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First published online December 15, 2003
doi: 10.1242/10.1242/jcs.00859

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Mutations in -tubulin promote basal body maturation and flagellar assembly in the absence of -tubulin

Sylvia Fromherz¹, Thomas H. Giddings, Jr¹, Natalia Gomez-Ospina^1,* and Susan K. Dutcher^1,2,

¹ Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Boulder, CO 80309-0347, USA
² Department of Genetics, Washington University School of Medicine, 660 South Euclid Ave, Box 8232, St Louis, MO 63110, USA

View larger version (16K): [in a new window]   Fig. 1. Survival of cells on varying concentrations of colchicine. The percentage of viable cells is plotted against the different concentrations of colchicine. , tua2-1; , tua2-7; , tua2-1; uni3-1; , wildtype; , TUA2; uni3-1, , tub2-1. Strains with a tua2 mutation are more sensitive to colchicine than TUA2 strains. Cells with tub2-1 mutation in ß2-tubulin are more resistant to colchicine.

View larger version (197K): [in a new window]   Fig. 2. Serial section electron microscopic images, showing cross-sections of the basal body region of tua2-6; uni3-1 (A-F) and two different tua2-6; UNI3 cells (G-J and K-N). Scale bars 0.4 µm. (A) Distal region of a basal body with doublet microtubules. (B,C) Distal region of a basal body with doublet and triplet microtubule blades, triplet blades are indicated by asterisks. This basal body has three extended triplet blades. (D) Distal region of a basal body with both doublet and triplet microtubule blades, triplet blades are indicated by asterisks. Most of the triplet blades are only present at the distal end of the basal body. (E,F) Transition zone of a basal body as indicated by the stellate fiber. As in wild-type basal bodies, only doublet microtubule blades are observed in the transition zone. G) A mature basal body is present and a probasal body (lower right) with triplet microtubule blades; the angles of the blades are indicative of a probasal body rather than of a mature basal body. (H) A mature basal body and probasal body are separated by rootlet microtubules that form a cross-shaped pattern. (I) A mature basal body with the distal striated fibers that are present only at the distal end. Transition fibers emanate from the basal body at the distal end. (J) Transition fibers elongate in a more distal region. The majority of the blades still have triplet microtubules. (K) Triplet microtubules have become doublet microtubules. Transition fibers are still present and are elongated on the right of the basal body, but on the left they have become connectors to the membrane. (L) Doublet microtubules with connectors to the membrane. (M) Transition zone with the appearance of the central stellate fibers that form a central ring. (N) Outer doublet microtubules and central pair microtubules of the flagellar axoneme.

View larger version (28K): [in a new window]   Fig. 3. Compared are ratios of sizes of daughter cells following mitotic cell division in wild-type and mutant cell populations. Histograms display the number of cells with a given large-/small-cell area ratio. uni3-1 cells produce daughters of unequal sizes, suggesting a defect in the placement of the cleavage furrow. This defect is not suppressed by either of the tua2 alleles. tua2 strains show wild-type ratios, which indicates that the positioning of the cleavage furrow is not affected by the mutations.

View larger version (49K): [in a new window]   Fig. 4. Western blot of proteins from cell bodies of wild-type and tua2-2R1 cells. Increasing amounts of protein were loaded to detect the predicted 39 kD truncated protein of tua2-2R1 cells. Lane 1: 7 µg of protein from wild-type cells. Lanes 2-5: 7 µg, 15 µg, 30 µg and 50 µg of protein from tua2-2R1 cells. Truncated tubulin protein is not detectable in tua2-2R1 extracts, only wild-type tubulin.

View larger version (99K): [in a new window]   Fig. 5. The alterations D205N and A208T are close to (D205N) and within (A208T) helix 6 of -tubulin. The Chlamydomonas amino acid sequence of -tubulin (red) and ß-tubulin (white) was modeled onto the bovine tubulin crystal structure. (A) Amino acid changes observed in tua2 alleles (eight blue) and in one revertant allele (blue). (B) Part of the predicted structure, showing helix 6 with A208 and the nearby D205 with respect to the adjacent helix 5 (with P173). (C) The increased length of the side chain in the P173L alteration in revertant tua2-3R1 has probably major effects on the structure of -tubulin. P173 is conserved in most tubulins.

View larger version (20K): [in a new window]   Fig. 6. The role of -tubulin in early maturation. Maturation of centrioles occurs at G2/M. Old centrioles are shown in red and young centrioles are shown in blue. Old centrioles are morphologically identifiable by the presence of subdistal appendages. Maturation of basal bodies is hypothesized to occur at two time points in the cell cycle: early maturation occurs at the end of mitosis and a later maturation event takes palce at G2/M. Young basal bodies that show early maturation are shown in purple. Old basal bodies are shown in red. Young basal bodies that have not matured are shown in blue. The hypothesis suggests that early maturation fails in uni3-1 cells as indicated by the lack of purple basal bodies. tua2 Mutations restore early maturation as indicated by the appearance of purple basal bodies.

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