtubular flagellar roots directly to particular triplets of the basal bodies. The four-stranded microtubular roots (s-roots) are attached to the basal bodies by two sets of s-fibers (sf¹ and sf²) and the two-stranded microtubular roots (d-roots) by the d-fibers (df). These fibers are predominantly associated with the C-tubules of the respective triplets (e.g. the sf¹ is exclusively linked to the C-tubule of triplet 4). For the related flagellate green alga S. similis, it has also been shown that the B- and (in particular) the C-tubules are the preferred attachment sites for bb-associated structures (Geimer et al., 1997).

The most significant single observation made during this study was the discovery of a structural component, the acorn, that confers rotational asymmetry on both bbs and pbbs. It has been previously noted that ciliary and flagellar basal bodies display axial as well as rotational polarities (e.g. Anderson and Brenner, 1971; Hoops and Witman, 1983; Sandoz et al., 1988; Beech and Melkonian, 1993; Geimer et al., 1997) (reviewed in Beisson and Jerka-Dziadosz, 1999). A marker for the axial polarity of bbs and centrioles is the highly conserved cartwheel, which is attached to the inner wall of the microtubular cylinder at its proximal end. The rotational polarity of basal bodies previously could only be inferred indirectly from the asymmetric association of various fibers and/or microtubules to specific bb triplets at the outside of the microtubular cylinder. Here, we describe a structural marker for the rotational asymmetry inherent in the bbs and pbbs: at the most distal end of the microtubular cylinder, a fiber outlining the shape of an acorn is attached to triplets 7, 8, 9, 1 and 2. This structure can also be recognized in intact cells in published micrographs [see Fig. 36 (Ringo, 1967); see supplemental videos (O'Toole et al., 2003)].

The ultrastructural data presented here show that the distal end of the pbb is already highly specialized. Other than tubulin, few proteins have been localized to pbbs. In the flagellate green alga S. similis, p210 (a component of the doublet outer projections in the tr), was localized to an asymmetric pattern at the distal end of the pbbs soon after their assembly (Lechtreck and Grunow, 1999). Centrin is also found in the pbbs of S. similis (Lechtreck and Grunow, 1999) and, in mammalian cells, centrin was detected at the site of procentriole formation even before tubulin was present (Middendorp et al., 1997). In Chlamydomonas, a kinesin-II protein, FLA10p (also called KHP1), is present at the pbbs (Vashishtha et al., 1996) and VFL1p, which is located within the lumen of bbs, was also shown to be present at the pbbs (Silflow et al., 2001). In the Chlamydomonas vfl1 mutant (Adams et al., 1985), the correct rotational orientation of the bbs is impaired (Silflow et al., 2001). By careful immunogold electron microscopy, Silflow et al. showed that the position of VFL1p in the basal body is rotationally asymmetric. VFL1p localizes to the most distal end of the bb near the microtubular triplets/doublets that face the dcf (triplets/doublets 9, 1 and 2) (Silflow et al., 2001). This localization corresponds well with the position of the acorn described here and suggests that VFL1p might be a component of this structure.

The asymmetric position of the acorn within the microtubular cylinder suggests to us that the acorn might play a role during probasal body maturation by controlling the asymmetric assembly of the various bb-associated fibers. Defects in or absence of the acorn could disturb the highly specific spatial pattern of the fibers attached to the bbs. The triplets to which the acorn is attached are the same triplets to which the centrin-containing fibers of the nucleus-bb connector (triplets 7, 8) (Salisbury et al., 1988) and the dcf (triplets 9, 1, 2) (Hoops and Witman, 1983) are attached (Fig. 7). In vfl1, the dcf is either absent or incomplete (Adams et al., 1985). Mispositioned or absent s- and d-fibers would probably result in a mispositioning of the microtubular roots. This is the case in vfl1 mutants, in which the normal cruciate array of microtubular flagellar roots has not been observed (Adams et al., 1985). In vfl1 mutants, cell motility is also severely impaired, with even biflagellate cells showing aberrant spinning or tumbling movements, indicating that the flagella do not beat in opposite directions (Adams et al., 1985).

Basal bodies have been shown to express intrinsic and autonomous polarities. During the development of the oral apparatus in the ciliates Tetrahymena and Stentor, bbs first proliferate and lie at random positions, but their appendages develop at a predetermined site (Williams and Frankel, 1973; Paulin and Bussey, 1971) (reviewed in Beisson and Jerka-Dziadosz, 1999). An analogous situation was described for bbs in the nasal epithelium of mouse embryos (Frisch and Farbman, 1968). The analysis of a Chlamydomonas vfl mutant (vfl3) affecting the proper positioning of the pbbs has led to the hypothesis that the bbs have an intrinsic rotational polarity (Wright et al., 1983).

To address the question of whether acorn-like structures are a universal feature of bbs/centrioles in eukaryotic cells, we performed an exhaustive literature search. An acorn-type structure attached to five or six bb triplets is visible at the distal end of bbs in the green flagellate S. similis [(Geimer et al., 1997) Fig. 3], in the parabasalid protist Pseudotrichonympha, [(Gibbons and Grimstone, 1960) plate 351, Fig. 7] and in the chytridiomycete fungus Phlyctochytrium irregulare Koch [(McNitt, 1974) Fig. 3]. Thus, acorn-type structures apparently occur in three different eukaryotic kingdoms.

In Bornens (Bornens, 1992), a cross section of a human (lymphoblast) centriole is shown in which an acorn-type fiber can be seen apparently attached to five triplets. Stubblefield and Brinkley (Stubblefield and Brinkley, 1967) described a structure in the distal lumen in centrioles in Chinese hamster fibroblasts that did not exhibit ninefold rotational symmetry. Vorobjev and Chentsov (Vorobjev and Chentsov, 1980) found a fine filament that formed an incomplete circle at the distal end of centrioles in various mammalian species. In the latter two studies, the acorn is not clearly revealed. However, internal structures in the centriole/bb of mammalian cells have always been ill-defined, which might be related to their susceptibility to divalent cations (Paintrand et al., 1992). The acorn was even overlooked for a long time in the otherwise well-characterized bb of Chlamydomonas. We conclude that the acorn might be the long-sought-after structural equivalent of the cartwheel at the distal end of bbs/centrioles, providing rotational asymmetry to this enigmatic constituent of eukaryotic cells.

This study was supported by the Deutsche Forschungsgemeinschaft (Me 658/22-1).