Characterization of Monoclonal Antibodies Against the Mitotic Apparatus Associated 51-kD Protein and the Effect of the Microinjection on Cell Division in Sand Dollar Eggs

Ohta, Kunihiro; Toriyama, Masaru; Hamaguchi, Y.; Maékawa, Shohei; Endo, Saburo; Sakai, Hikoichi

doi:10.1007/978-3-7091-9008-1_14

Cited by 18 publications

(11 citation statements)

References 19 publications

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“…lndirect evidence indicates that EF-lu associates with MTs of the mitotic apparatus and that it may play a role in nucleating MTs (Ohta et al, 1988a(Ohta et al, , 1990Toriyama et al, 1988;Kuriyama et al, 1990;Marchesi and Ngo, 1993). However, a direct interaction of EF-lu with MTs has not been demonstrated; it is unclear whether previous investigators have attained the high leve1 of purity for EF-la preparations that we show here (Figure 3, lane c).…”

Section: Nove1 Evidence For the Direct Binding Between An E F L A Hommentioning

confidence: 50%

“…For example, one Xenopus EF-1 a homolog forms a phylogenetic outgroup to all eukaryotic EF-las (including three others also found in Xenopus), and this homolog's biochemical properties are distinct from those of EF-1 u (Viel et al, 1991). In this same analysis, sea urchin EF-1 a homologs implicated in nucleating mitotic apparatus MTs (see above) also appear divergent from other eukaryotic EF-1as; the amino acid composition (Ohta et al, 1990) and sequence comparisons (Kuriyama et al, 1990) are consistent with this finding. In fact, Kuriyama et al (1990) discuss that their EF-la homolog may not be a typical EF-ia in sea urchin, but rather a gene family member serving the additional function in the mitotic apparatus.…”

Section: Methods E F L a Gene Families Solutionsmentioning

confidence: 67%

“…Binding between EF-1 as and Ca2+/CaM has not been described previously, and evidence for interactions between EF-1as and tubulin or MTs has been indirect (Ohta et al, 1988a(Ohta et al, , 1990Toriyama et al, 1988; Kuriyama et ai., 1990;Hasezawa and Nagata, 1993;Marchesi and Ngo, 1993). A direct binding between EF-la and MTs is demonstrated in this report.…”

Section: Dlscusslonmentioning

confidence: 91%

“…An antibody raised against a mitotic apparatus-associated EF-1 a homolog from sea urchins (Ohta et al, 1988a(Ohta et al, , 1988b(Ohta et al, , 1988c(Ohta et al, , 1990Toriyama et ai., 1988) reacts with a 49-kD tobacco homolog that appears to be localized to MT structures of cultured tobacco cells (Hasezawa and Nagata, 1993). Significantly, CaM has been immunocytochemically localized to bundled MTs in the cortical array (Fisher and Cyr, 1993) and mitotic array (Vantard et ai., 1985;Wick, 1985;Wicket ai., 1985;Fisher and Cyr, 1993) in plant cells.…”

Section: Physiological Significance Of Pp5o's Propertiesmentioning

confidence: 99%

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A calmodulin-sensitive interaction between microtubules and a higher plant homolog of elongation factor-1 alpha.

1994

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The microtubules (MTs) of higher plant cells a= organized into arrays with essential functions in plant cell growth and differentiation; however, molecular mechanisms underlying the organization and regulation of these arrays remain largely unknown. We have approached this problem using tubulin affinity chromatography to isolate carrot proteins that interact with MTs. From these proteins, a 50-kD polypeptide was selectively purified by exploiting its Ca2+-dependent binding to calmodulin (CaM). This polypeptide was identified as a homolog of elongation factor-lu (EF-1a)-a highly conserved and ubiqultous protein translation factor. The carrot EF-la homolog bundles MTs in vitro, and moreover, this bundling is modulated by the addition of Ca2+ and CaM together (Ca2+/CaM). A direct binding between the EF-la homolog and MTs was demonstrated, providing nove1 evidence for such an interaction. Based on these findings, and others discussed herein, we propose that an EF-la homolog mediates the lateral association of MTs in plant celk by a Ca2+/CaM-sensitive mechanism. INTRODUCTIONMicrotubules (MTs) participate in a number of essential processes in eukaryotes (MacRae, 1992b). In cells of higher plants, MTs are generally arranged into four structurally and functionally distinct, cell cycle-dependent MT arrays (Goddard et al., 1994;Lambert and Lloyd, 1994). During interphase, the MT array in a cell's cortex is involved in the extracellular deposition of cellulose microfibrils (Giddings and Staehelin, 1991). During G2 of the cell cycle, this array is succeeded by a narrower preprophase band involved in establishing the site of the incipient division plane (Palevitz, 1991; Wick, 1991). The preprophase band resolves into the mitotic spindle apparatus as M phase progresses Palevitz, 1993). The fourth MT array, the phragmoplast, appears as cytokinesis begins and is involved in depositing the new cell plate at the position previously marked by the preprophase band (Lloyd, The bases by which plant MTs are organized into higher order arrays and by which those arrays are regulated remain largely unknown (Lambert, 1993). MTs are hollow cylinders with 25-nm diameters, and tubulin is the principal protein subunit of MTs. MTs assembled from pure tubulin have a limited range of behavior-they assemble or disassemble. This limited range of behavior can only partly account for the diverse structures and functions of MT arrays.Nearly all species have multiple tubulin genes encoding various tubulin isotypes (for plant genes, see Goddard et al., 1994), and it has been proposed that the isotypes may provide a 1991).To whom correspondence should be addressed. means for serving different MT functions (Fulton and Simpson, 1976). However, nearly all existing evidence indicates that tubulin isotypes are functionally interchangeable (LudueAa, 1993). In general, tubulins are COnseNed even between kingdoms (Fosket and Morejohn, 1992; Burns and Surridge, 1994). In fact, animal brain tubulin that is microinjected into living plant cells incorporates into t...

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Section: Nove1 Evidence For the Direct Binding Between An E F L A Hommentioning

confidence: 50%

Section: Methods E F L a Gene Families Solutionsmentioning

confidence: 67%

Section: Dlscusslonmentioning

confidence: 91%

Section: Physiological Significance Of Pp5o's Propertiesmentioning

confidence: 99%

See 2 more Smart Citations

A calmodulin-sensitive interaction between microtubules and a higher plant homolog of elongation factor-1 alpha.

1994

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“…Addition of purified tubulin to the dialyzed fraction allowed nucleation of microtubules focused on the granular aggregates, eventually forming asters (18). Either monoclonal antibodies (13) or specific polyclonal antibodies against the 51 kDa protein (12,18) stained the centrosome in the sea urchin egg as well as the centers of reconstituted asters in vitro. The antibodies totally inhibited the formation of asters.…”

mentioning

confidence: 99%

GDP Inhibits the Activity of the Centrosome to Nucleate Microtubules.

Nakazawa

Motai

Sakai

1994

Cell Struct. Funct.

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ABSTRACT. Microtubule-nucleating ability of the centrosome in the isolated mitotic apparatus was investigated under the condition that the centrosomal fragment was preincubated with guanine nucleotides or its derivatives. GTPand non-hydrolyzable GTP analogues such as GTP-fS or GMP-PNPconsistently supported high activity of the centrosome to nucleate microtubules. On the other hand, GDPsubstantially suppressed the microtubule-nucleating activity of the centrosomes. Evidence has been presented that this is not simply due to the inhibitory effect of GDPon microtubule assembly. This result is consistent with the idea that GTPbinding proteins such as 51 kDa protein and f-tubulin localized to the pericentriolar materials in the centrosome are competent for the nucleation of microtubules when it is saturated with GTP.

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Formation of miniasters in the cytoplasm of hexyleneglycol‐treated sea urchin eggs

Endo

Toriyama

Ohta

et al. 1990

Cell Motil. Cytoskeleton

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Miniasters formed in mitotic sea urchin egg after treatment with 5% hexyleneglycol were investigated with the combined techniques of indirect immunofluorescence using anti-tubulin and anti-5 1 kD protein antibodies and electron microscopy.The formation of miniasters was dependent on the mitotic cycle. In the cytoplasm of eggs treated with hexyleneglycol at early prometaphase, a small number of microtubule fragments was observed, whereas in those treated at prometaphase, many miniasters and microtubule fragments were seen. When treated at metaphase, we found a great number of miniasters: 250-350 in one egg. In contrast, no miniasters were seen in eggs treated at anaphase, although many long microtubules that spread throughout the iy-toplasm were observed. In the egg> treated at telophase, we scarcely noticed microtubule structures in the cytoplasm. In the center of miniasters, granules were found, showing the same size and electron density as those of the microtubule-organizing granules (MTOGs). Furthermore, the 51 kD protein, a component of the centrosome and mitotic spindle, was observed to be localized in the region of miniasters.

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Characterization of Monoclonal Antibodies Against the Mitotic Apparatus Associated 51-kD Protein and the Effect of the Microinjection on Cell Division in Sand Dollar Eggs

Cited by 18 publications

References 19 publications

A calmodulin-sensitive interaction between microtubules and a higher plant homolog of elongation factor-1 alpha.

A calmodulin-sensitive interaction between microtubules and a higher plant homolog of elongation factor-1 alpha.

GDP Inhibits the Activity of the Centrosome to Nucleate Microtubules.

Formation of miniasters in the cytoplasm of hexyleneglycol‐treated sea urchin eggs

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