Tubulin antibody inhibits in vitro polymerization independently of microtubule‐associated proteins

Morgan, Janet L.; Holladay, Carter R.; Spooner, Brian S.

doi:10.1016/0014-5793(78)80823-0

Cited by 8 publications

(2 citation statements)

References 22 publications

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“…An anti-tubulin antibody has been reported to diminish the assembly of, and disrupt, cellular microtubules (25), but these observations are difficult to interpret because the tubulin epitope recognized by this particular anti-tubulin antibody is not known. Another polyclonal anti-tubulin antibody, when present in a stoichiometry of 3:1 with respect to tubulin, reduced assembly by 40% (26). In contrast, f3Ab2 decreased assembly by 50% at a concentration of only 7 gg/ml (stoichiometry of 1:8.5 with respect to MAPs) and by 95% at a concentration of 50 ,tg/ml.…”

Section: Resultsmentioning

confidence: 99%

Anti-idiotypic antibodies that react with microtubule-associated proteins are present in the sera of rabbits immunized with synthetic peptides from tubulin's regulatory domain.

Rivas

Vera

Maccioni

1988

Proc. Natl. Acad. Sci. U.S.A.

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A fundamental question in microtubule research is how the interactions of tubulin subunits with microtubule-associated proteins (MAPs) are controlled. The answer should provide insight into the regulation of the cellular processes in which microtubules are implicated. Previous work demonstrated the interaction ofMAPs with a 4-kDa C-terminal domain of tubulin a and 13 subunits. Synthetic peptides from the variable region of the 4-kDa C-terminal moiety of tubulin subunits, a-(430-441) and 13-(422-434), bind to MAP-2 and to the MAP tau, and a preferential interaction of the 13 peptide is observed. To define the regulatory significance of the substructure of the C-terminal tubulin domain, we produced rabbit antisera against these MAP-interacting peptides. We found that these antisera contained not only antibodies to the original synthetic peptides but also antibodies to MAPs. Here, we report that these antibodies, which react with MAP-1, MAP-2, and tau, appear to be a population of anti-idiotypic antibodies directed to the anti-peptide antibodies. They can inhibit MAPinduced tubulin assembly into microtubules in vitro, and the addition of MAPs overcomes the inhibition. The recognition by these anti-idiotypic antibodies of the tubulin-binding domain on MAPs provides unequivocal evidence that the tubulin region defined by the synthetic peptides is directly involved in the interaction with MAPs.The functional versatility of microtubules, in addition to their cellular dynamics, indicates the existence of fine regulatory signals modulating their assembly and intracellular orientation (1). The C-terminal moiety of tubulin plays a role in modulating the interactions responsible for tubulin selfassociation into microtubules (2, 3). Limited proteolysis of tubulin with subtilisin (between Glu-417 and Phe-418 in the a subunit; between Glu-407 and Phe-408 in the (3 subunit) produces the cleaved heterodimer and two 4-kDa peptide fragments containing the C-terminal domains of the a and (3 subunits, rendering tubulin assembly independent of microtubule-associated proteins (MAPs) (4). This 4-kDa domain appears to contain an essential site for MAP interactions (5, 6). Studies with exopeptidases that remove the last eight amino acid residues from tubulin subunits (7) and with antibodies that recognize the C-terminal tyrosine ofa-tubulin (8) indicate that the acidic, -1-kDa C-terminal subdomain of tubulin subunits is not directly involved in the binding of MAPs. Two synthetic peptides from the variable subdomain within the 4-kDa moiety of tubulin subunits were used in our studies, a-(430-441) (Lys-Asp-Tyr-Glu-Glu-Val-Gly-ValAsp-Ser-Val-Glu) and (3-(422-434) (Tyr-Gln-Gln-Tyr-GlnAsp-Ala-Thr-Ala-Asp-Glu-Gln-Gly). peptide exhibited a relatively strong interaction with two purified MAPs, MAP-2 and tau, whereas a-(430-441) interacted with these proteins with a rather low affinity, suggesting a differential capacity of the two tubulin subunits to bind selectively to MAPs (7). We report here the presence of MAP-reacting anti-idiotypi...

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Section: Resultsmentioning

confidence: 99%

Anti-idiotypic antibodies that react with microtubule-associated proteins are present in the sera of rabbits immunized with synthetic peptides from tubulin's regulatory domain.

Rivas

Vera

Maccioni

1988

Proc. Natl. Acad. Sci. U.S.A.

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“…Antibodies reported to interact with tubulin do not appear to be effective in blocking microtubule formation in vitro (13,21) and, accordingly, would not affect cellular microtubules. A polyclonal anti-tubulin antibody used with a stoichiometry of3:1 with respect to tubulin reduced assembly by 40% (22). The anti-peptide antibodies aAbl and /3Abl reported here blocked the assembly and interact with a functionally significant epitope on tubulin, which is the site for the binding of MAPs.…”

Section: Discussionmentioning

confidence: 99%

Antibodies to synthetic peptides from the tubulin regulatory domain interact with tubulin and microtubules.

Vera

Rivas

Maccioni

1988

Proc. Natl. Acad. Sci. U.S.A.

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The carboxyl-terminal region of tubulin a and ( subunits plays a major role in regulating its assembly into microtubules and constitutes an essential domain for the selective interaction of microtubule-associated proteins (MAPs). With the goal of understanding the structural basis of the regulatory function of the carboxyl-terminal domains of tubulin subunits, we have produced rabbit antisera against two MAP-interacting peptides Lys-Asp-Tyr-Glu-Glu-Val-Gly-ValAsp-Ser-Val-Glu of a-tubulin and Tyr-Gln-Gln-Tyr-Gln-AspAla-Thr-Ala-Asp-Glu-Gln-Gly of fl subunit. The affinitypurified a and (3 anti-peptide antibodies interacted specifically with tubulin and with the respective peptide antigens but did not interact with MAPs. Substoichiometric amounts of both antibodies showed the capacity to inhibit in vitro MAP-induced tubulin assembly and to promote a fast depolymerization of preassembled microtubules. Taxol-promoted assembly of pure tubulin was not inhibited by the antibodies. In the presence of MAP-2 and taxol, the antibodies decreased the MAP-2 content of taxol-promoted microtubules. The interaction with microtubules was corroborated by immunofluorescence experiments in HeLa and NE-18 lung carcinoma cells. The epitopes recognized by the a and .8 anti-peptide antibodies appear to be located in the outer surface of the microtubular structure.A general overview of the essential features of tubulin structure and functions has emerged over the past two decades (1, 2). One question of current interest is the elucidation of the regulatory pathways for the assembly of tubulin into microtubules. Understanding of the control of microtubule assembly should provide clues about the functional versatility of microtubules. We have demonstrated that the C-terminal moieties of tubulin subunits constitute essential sites for the interaction of microtubule-associated proteins (MAPs) (3). Limited proteolysis of tubulin with subtilisin produces the cleaved tubulin dimer and two 4-kDa fragments containing the C-terminal domain of a and ,8 subunits. Removal of the 4-kDa fragment facilitates tubulin self-association and makes the assembly independent of the MAPs (4, 5). This 4-kDa tubulin domain appears to hinder the interactions responsible for its self-assembly into microtubules (4-6) and plays a regulatory role in microtubule formation (7). Exopeptidases that remove the last eight amino acid residues from the C-terminal moiety of both tubulin subunits do not relieve the modulatory effect of the 4-kDa C-terminal region and the MAP dependence of the assembly (8), demonstrating that these residues are not directly implicated in the interaction of tubulin with MAPs. This is further supported by observations indicating that antibodies that recognize the C-terminal tyrosine ofa tubulin (9) do not affect microtubule assembly (10).Peptides corresponding to short discrete sequences of the C-terminal domain, a(430-441) and 1(422-434) bind to MAP-2 and tau, but a preferential interaction of the 1 peptide with these MAPs components was obs...

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Distribution of tubulin and actin in neurites and growth cones of differentiating nerve cells

Spooner¹,

Holladay²

1981

Cell Motility

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Embryonic chick nerve cells, from dissociated dorsal root ganglia, were cultured on polylysine substrata and examined for tubulin and actin distribution by indirect immunofluorescence. Antibodies generated against chick brain tubulin produced specific fluorescence in growth cones, neurites, and cell bodies without revealing distribution differences or substructure in the nerve cells. However, at reduced antitubulin concentrations, differences were resolved. Tubulin fluorescence remained uniform and intense in neurites and cell bodies, but exhibited reduced intensity and patterning in growth cones. Nonneuronal cells in the cultures served as controls for typical cytoplasmic tubulin fluorescence distribution. Straining controls demonstrated that fluorescence resulted from tubulin-antitubulin binding. Analogous studies, using antibodies generated against chick brain actin, demonstrated distribution differences at reduced antiactin concentrations, including "hot spots" of intense fluorescence in growth cones and a paucity of fluorescence in neurites.

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Tubulin antibody inhibits in vitro polymerization independently of microtubule‐associated proteins

Cited by 8 publications

References 22 publications

Anti-idiotypic antibodies that react with microtubule-associated proteins are present in the sera of rabbits immunized with synthetic peptides from tubulin's regulatory domain.

Anti-idiotypic antibodies that react with microtubule-associated proteins are present in the sera of rabbits immunized with synthetic peptides from tubulin's regulatory domain.

Antibodies to synthetic peptides from the tubulin regulatory domain interact with tubulin and microtubules.

Distribution of tubulin and actin in neurites and growth cones of differentiating nerve cells

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