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First published online 11 May 2004
doi: 10.1242/jcs.01120

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

Botanisches Institut, Universität zu Köln, Gyrhofstr. 15, 50931 Köln, Germany

View larger version (54K): [in a new window]   Fig. 7. The basal apparatus of Chlamydomonas reinhardtii (transverse sections, from distal to proximal, doublets/triplets are numbered). (A-F) The transitional region and distal part of a bb. Centrin-containing filaments are depicted in blue and triplets to which the dcf is attached are filled with blue stripes. (A-C) The transitional region. The doublet outer projections attached to the A/B-tubule junction at the outside of the doublets are shown in light gray. (B) The region containing the transitional plate (tp). (D-F) Transition between transitional region and bb, and distal part of the bb with the attached transitional fibers (tf, dark green). (D) Filaments are attached in an asymmetrical way at the inner surface of the microtubular cylinder exactly at the transition between transitional region and bb. A filament outlining the shape of an acorn (red) is attached to doublets (rarely triplets) 7, 8, 9, 1 and 2. The lumenal side of the acorn is crossed by a V-shaped filament system; two filaments extend from triplets 4 and 5 to fuse in the center of the bb lumen and continue to the other side of the bb, making contact with triplet 9. The acorn and V-shaped filament system are already present at the distal end of the pbbs (G, pbb). (G-I) The bb root complex. (G) Median part of the bb root complex at a level where the microtubular roots and striated microtubule associated fibers (SMAFs) are present. The distal connecting fiber has been omitted for clarity but its position is outlined by dotted blue lines and the triplets to which the dcf is attached are filled with blue stripes. At this level, the acorn is actually not present in the bb but it is given in this schematic diagram for a better understanding of its positional relationship with the other elements of the basal apparatus. The cruciate microtubular roots are labeled 1d, 1s, 2d and 2s. Each microtubular root is associated with a SMAF, which are shown in green. The microtubular roots and SMAFs are also shown in cross sectional view, with individual root microtubules numbered. The first set of s-fibers (1sf1, 2sf1) connecting the bbs to their corresponding four-stranded microtubular roots (s-roots, 1s, 2s) are shown in red. The d-fibers (1df, 2df) that interconnect the bbs with their corresponding two-stranded microtubular roots (d-roots, 1d, 2d) are given in purple. The nucleus-bb connectors (NBBC, blue) are seen attached to the bbs in this region. At the distal ends of the pbbs (pbb, shades of gray) the acorn (red) and the V-shaped filament system are present (compare with D). (H) Proximal part of the bb root complex at a level below the microtubular roots and SMAFs (for a better orientation outlined in gray and green, respectively). The second set of s-fibers (1sf2, 2sf2) connecting the bbs to their corresponding four-stranded microtubular roots (s-roots, 1s, 2s) is shown in red. The proximal connecting fibers (pcf, yellow) interconnect the bbs and also make contact with the adjacent pbb (with pbb triplets 8 and 9). The pbbs (their microtubular triplets are numbered) are attached by the lateral striated fiber (arrows) to their corresponding d-root and the sf2 of the neighboring bb. In this region, the median proximal connecting fiber is attached to triplets 5 and 6 (mpcf, yellow). (I) Structures lying below the proximal ends of the bbs (faintly outlined) are the median proximal connecting fiber (mpcf, yellow) and the proximal parts of the sf2 (red). Of the pbbs, only the hub and spoke complex of the cartwheels are visible at this level.

View larger version (135K): [in a new window]   Fig. 1. The axoneme, transitional region and basal body. (A-J) Series of transverse sections (section numbers 1, 10, 13-20) showing the axoneme (A,B), transitional region with the distal stellate structure (C,D), transitional plate (E) and proximal stellate structure (F), and distal part of the bb (G-J). Within the axoneme, doublet 1 (A,B, arrowhead) can be identified by a bridge extending from its A-tubule to the B-tubule of doublet 2. This doublet (doublet/triplet 1 is marked with an arrowhead in C-J) is continuous with the middle of the three triplets to which the distal connecting fiber (dcf; J) is attached (J, arrowhead). In the transitional region, short, T-shaped fibers (the doublet outer projections; C,D,F,K, small arrows) are attached to the A/B-tubule junction on the outside of the doublets. In the area of the proximal stellate structure, the doublet outer projections are sometimes interconnected, forming a ring (F, small arrows). In the region of the transitional plate, the lumen of the transitional region is filled with electron-dense material that extends between the microtubular doublets into the doublet outer projections, which, at this level, form saw-tooth-like structures projecting from the microtubular cylinder (E). The transitional fibers (G-I,K, large arrows) are attached to the distal half of the bb. Within the microtubular cylinder – exactly at the transition between the microtubular doublets of the transitional region and the microtubular triplets of the bb – filaments are attached in a rotationally asymmetric pattern to the microtubular doublets/triplets (G) (Fig. 2D,E). A filament outlining the shape of an acorn is attached to the A-tubules of doublets 7, 8, 9, 1 and 2 (G, doublets 7, 9 and 2 are numbered). The lumenal side of the acorn is crossed by a V-shaped filament system; two filaments extend from triplets 4 and 5, 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 (F,G,K, small arrowheads). The acorn and V-shaped filament system are already present at the distal end of the pbbs (Fig. 2A). (K) Median longitudinal section through the axoneme and bb showing the transitional region (vertical line labeled tr) and doublet outer projections (small arrows), the transitional fibers (large arrows), and the cartwheel (vertical line labeled cw). Sectional planes corresponding to transverse sections B-J are marked by dotted lines. Two rows of projections can be seen attached to the triplets and extending into the lumen of the bb (between two pairs of dashes). A fine filament (small arrowhead) interconnects the proximal end of the proximal stellate structure with the system of filaments present in the distal lumen of the bb (compare with F and G, showing this filament in transverse section, small arrowheads). Scale bar, 250 nm.

View larger version (138K): [in a new window]   Fig. 2. The probasal body. (A-C) Consecutive transverse sections through a pbb; the two bbs are sectioned at an oblique angle. The microtubular roots (A-C, d for d-roots, s for s-roots) and the distal connecting fiber (B, dcf) are visible. At the distal end of the pbb (A, open arrow), the acorn and V-shaped filament system, which are also present at the distal end of the bb, can be seen (compare to bbs shown D and E; see also Fig. 1G). More proximally (B, pbb triplets are numbered), the microtubular triplets and the hub and spoke complex of the cartwheel are visible. The B- and C-tubules are filled with electron-dense material. An electron-dense patch is overlying the C-tubule of triplet 9 and filaments of the lateral striated fiber link at least triplets 7, 8 and 9 to the corresponding d-root (B, small arrowhead; see also Fig. 6E,F). Triplets 1 and 2 lie close to the d-root and corresponding striated microtubule-associated fiber (SMAF), and are connected to the SMAF by fine filaments (B, double arrowhead; see also Fig. 6H). At the most proximal end of the pbb, only the hub and spoke complex of the cartwheel, protruding from the microtubular cylinder, is visible (C, compare with longitudinal sectioned pbb in F, open arrows). The lateral striated fiber is labeled (C, large arrowhead). (D,E) Transverse sections through the most distal end of bbs showing the acorn and V-shaped filament system (triplet numbers are indicated, compare with Fig. 1G). The transitional fibers are marked (large arrows). In E, the two filaments that extend from triplets 4 and 5, and fuse in the center of the bb, continue and make contact with doublet 9 (small arrowhead). (F) Longitudinal section through a bb and pbb (open arrow). The cartwheel of the pbb consists of six tiers, of which only three are bound by the short microtubular triplets. Also, in longitudinal section, the filamentous system present at the distal end of the pbb clearly resembles that present within the lumen at the distal end of the bb. This becomes clear from a comparison of the pbb shown in F with the bb shown in G. The acorn is seen as two asymmetrically positioned electron-dense dots in the bb (Fig. 2G, small arrowheads) and pbb (Fig. 2F, small arrowheads). The filaments extending from triplets 4 and 5 at the level of the acorn (small arrow at the left side of the pbb in F and bb in G) bend downwards with an angle of about 35° (ref erring to the transverse axis of the pbb/bb) after reaching the lumenal side of the acorn and continue to the other side of the pbb/bb. In the pbb, these filaments make contact with triplets and the cartwheel (F, small arrow at the right side of the pbb). In the bb, these filaments make contact with triplets at the level of the dcf attachment with the bb (G, small arrow at the right side of the bb). (H) Section through a basal apparatus; one bb is sectioned longitudinally, the other one transversely. The proximal connecting fibers interconnecting the bbs are visible (pcf). The transversely sectioned pbb (open arrow) is attached by the lateral striated fiber (see also C, large arrowheads) to the corresponding d-root (d). The lateral striated fiber also interconnects the pbb with parts of the sf2 and probably the pcf. The limits of the sf2 to the pcf are outlined by white squares. Scale bar, 250 nm.

View larger version (133K): [in a new window]   Fig. 3. The older and younger basal bodies. (A-D) Consecutive longitudinal sections through a basal apparatus. The cartwheel of the right bb consists of four tiers (A) and so this bb is assumed to be the developmentally older (A, 1), whereas the cartwheel of the left bb consists of seven tiers (C) and so this bb is assumed to be developmentally younger (A, 2). Membrane fragments (B,C, double arrowheads) associated with the doublet outer projections (B,C, small arrows) make contact with the distal end of the transitional fibers (B,C, large arrows). Above the center of the distal connecting fiber, a triangular structure is visible (A-D, asterisks) that is connected by a fine filament to the distal end of at least the transitional fiber attached to triplet 9 (B, open triangle). Lying below the dcf, the SMAFs (A-D, small arrowheads) seem to form a continuous fiber in the center region of the basal apparatus (B,C, small arrowheads) and are connected to the outer bands of the dcf by fine filaments (visible in C). The median proximal connecting fiber (A-D, large arrowheads) and the proximal connecting fiber of bb 2 (D, pcf) are visible. (E) Longitudinal section through a basal apparatus (the bbs are numbered). The cartwheel of bb 1 consist of three tiers, whereas the cartwheel of bb 2 exhibits four tiers. The complex system of filaments present in the lumen of the bbs is clearly visible in bb 1 and seems to be more dense in bb 1 than in bb 2. This is also the case for the bbs shown in A-D. (F) Transverse section through the proximal end of a bb showing the hub and spoke complex of the cartwheel (triplet numbers are indicated). Parts of the pcf and sf2 are visible. Notice that the C-tubules are filled with electron-dense material. Scale bar, 250 nm.

View larger version (145K): [in a new window]   Fig. 4. Consecutive transverse sections through the basal apparatus showing the two bbs (A; the upper bb is sectioned transversely, the lower bb at an oblique angle) and probasal bodies (C-F, open arrows; the left pbb is sectioned longitudinally, the right pbb transversely). Within the lumen of the bb, electron-dense patches are associated with the A- and B-tubules of the microtubular triplets (A, small arrowheads; see also Fig. 1K). These patches are interconnected by a fine filament (A, small arrow). The distal connecting fiber (A,B, dcf) is attached to triplets 9, 1 and 2 of each bb, starting at the level where the transitional fibers (A, large arrows) terminate. The nucleus-bb connector is attached to triplets 7 and 8 (B-D, curved arrows). The cruciate microtubular root system is labeled in C [d for the dexter (right) roots, s for the sinister (left) roots]. The s-roots are connected to the bbs by two sets of fibers: the more distal sf1 is attached to the C-tubule of triplet 4 (C, sf1) and makes contact with the upper surface of the corresponding s-root; the more proximal sf2 (D-F, sf2) makes contact with the lower surface of the corresponding s-root. The main attachment site of the sf2 with the bb is triplet 3, but projections are also in contact with triplets 2 and 4 (see also Fig. 3F). Material of the sf2 is underlying at least triplets 2 and 3 below the proximal end of the bbs (F, sf2). The d-roots are linked to the bbs by the d-fibers; the df is attached to triplets 8 and 9 (C, triplets numbered). At their proximal ends, the bbs are interconnected by two proximal connecting fibers and a median proximal connecting fiber. The main attachment site of the proximal connecting fiber (D,E, arrows) is triplet 8, but it also makes contact with triplets 7 and 9 (see also Fig. 3F). Attachment sides of the median proximal connecting fiber are, more distal at the bb, triplets 5 and 6 (D,E, arrowheads); more proximal at the bb, the mpcf is also attached to triplets 4, 7 and 8. Parts of the median proximal connecting fiber underlying the proximal ends of the bbs are visible in F (arrowheads, see also Fig. 3A-D). For the proximal connecting fibers and median proximal connecting fibers, see also Fig. 6I,J. Scale bar, 250 nm.

View larger version (143K): [in a new window]   Fig. 5. Consecutive series showing the d- and s-roots, SMAFs and pbb. (A,B) The distal connecting fiber (dcf), both d-roots (d; their proximal ends are visible) and the corresponding SMAFs (double arrowheads) are marked. Notice that, at their proximal ends, the d-root microtubules are detached from their corresponding SMAFs. (C-F) The s-root in transverse sections. The SMAF (C-F, large arrowheads) overlying the s-root is very small and only associated with root microtubule 4. Near the proximal end of the s-root, microtubules 2 and 3 are attached by some electron-dense material to the bb (C). The sf1 (D, sf1) is attached to the upper surface of the s-root to root microtubules 2 and 3. The sf2 (D,E, sf2) makes contact with the lower surface of the s-root to root microtubules 1, 2 and 3. Material of the sf2 is underlying the bb (B,C, sf2). Filaments of the lateral striated fiber interconnecting the pbb (D-F, open arrows) to the sf2, the s-root microtubules and probably the bb proper are labeled (D-F, small arrowheads). Scale bar, 250 nm.

View larger version (125K): [in a new window]   Fig. 6. The d-root, SMAFs, pbb and proximal connecting fibers. (A-H) Consecutive series showing the d-root in transverse section. The two bbs are sectioned at an oblique angle and the pbb is sectioned longitudinally. (A,B) The distal connecting fiber (B, dcf) and one proximal connecting fiber (A, pcf) are marked. The d-root is linked to the corresponding bb by the df (A,D and inset in D, small arrows). In the more proximal region of the d-root, the corresponding SMAF (E-H, large arrowheads) is overlying both root microtubules (D-G), whereas, more distally, it is overlying only root microtubule 2 (H). The striation pattern of the SMAF is clearly visible in F and G. (E,F) The pbb is connected to the d-root by the lateral striated fiber (see also Fig. 2C,H, large arrowheads). Filaments of the lateral striated fiber (E,F, small arrowheads) extend from an electron-dense plate underlying both microtubules of the d-root (E,F, open triangles) to the side of the pbb (F, open arrow), and some of these filaments (E, small arrowheads) seem also to be connected to the s-root (E,F, s). More distal at the d-root, filamentous material interconnects the pbb with the SMAF (H, double arrowheads). (I,J) Two consecutive serial sections showing the two sets of proximal connecting fibers. The left bb is seen in transverse, the right in longitudinal section. The proximal connecting fiber (I, pcf) shows a complex cross striation. The median proximal connecting fiber (J, arrowhead) is not cross striated and is attached to a hook-like structure (I,J, small arrow). Scale bar, 250 nm.

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