Duplication of Basal Bodies in Chlamydomonas

In mammalian cells, centriole duplication begins at the G1 to S transition [26]. In contrast, Chlamydomonas cells exiting mitosis have already assembled probasal bodies [27]. Electron microscopy of Chlamydomonas cells in G1, as judged by FACS analysis, shows that they have two probasal bodies adjacent to the two mature basal bodies. These probasal bodies are 40-90 nm in length [5] and are often missed by conventional electron microscopy [3, 4]. This is likely to be due to their short length relative to the normal depth of thin sections used for electron microscopy. The probasal bodies contain the amorphous material that is present in the mature basal bodies, which suggests that this material is important for early events in basal body assembly. The pinwheel structure and the knob-like elaborations are also present. The microtubule blades in these probasal bodies have complete triplet blades. Elongation of the probasal bodies to new, full length basal bodies occurs just prior to prophase (Figure 5.5B). Concurrently, the centrin-containing striated fibers that connect the two mature basal bodies are lost [3] breaking the link be-

Chlamydomonas

Figure 5.6 Drawings from 1929 showing cell division in Chlamydomonas nasuta closely resemble the current view of the cell cycle in Chlamydomonas reinhardtii. (A) Interphase cells are flagellated and the basal bodies are connected by a fiber that can be visualized with Schaudinn's fixative. This is likely to be the centrin connection between the basal bodies and the nucleus. (B) Pre-prophase is evidenced by increased granularity in the nucleus. (C) The basal bodies begin to move apart. (D) Basal bodies are found at the poles of the spindle and a more darkly staining fiber connects them. This fiber is likely to be the rootlet microtubules [19]. (E) At telophase, the beginning of the cleavage furrow is observed. (F) The cleavage furrow is extended from one end of the cell towards the other. (G) Cytokinesis is complete. (H) The new daughter cells will re-grow flagella and then emerge from the old cell wall, which is discarded. Reproduced from [25].

Figure 5.6 Drawings from 1929 showing cell division in Chlamydomonas nasuta closely resemble the current view of the cell cycle in Chlamydomonas reinhardtii. (A) Interphase cells are flagellated and the basal bodies are connected by a fiber that can be visualized with Schaudinn's fixative. This is likely to be the centrin connection between the basal bodies and the nucleus. (B) Pre-prophase is evidenced by increased granularity in the nucleus. (C) The basal bodies begin to move apart. (D) Basal bodies are found at the poles of the spindle and a more darkly staining fiber connects them. This fiber is likely to be the rootlet microtubules [19]. (E) At telophase, the beginning of the cleavage furrow is observed. (F) The cleavage furrow is extended from one end of the cell towards the other. (G) Cytokinesis is complete. (H) The new daughter cells will re-grow flagella and then emerge from the old cell wall, which is discarded. Reproduced from [25].

tween the two mature basal bodies. This process allows separation of two centriole pairs. Each pair consists of an old basal body/centriole and a new basal body/cen-triole. Each pair segregates to a pole of the mitotic spindle. The pairs of centrioles are found outside of the perforated nuclear envelope and the spindle microtubules are inserted into the nucleus through these perforations [28].

Centriole Basal Body Spindle Pole

Figure 5.7 Migration of centrioles at mitosis. Differential interference contrast micrograph of Chlamydomonas centrioles in preprophase of mitosis. (A) The four centrioles are arranged in a diamond configuration and can be oriented relative to the contractile vacuole that appears as a large crater below the centrioles. Large black arrows point to the mother centrioles and the smaller arrowheads indicate the daughter centrioles (t = 0 min.). (B) The mother-daughter pairs of centrioles have separated and are moving towards the spindle poles (t = 7 min.). (Reprinted with permission from the Journal of Cell Science [18]).

Figure 5.7 Migration of centrioles at mitosis. Differential interference contrast micrograph of Chlamydomonas centrioles in preprophase of mitosis. (A) The four centrioles are arranged in a diamond configuration and can be oriented relative to the contractile vacuole that appears as a large crater below the centrioles. Large black arrows point to the mother centrioles and the smaller arrowheads indicate the daughter centrioles (t = 0 min.). (B) The mother-daughter pairs of centrioles have separated and are moving towards the spindle poles (t = 7 min.). (Reprinted with permission from the Journal of Cell Science [18]).

Studies of basal body duplication in Paramecium by Dippell [29] elegantly showed intermediates in basal body assembly. A ring of nine singlet microtubules was formed, the B tubule was added, and before all of the B subfiber was added, the C subfiber would begin to be added. Cross-sectional images thus reveal a cylinder of microtubules with varying numbers of B and C subfibers (Figure 5.8A). This type of image has not been observed in Chlamydomonas.

Several lines of evidence suggest that the assembly pathway may differ from that proposed by Dippell [29]. The localization of two proteins (p210 and Vfl1) suggests a different pathway. In mature basal bodies, p210 co-localizes with the Y-shaped fibers at the distal end of the transitional fibers in mature basal bodies in Sperma-tozopsis [7] and the Vfl1 protein localizes to the distal end [10]. If basal body duplication were to proceed strictly by the model proposed by Dippell in Figure 5.8A, then one would not expect to see p210 or Vfl1 on probasal bodies. However, in both cases, these proteins are present on probasal bodies and end up at the distal end of the basal bodies. As shown in the diagram in Figure 5.8B, it is suggested that several of these proteins may sit on the plus end of the assembling micro-tubules and move outward during elongation much like the attachment between kinetochores and microtubules [30]. The phenotype of basal bodies in bld2; rgn1-1 cells also suggest that disassembly may occur from the minus end of the microtubules of the basal body in these mutant cells. Electron micrographs of these cells show basal bodies with blades missing at the proximal end but intact at the distal end [31].

Chlamydomonas Cytokinesis

Figure 5.8 (A) Schematic diagram of basal body assembly as suggested by Dippell [29]. The first sign of basal body assembly is the appearance of an amorphous disk. This is followed by singlet microtubules that correspond to the A-tubule. These are followed by B- and, finally, C-tubules. Each stage is not completed simultaneously for all nine blades, so it is possible to observe mixtures of singlet, doublet and triplet microtubules within a given cross-section. (B) Schematic diagram of basal body assembly with a traveling cap model of regulation. Proteins (black) destined for fibers at the distal end (such as p210) travel at the tip of elongating microtubule blades. This complex could also serve as a negative regulator to prevent premature elongation of pro-basal bodies. These proteins leave the distal end of the mature basal body and move to various fibers once elongation is complete.

Figure 5.8 (A) Schematic diagram of basal body assembly as suggested by Dippell [29]. The first sign of basal body assembly is the appearance of an amorphous disk. This is followed by singlet microtubules that correspond to the A-tubule. These are followed by B- and, finally, C-tubules. Each stage is not completed simultaneously for all nine blades, so it is possible to observe mixtures of singlet, doublet and triplet microtubules within a given cross-section. (B) Schematic diagram of basal body assembly with a traveling cap model of regulation. Proteins (black) destined for fibers at the distal end (such as p210) travel at the tip of elongating microtubule blades. This complex could also serve as a negative regulator to prevent premature elongation of pro-basal bodies. These proteins leave the distal end of the mature basal body and move to various fibers once elongation is complete.

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