The Dynamics of Filamentous Structures in the Apical Band, Oral Crescent, Fission Line and the Postoral Meridional Filament in Tetrahymena thermophila Revealed by Monoclonal Antibody 12G9

Jerka‐Dziadosz, Maria; Strzyżewska-Jówko, Izabela; Wojsa-Lugowska, Urszula; Krawczynska, W.; Krzywicka, Anna

doi:10.1078/1434-4610-00043

Cited by 22 publications

(24 citation statements)

References 46 publications

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“…First, a detailed analysis of the placement of the CVP sets revealed that (i) the midpoint of the two CVP sets was halfway between the two sets of oral structures when both were present and (ii) the positions of these CVP sets were the same irrespective of whether a sOA was present or not (42). More direct evidence followed, with the discovery of a longitudinally oriented "postoral meridional filament" (pmf) in cells stained by the highly informative monoclonal antibody 12G9 (78). In wild-type cells, the pmf marked the oral meridian and was not found elsewhere.…”

Section: The Mutationsmentioning

confidence: 95%

What Do Genic Mutations Tell Us about the Structural Patterning of a Complex Single-Celled Organism?

Frankel

2008

Eukaryot Cell

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PROLOGUE: FROM THE CYTOPLASM TO THE NUCLEUSStructural inheritance in the ciliate cortex. Ciliates make up a distinctive group of unicellular organisms characterized by dualism of germ line and somatic nuclei, sex by reciprocal exchange of gametic nuclei with subsequent replacement of the somatic nucleus, and an extraordinarily complex organization of the cell surface layer. This organization is dominated by cytoskeletal structures, including rows of basal bodies, cilia, and accessory fibrillar structures, and is perpetuated by longitudinal extension of these structural ensembles during clonal growth.This mode of perpetuation provides an ideal opportunity for the demonstration and analysis of cellular heredity at a nongenic level. This opportunity was seized by three gifted biologists, the comparative zoologist Emmanuel Fauré-Fremiet, the experimental embryologist Vance Tartar, and the geneticist Tracy Sonneborn, who together led the way in establishing the principle that structural variations of nongenic origin in the ciliate cortex can indeed be faithfully transmitted to progeny over numerous cell generations.Although this review is primarily devoted to the contribution of genic mutations to the understanding of the ciliate cortex, the interpretation of some of the more interesting of these mutations becomes more meaningful against a background of the three major arenas of nongenic structural inheritance in the ciliate cortex, all of which were solidly established before the genetic approach was initiated.The best-known form of structural inheritance is the organization of the longitudinal ciliary rows that cover the surfaces of most ciliates. This was first demonstrated by Beisson and Sonneborn (12), who showed that an inversion (180°rotation) of one or more ciliary rows of Paramecium tetraurelia (then Paramecium aurelia, syngen 4) (Fig. 1A) could be nongenically inherited; Sonneborn gave the name "cytotaxis" to "this ordering and arranging of new structures under the influence of pre-existing cell structure" (137). This demonstration was later repeated for Tetrahymena thermophila (then Tetrahymena pyriformis, syngen 1) (118) and for other ciliates (62,63,71). It was extended by Nanney's observation that the preexisting number of ciliary rows in T. thermophila tended to be conserved (103, 104), presumably due to the same structural constraints that conserve the geometrical organization of these rows.A second form of structural inheritance is demonstrated by the propagation of the number of complete sets of cortical structures. This was investigated in detail by 32), who noted that ciliates that became fused side by side, as a consequence of blockage of division followed by anterior sliding of the presumptive posterior daughter cell, could propagate their duality. Later, Vance Tartar (147) demonstrated that microsurgically constructed Siamese-twin doublets in the large ciliate Stentor coeruleus could perpetuate their doublet condition. In both cases, the way in which the doublets were created virtually rules o...

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Section: The Mutationsmentioning

confidence: 95%

What Do Genic Mutations Tell Us about the Structural Patterning of a Complex Single-Celled Organism?

Frankel

2008

Eukaryot Cell

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“…Cells were viewed in a Leica TCS SP confocal microscope. For transmission electron microscopy, cells were prepared as described previously (30). To assay phagocytosis, cells were fed with 0.2% India ink in SPP for 2 to 10 min, fixed by adding an equal volume of 2% paraformaldehyde, and scored for the presence of labeled food vacuoles.…”

Section: Methodsmentioning

confidence: 99%

Glutamylation on α-Tubulin Is Not Essential but Affects the Assembly and Functions of a Subset of Microtubules in Tetrahymena thermophila

et al. 2008

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Tubulin undergoes glutamylation, a conserved posttranslational modification of poorly understood function. We show here that in the ciliate Tetrahymena, most of the microtubule arrays contain glutamylated tubulin. However, the length of the polyglutamyl side chain is spatially regulated, with the longest side chains present on ciliary and basal body microtubules. We focused our efforts on the function of glutamylation on the ␣-tubulin subunit. By site-directed mutagenesis, we show that all six glutamates of the C-terminal tail domain of ␣-tubulin that provide potential sites for glutamylation are not essential but are needed for normal rates of cell multiplication and cilium-based functions (phagocytosis and cell motility). By comparative phylogeny and biochemical assays, we identify two conserved tubulin tyrosine ligase (TTL) domain proteins, Ttll1p and Ttll9p, as ␣-tubulin-preferring glutamyl ligase enzymes. In an in vitro microtubule glutamylation assay, Ttll1p showed a chain-initiating activity while Ttll9p had primarily a chain-elongating activity. GFP-Ttll1p localized mainly to basal bodies, while GFP-Ttll9p localized to cilia. Disruption of the TTLL1 and TTLL9 genes decreased the rates of cell multiplication and phagocytosis. Cells lacking both genes had fewer cortical microtubules and showed defects in the maturation of basal bodies. We conclude that glutamylation on ␣-tubulin is not essential but is required for efficiency of assembly and function of a subset of microtubule-based organelles. Furthermore, the spatial restriction of modifying enzymes appears to be a major mechanism that drives differential glutamylation at the subcellular level.The principal components of microtubules, heterodimers of ␣-and ␤-tubulin, are known to undergo several types of conserved posttranslational modifications (PTMs), including acetylation, detyrosination, phosphorylation, palmitoylation, glutamylation, and glycylation (61). Some of these PTMs strongly influence interactions between microtubules and microtubuleassociated proteins, including motors and plus-end tracking proteins (27,32,34,43,49). Specific PTMs are enriched on microtubules in restricted subcellular areas, suggesting that these mechanisms act as marks that locally adapt the microtubule polymer for specific functions (reviewed in reference 58). How the spatially restricted modified microtubules are generated within the cell is not well understood.Glutamylated microtubules are generated by the sequential addition of multiple glutamates to the ␥-carboxyl group of specific glutamic acids of the primary sequence of the C-terminal tail (CTT) domain of ␣-and ␤-tubulin (14). This reversible PTM creates glutamyl side chains of variable length and is enriched on microtubules in cilia, centrioles/basal bodies, the mitotic spindle, microtubules of nerve projections, and pellicular arrays in protists (2,5,6,31,35,45,63). Recently, we have identified tubulin glutamylases as proteins with a tubulin tyrosine ligase homology (28). The identification of forward enzymes for...

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“…The lengths of the cilia were measured by using ImageJ 1.37. For transmission electron microscopy (TEM), the cells were prepared as described previously (24).…”

Section: Methodsmentioning

confidence: 99%

Hyperglutamylation of Tubulin Can either Stabilize or Destabilize Microtubules in the Same Cell

et al. 2010

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In most eukaryotic cells, tubulin is subjected to posttranslational glutamylation, a conserved modification of unclear function. The glutamyl side chains form as branches of the primary sequence glutamic acids in two biochemically distinct steps: initiation and elongation. The length of the glutamyl side chain is spatially controlled and microtubule type specific. Here, we probe the significance of the glutamyl side chain length regulation in vivo by overexpressing a potent side chain elongase enzyme, Ttll6Ap, in Tetrahymena. Overexpression of Ttll6Ap caused hyperelongation of glutamyl side chains on the tubulin of axonemal, cortical, and cytoplasmic microtubules. Strikingly, in the same cell, hyperelongation of glutamyl side chains stabilized cytoplasmic microtubules and destabilized axonemal microtubules. Our observations suggest that the cellular outcomes of glutamylation are mediated by spatially restricted tubulin interactors of diverse nature.Microtubules are dynamic elements of the cytoskeleton that are assembled from heterodimers of ␣-and ␤-tubulin. Once assembled, tubulin subunits undergo several conserved posttranslational modifications (PTMs) that diversify the external and luminal surfaces of microtubules (51). Two tubulin PTMs, glycylation and glutamylation, collectively known as polymodifications, form peptide side chains that are attached to the ␥-carboxyl groups of glutamic acids in the primary sequence of the C-terminal tails (CTTs) of ␣-and ␤-tubulin (14, 36). Glutamylated microtubules are abundant in projections of neurons (14), axonemes (8,15,17), and centrioles/basal bodies (5, 31) and are detectable in the mitotic spindle and on a subset of cytoplasmic network microtubules (1, 5). The modifying enzymes, tubulin glutamic acid ligases (tubulin E-ligases), belong to the family of proteins related to the tubulin tyrosine ligase (TTL), known as TTL-like (TTLL) proteins (22,50,53). Tubulin glutamylation appears to be important in vivo. A knockdown of the TTLL7 E-ligase mRNA in cultured neurons inhibits the outgrowth of neurites (20). A loss of PGs1, a protein associated with TTLL1 E-ligase (22, 37), disorganizes sperm axonemes in the mouse (11), and a morpholino knockdown of TTLL6 E-ligase expression in zebrafish inhibits the assembly of olfactory cilia (33). The biochemical consequences of tubulin glutamylation in vivo are poorly understood, but the emerging model is that this PTM regulates interactions between microtubules and microtubuleassociated proteins (MAPs) (6,7,19,27).The ciliate Tetrahymena thermophila has 18 types of diverse microtubules that are all assembled in a single cell. Although most, if not all, of these microtubules are glutamylated, the length of glutamyl side chains is spatially regulated (8, 53). Minimal side chains composed of a single glutamic acid (monoglutamylation) are present on the cytoplasmic and nuclear microtubules, whereas elongated side chains are present on the basal bodies and axonemes (53). In Tetrahymena, Ttll6Ap is a ␤-tubulin-preferring E-ligase (22), wi...

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The Dynamics of Filamentous Structures in the Apical Band, Oral Crescent, Fission Line and the Postoral Meridional Filament in Tetrahymena thermophila Revealed by Monoclonal Antibody 12G9

Cited by 22 publications

References 46 publications

What Do Genic Mutations Tell Us about the Structural Patterning of a Complex Single-Celled Organism?

What Do Genic Mutations Tell Us about the Structural Patterning of a Complex Single-Celled Organism?

Glutamylation on α-Tubulin Is Not Essential but Affects the Assembly and Functions of a Subset of Microtubules in Tetrahymena thermophila

Hyperglutamylation of Tubulin Can either Stabilize or Destabilize Microtubules in the Same Cell

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