Acetylated α‐tubulin in spermatogenic cells of the crane fly <i>Nephrotoma suturalis</i>: Kinetochore microtubules are selectively acetylated

Wilson, Paula J.; Forer, Arthur

doi:10.1002/cm.970140210

Cited by 46 publications

(32 citation statements)

References 45 publications

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“…Recently we have revealed by immunofluorescence using monoclonal anti-tubulin antibodies that the tubulin isotypes of the spindle differ from those of the aster (13). Heterogeneity in spindle microtubules has been reported (5, 8,14,16, 17,21). These findings substantiate the idea that functionally differentiated microtubules are composed of different tubulin isotypes (1, 2, 4, 7).…”

Section: Japansupporting

confidence: 82%

Microinjection of the monoclonal anti-tubulin antibody YL1/2 inhibits cleavage of sand dollar eggs.

Oka

Arai

Hamaguchi

1990

Cell Struct. Funct.

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JapanKeywords: cleavage/microinjection/mitotic apparatus/monoclonal antibody/sand dollar/tubulin ABSTRACT. Two monoclonal antibodies against a-tubulin (YL1/2 and D2D6) were microinjected into the egg of the sand dollar Clypeasterjaponicus, and their effects on cleavage of the egg were investigated. They had already been shown by immunobloting to react specifically with egg tubulin and by immunofluorescence to stain the mitotic apparatus [OKA et a/. , (1990). CellModi CytoskeL 16: 239-250]. Injection of YL1/2 prevented chromosomemovementand cleavage, although the cleavage furrow developed in some cases. In all eggs injected at prometaphase, metaphase, or anaphase, the birefringence of the mitotic apparatus disappeared immediately after injection. Injection of D2D6had no significant effect on mitosis or cleavage of whole eggs injected after nuclear disappearance, although it prevented the disappearance of the nuclear envelope in 54% of the eggs injected before the disappearance. FITC-conjugated D2D6did not accumulate in the spindle when injected into the dividing sand dollar egg. These results indicate that YL1/2 disassembled microtubules, whereas D2D6did not bind to microtubules in the living cell.Microtubules play very important roles in mitotic mechanisms: Spindle and astral microtubules are involved in chromosome movement, spindle elongation, and the establishment of the cleavage plane. Recently we have revealed by immunofluorescence using monoclonal anti-tubulin antibodies that the tubulin isotypes of the spindle differ from those of the aster (13). Heterogeneity in spindle microtubules has been reported (5, 8,14,16, 17,21). These findings substantiate the idea that functionally differentiated microtubules are composed of different tubulin isotypes (1, 2, 4, 7). Because monoclonal anti-tubulin antibodies are specific to tubulin isotypes, they are useful not only for studying microtubules in the fixed cell by immunofluorescence, but also for investigating microtubule function in the living cell. However, it is not clear whether they react with tubulin in the living cell: some monoclonal antitubulin antibodies were found to bind to microtubules when injected into living cells (3,6, 19, 20), but others were not (3, 6). In this study, two monoclonal antibodies, YL1/2 and D2D6,were injected into the sand dollar egg to examine the roles of microtubules in mitosis in vivo. That both reacted specifically with egg tubulin was determined by analyzing their reactivity by immunobloting, and that both stained the mitotic apparatus was demonstrated byTo whomcorrespondence should be addressed. indirect immunofluorescence (13). We found that YL1/2 induced microtubule depolymerization and inhibited cell division, whereas D2D6did not cause any significant effects. MATERIALS AND METHODSMaterials. Eggs of the sand dollar Clypeaster japonicus were obtained by intracoelomic injection of 1 mMacetylcholine in sea water, rinsed three times with artificial sea water (Jamarin U, Jamarin Laboratory, Osaka), and stored at 15°C until use....

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Section: Japansupporting

confidence: 82%

Microinjection of the monoclonal anti-tubulin antibody YL1/2 inhibits cleavage of sand dollar eggs.

Oka

Arai

Hamaguchi

1990

Cell Struct. Funct.

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“…That is, not only are certain microtubules modified, but acetylated tubulin is often in discrete polymer domains tending to turn over more slowly than other regions [ 891. The results reflect tubulin acetyltransferase function and posit, reinforced by the coorelation between acetylation and increased stability [78,86,87,901, that binding of the enzyme to microtubules is regulated [89]. There may be a factor(s) limiting enzyme activity to certain cellular microtubules and to restricted regions of these structures.…”

Section: Non-tyrosinatable Tubulinmentioning

confidence: 98%

Tubulin Post‐Translational Modifications

MacRae

1997

European Journal of Biochemistry

279

191

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This review describes the enzymes responsible for the post-translational modifications of tubulin, including detyrosinationltyrosination, acetylationldeacetylation, phosphorylation, polyglutamylation, polyglycylation and the generation of non-tyrosinatable a-tubulin. Tubulin tyrosine-ligase, which reattaches tyrosine to detyrosinated tubulin, has been extensively characterized and its gene sequenced. Enzymes such as tubulin-specific carboxypeptidase and a-tubulin acetyltransferase, required, respectively, for detyrosination and acetylation of tubulin, have yet to be purified to homogeneity and examined in defined systems. This has produced some conflicting results, especially for the carboxypeptidase. The phosphorylation of tubulin by several different types of kinases has been studied in detail but drawing conclusions is difficult because many of these enzymes modify proteins other than their actual substrates, an especially pertinent consideration for in vitro experiments. Tubulin phosphorylation in cultured neuronal cells has proven to be the best model for evaluation of kinase effects on tubulinlmicrotubule function. There is little information on the enzymes required for polyglutamylation, polyglycylation, and production of non-tyrosinatable tubulin, but the available data permit interesting speculation of a mechanistic nature. Clearly, to achieve a full appreciation of tubulin post-translational changes the responsible enzymes must be characterized. Knowing when the enzymes are active in cells, if soluble or polymerized tubulin is the preferred substrate and the amino acid residues modified by each enzyme are all important. Moreover, acquisition of purified enzymes will lead to cloning and sequencing of their genes. With this information, one can manipulate cell genomes in order to either modify key enzymes or change their relative amounts, and perhaps reveal the physiological significance of tubulin post-translational modifications.Keywords: tubulin; microtubule ; post-translational modification ; tyrosination ; detyrosination ; non-tyrosinatable ; acetylation; phosphorylation; polyglutamylation ; polyglycylation.Of the three major isotypes of tubulin in eukaryotic cells, atubulin and P-tubulin form heterodimeric complexes which associate head-to-tail into protofilaments and then laterally to make up the wall of the cylindrical microtubule [I-31. The third isotype, y-tubulin, appears in the cytosol and in microtubule-organizing centers as ring-shaped structures where it nucleates microtubules [4-61. Both a-tubulin and P-tubulin, and perhaps y-tubulin [7], exist within most eukaryotic cells as families of closely related isoforms. Isotubulin composition varies spatially and temporally, determined by differential transcription of a-tubulin and P-tubulin genes within small families [2,8,91 and by post-translational modifications of tubulin [I, 8, lo].

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“…Acetylation seems to occur in tubulin after it has been incorporated into microtubules (Sasse andGull, 1988 andWilson andForer, 1989). It has been correlated with flagellar assembly (L'Hernault andRosenbaum, 1985 andHuitorel et al, 2002).…”

Section: Discussionmentioning

confidence: 99%

“…There are few studies about the tubulin composition of the insect sperm axoneme (Wilson and Forer, 1989, Fernandes and Báo, 1996, Fernandes and Báo, 2001, Mencarelli et al, 2000and Taddei et al, 2000. Here, we made a comparative analysis of the different tubulin distribution in apyrene and eupyrene sperm of Euptoieta hegesia butterflies and a verification of the possible presence of tubulin in lacinate appendages.…”

Section: Introductionmentioning

confidence: 99%

Immunocytochemical localization of tubulins in spermatids and spermatozoa of Euptoieta hegesia (Lepidoptera: Nymphalidae)

et al. 2005

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A comparative analysis of the distribution of tubulin types in apyrene and eupyrene sperm of Euptoieta hegesia butterflies was carried out, also verifying the presence of tubulin in lacinate appendages of the eupyrene sperm. Ultrathin sections of LR White embedded spermatids and spermatozoa were labeled for alpha, beta, gamma, alpha-acetylated and alpha-tyrosinated tubulins. Apyrene and eupyrene spermatids show the same antibody recognition pattern for tubulins. All tubulin types were detected in axonemal microtubules. Alpha and gamma tubulins were also detected on the cytoplasmic microtubules. However, for beta and tyrosinated tubulins only scattered labeling was detected on cytoplasmic microtubules and acetylated tubulin was not detected. In apyrene and eupyrene spermatozoa only the axoneme labeling was analyzed since cytoplasmic microtubules no longer exist in these cells. Alpha, beta and tyrosinated tubulins were easily detected on the apyrene and eupyrene axoneme; gamma tubulin was strongly marked on eupyrene axonemes but was scattered on the apyrene ones. Acetylated tubulin appeared with scattered labeling on the axoneme of both sperm types. Our results demonstrate significant differences in tubulin distribution in apyrene and eupyrene axonemal and cytoplasmic microtubules. Extracellular structures, especially the lacinate appendages, were not labeled by antibodies for any tubulin.

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Acetylated α‐tubulin in spermatogenic cells of the crane fly Nephrotoma suturalis: Kinetochore microtubules are selectively acetylated

Cited by 46 publications

References 45 publications

Microinjection of the monoclonal anti-tubulin antibody YL1/2 inhibits cleavage of sand dollar eggs.

Microinjection of the monoclonal anti-tubulin antibody YL1/2 inhibits cleavage of sand dollar eggs.

Tubulin Post‐Translational Modifications

Immunocytochemical localization of tubulins in spermatids and spermatozoa of Euptoieta hegesia (Lepidoptera: Nymphalidae)

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