Cell Context-specific Effects of the β-Tubulin Glycylation Domain on Assembly and Size of Microtubular Organelles

Tubulin glycylation is a posttranslational modification found in cells with cilia or flagella. The ciliate Tetrahymena has glycylation on ciliary and cortical microtubules. We showed previously that mutating three glycylation sites on β-tubulin produces immotile 9 + 0 axonemes and inhibits cytokinesis. Here, we use an inducible glycylation domain mutation and epitope tagging to evaluate the potential of glycylation-deficient tubulin for assembly and maintenance of microtubular systems. In axonemes, the major defects, including lack of the central pair, occurred during assembly, and newly made cilia were abnormally short. The glycylation domain also was required for maintenance of the length of already assembled cilia. In contrast to the aberrant assembly of cilia, several types of cortical organelles showed an abnormally high number of microtubules in the same mutant cells. Thus, the consequences of deficiency in tubulin glycylation are organelle type specific and lead to either insufficient assembly (cilia) or excessive assembly (basal bodies and cortical microtubules). We suggest that the diverse functions of the β-tubulin glycylation domain are executed by spatially restricted microtubule-associated proteins.

Post-translational Tubulin Modifications in the Ciliate Tetrahymena thermophila: Role in Assembly and Maintenance of Complex Microtubular Organelles

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

Tubulin post-translational modifications and the construction of microtubular organelles in Trypanosoma brucei

We have used specific monoclonal antibodies to facilitate a study of acetylated and tyrosinated alpha-tubulin in the microtubule (MT) arrays in the Trypanosoma brucei cell. Acetylated alpha-tubulin is not solely located in the stable microtubular arrays but is present even in the ephemeral microtubules of the mitotic spindle. Moreover, there is a uniform distribution of this isoform in all arrays. Studies of flagella complexes show that acetylation is concomitant with assembly of MTs. There is no subsequent major modulation in the content of acetylated alpha-tubulin in MTs. Conversely, polymerizing flagellar MTs have a high tyrosinated alpha-tubulin content, which is subsequently reduced to a basal level at a discrete point in the cell cycle. The MTs of the intranuclear mitotic spindle appear never to contain tyrosinated alpha-tubulin, suggesting that they are actually constructed of detyrosinated alpha-tubulin or that detyrosination is extremely rapid at this time in the cell cycle. T. brucei therefore, represents a cell type with extremely active mechanisms for the post-translational modification of alpha-tubulin. Our analyses of the timing of acquisition and modulation in relation to MT construction in T. brucei, suggest that acetylation and detyrosination of alpha-tubulin are two independently regulated post-translational modifications, that are not uniquely associated with particular subsets of MTs of defined lability, position or function. Post-assembly detyrosination of alpha-tubulin may provide a mechanism whereby the cell could discriminate between new and old MTs, during construction of the cytoskeleton through the cell cycle. However, we also suggest that continuation of detyrosination, allows the cell, at cell division, to partition into daughter cells two equivalent sets of cytoskeletal MTs.

A gene encoding the major beta tubulin of the mitotic spindle in Physarum polycephalum plasmodia.

The multinucleate plasmodium of Physarum polycephalum is unusual among eucaryotic cells in that it uses tubulins only in mitotic-spindle microtubules; cytoskeletal, flagellar, and centriolar microtubules are absent in this cell type. We have identified a beta-tubulin cDNA clone, beta 105, which is shown to correspond to the transcript of the betC beta-tubulin locus and to encode beta 2 tubulin, the beta tubulin expressed specifically in the plasmodium and used exclusively in the mitotic spindle. Physarum amoebae utilize tubulins in the cytoskeleton, centrioles, and flagella, in addition to the mitotic spindle. Sequence analysis shows that beta 2 tubulin is only 83% identical to the two beta tubulins expressed in amoebae. This compares with 70 to 83% identity between Physarum beta 2 tubulin and the beta tubulins of yeasts, fungi, alga, trypanosome, fruit fly, chicken, and mouse. On the other hand, Physarum beta 2 tubulin is no more similar to, for example, Aspergillus beta tubulins than it is to those of Drosophila melanogaster or mammals. Several eucaryotes express at least one widely diverged beta tubulin as well as one or more beta tubulins that conform more closely to a consensus beta-tubulin sequence. We suggest that beta-tubulins diverge more when their expression pattern is restricted, especially when this restriction results in their use in fewer functions. This divergence among beta tubulins could have resulted through neutral drift. For example, exclusive use of Physarum beta 2 tubulin in the spindle may have allowed more amino acid substitutions than would be functionally tolerable in the beta tubulins that are utilized in multiple microtubular organelles. Alternatively, restricted use of beta tubulins may allow positive selection to operate more freely to refine beta-tubulin function.

A gene encoding the major beta tubulin of the mitotic spindle in Physarum polycephalum plasmodia

The multinucleate plasmodium of Physarum polycephalum is unusual among eucaryotic cells in that it uses tubulins only in mitotic-spindle microtubules; cytoskeletal, flagellar, and centriolar microtubules are absent in this cell type. We have identified a beta-tubulin cDNA clone, beta 105, which is shown to correspond to the transcript of the betC beta-tubulin locus and to encode beta 2 tubulin, the beta tubulin expressed specifically in the plasmodium and used exclusively in the mitotic spindle. Physarum amoebae utilize tubulins in the cytoskeleton, centrioles, and flagella, in addition to the mitotic spindle. Sequence analysis shows that beta 2 tubulin is only 83% identical to the two beta tubulins expressed in amoebae. This compares with 70 to 83% identity between Physarum beta 2 tubulin and the beta tubulins of yeasts, fungi, alga, trypanosome, fruit fly, chicken, and mouse. On the other hand, Physarum beta 2 tubulin is no more similar to, for example, Aspergillus beta tubulins than it is to those of Drosophila melanogaster or mammals. Several eucaryotes express at least one widely diverged beta tubulin as well as one or more beta tubulins that conform more closely to a consensus beta-tubulin sequence. We suggest that beta-tubulins diverge more when their expression pattern is restricted, especially when this restriction results in their use in fewer functions. This divergence among beta tubulins could have resulted through neutral drift. For example, exclusive use of Physarum beta 2 tubulin in the spindle may have allowed more amino acid substitutions than would be functionally tolerable in the beta tubulins that are utilized in multiple microtubular organelles. Alternatively, restricted use of beta tubulins may allow positive selection to operate more freely to refine beta-tubulin function.

Temperature-induced modifications in size and pattern of microtubular organelles in a ciliate, Dileptus

Supernumerary microtubules were found in the so-called sensory cilia, in addition to a sensory axoneme. The supernumerary microtubules were not structurally connected to a basal body, but were probably anchored to clusters of dense material inside the ciliary shaft. The frequency of appearance of the supernumerary microtubules was found to be temperature-dependent: the higher the temperature during formation of sensory cilia, the greater was the number of supernumerary microtubules in cross-sections, and the more cross-sections contained them. The possibility is discussed that the formation of the supernumerary microtubules is not due to formation of new nucleating sites inside the cilium. Instead, the microtubules may be remnants of a previously existing axoneme, separated from the basal body during the formation of a sensory cilium. Some of the microtubules of the released axoneme may persist as the supernumerary microtubules, if capped with dense material or some other structure within the ciliary shaft.

Acetylated alpha-tubulin in Physarum: immunological characterization of the isotype and its usage in particular microtubular organelles.

We have used monoclonal antibodies specific for acetylated and unacetylated alpha-tubulin to characterize the acetylated alpha-tubulin isotype of Physarum polycephalum, its expression in the life cycle, and its localization in particular microtubular organelles. We have used the monoclonal antibody 6-11B-1 (Piperno, G., and M. T. Fuller, 1985, J. Cell Biol., 101:2085-2094) as the probe for acetylated alpha-tubulin and have provided a biochemical characterization of the monoclonal antibody KMP-1 as a probe for unacetylated tubulin in Physarum. Concomitant use of these two probes has allowed us to characterize the acetylated alpha-tubulin of Physarum as the alpha 3 isotype. We have detected this acetylated alpha 3 tubulin isotype in both the flagellate and in the myxameba, but not in the plasmodium. In the flagellate, acetylated tubulin is present in both the flagellar axonemes and in an extensive array of cytoplasmic microtubules. The extensive arrangement of acetylated cytoplasmic microtubules and the flagellar axonemes are elaborated during the myxameba-flagellate transformation. In the myxameba, acetylated tubulin is not present in the cytoplasmic microtubules nor in the mitotic spindle microtubules, but is associated with the two centrioles of this cell. These findings, taken together with the apparent absence of acetylated alpha-tubulin in the ephemeral microtubules of the plasmodium suggest a natural correspondence between the presence of acetylated alpha-tubulin and microtubule organelles that are intrinsically stable or cross-linked.

Modifications of size and pattern of microtubular organelles in overfed cells of a ciliate Dileptus

The size of mouthparts and their constituent organelles was studied in cells of Dileptus anser, enlarged by overfeeding. The oral structures of large cells were either of normal dimensions and appearance, or they were enlarged and deformed in shape. The increase in size of mouthparts was accompanied by an increase in number of their microtubular organelles. There was, however, no net increase in size of the organelles, as defined by length and number of micro tubules they contained. It is concluded that there exists an upward regulation in the size of the mouthparts as a whole, whilst the size of their constituent organelles is probably not so regulated. It is supposed that the patterning of normal-size organelles into large oral structure may lead to the observed deformations in the shape of the mouthparts.