Tubulin evolution: Ciliate-specific epitopes are conserved in the ciliary tubulin of metazoa

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

et al. 1990

Incorporation of Paramecium axonemal tubulin into lysed endosperm cells of the higher plant Haemanthus enabled us to identify sites of microtubule assembly. This exogenous Paramecium tubulin could be traced by specific antibodies that do not stain endogenous plant microtubules. Intracellular copolymerization of protozoan and higher plant tubulins gave rise to hybrid polymers that were visualized by immunofluorescence and by immunoelectron microscopy. The addition of exogenous tubulin revealed many free ends of endogenous microtubules that were competent to assemble ciliate tubulin. The functional roles of the nuclear surface and the equatorial region of the phragmoplast as plant microtubuleorganizing centers, which were revealed by the intense incorporation of exogenous tubulin, are-discussed. These data shed light on the present debate on higher plant microtubule organizing centers.Temporal and spatial regulation of microtubule (MT) assembly in acentriolar higher plant cells remains poorly understood, because of the lack of data from a functional assay of nucleation capacity that would unambiguously identify plant microtubule-organizing centers (MTOCs). Indirect information has been obtained through immunocytochemical and ultrastructural approaches. Human autoantibodies that stain pericentriolar nucleating material in animal cells (1, 2) decorate the periphery of the nucleus and the spindle poles of higher plant cells (3, 4). However, the significance of this cross-reactivity has been questioned (5). It has also been suggested that sites of MT anchoring, detected by antitubulin labeling (6-8), may correspond to nucleation centers since electron dense material resembling pericentriolar material is usually associated with them (9-12). Our specific aims were to obtain functional evidence that the nuclear envelope in higher plants acts as a MT nucleation center and to study the origin of phragmoplast MT assembly during anaphase-telophase.For this purpose we used a method that is derived from the analysis of MT dynamics and nucleation sites in animal cells in which a "reporter" tubulin is microinjected. The reporter tubulin can either be labeled tubulin (13,14) or tubulin from a distant organism for which a species-specific anti-tubulin antibody is available (15,16). In this context, we have developed an in vitro system of lysed Haemanthus endosperm cells (17) in which unmodified Paramecium axonemal (PA) tubulin is incorporated and then selectively labeled by its homologous antibody (15,18,19). Haemanthus endosperm cells are remarkably suited for investigating plant mitosis and cytoskeleton (6)(7)(8)20), and their lack of cell wall makes them ideal for studies involving permeabilization.In this report, we show (i) the intracellular copolymerization of ciliate and higher plant tubulins leading to hybrid MTs, (ii) the assembly of exogenous tubulin around the plant nucleus in interphase and telophase, and (iii) an intense incorporation of PA tubulin at the cell equator in the early stage of phragmoplast develop...

“…By immunoblot analysis, we demonstrated that antibodies directed against PA tubulin (18,19) did not react with higher plant tubulin (Fig. 1A).…”

Section: Resultsmentioning

confidence: 96%

“…Tubulin concentrations were determined according to Bradford (22), and purity was assessed by SDS/PAGE (23). Anti-PA-tubulin-specific antibodies (18,19) were affinity-purified (15).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Incorporation of Paramecium axonemal tubulin into higher plant cells reveals functional sites of microtubule assembly.

Vantard

Levilliers

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

et al. 1990

“…Paramecium axonemal tubulin and the specific, affinity-purified antibody against this tuhniin were prepared as previously described (Adoutte et at., 1985;Cohen et at., 1982;Geuens et at., 1989).…”

Section: Tubulin and Antitubulin Preparationmentioning

confidence: 99%

Regulation of microtubule dynamics and nucleation during polarization in MDCK II cells.

Bré

Pepperkok

The Journal of Cell Biology

et al. 1990

Abstract. MDCK cells form a polarized epithelium when they reach confluence in tissue culture. We have previously shown that concomitantly with the establishment of intercellular junctions, centrioles separate and microtubules lose their radial organization (Bacallao, R., C. Antony, C. Dotti, E. Karsenti, E. H. K. Stelzer, and K. Simons. 1989. J. Cell Biol. 109:2817-2832. Buendia, B., M. H. Brt, G. Griffiths, and E. Karsenti. 1990Karsenti. . 110:1123Karsenti. -1136. In this work, we have examined the pattern of microtubule nucleation before and after the establishment of intercellular contacts. We analyzed the elongation rate and stability of microtubules in single and confluent cells. This was achieved by microinjection of Paramecium axonemal tubulin and detection of the newly incorporated subunits by an antibody directed specifically against the Paramecium axonemal tubulin. The determination of newly nucleated microtubule localization has been made possible by the use of advanced doubleimmunofluorescence confocal microscopy. We have shown that in single cells, newly nucleated microtubules originate from several sites concentrated in a region localized close to the nucleus and not from a single spot that could correspond to a pair of centrioles. In confluent cells, newly nucleated microtubules were still more dispersed. The microtubule elongation rate of individual microtubules was not different in single and confluent cells (4/xm/min). However, in confluent cells, the population of long lived microtubules was strongly increased. In single or subconfluent cells most microtubules showed a tla of < 30 min, whereas in confluent monolayers, a large population of microtubules had a t~a of >2 h. These results, together with previous observations cited above, indicate that during the establishment of polarity in MDCK cells, microtubule reorganization involves both a relocalization of microtubule-nucleating activity and increased microtubule stabilization.

“…We have used, however, a newly developed tool consisting of unmodified Paramecium ciliary tubulin and a polyclonal antibody (Cohen et al, 1982) which reacts strongly with this tubulin but is totally unreactive with vertebrate cytoplasmic or mitotic tubulins (Adoutte et al, 1985). It appeared, therefore, that we might be able to follow specifically the in vivo incorporation of Paramecium tubulin into the MTs of a vertebrate cell by using this specific antibody.…”

mentioning

confidence: 99%

Microtubule dynamics investigated by microinjection of Paramecium axonemal tubulin: lack of nucleation but proximal assembly of microtubules at the kinetochore during prometaphase.

Geuens

The Journal of Cell Biology

Levilliers

et al. 1989

Abstract. Microtubule (MT) dynamics in PtK2 cells have been investigated using in vivo injection of unmodified Paramecium ciliary tubulin and time-lapse fixation. The sites of incorporation of the axonemal tubulin were localized using a specific antibody which does not react with vertebrate cytoplasmic tubulin (Adoutte, A., M. Claisse, R. Maunoury, and J. Beisson. 1985. J. Mol. EvoL 22:220-229), followed by immunogold labeling, Nanovid microscopy, and ultrastructural observation of the same cells. We confirm data from microinjection of labeled tubulins in other cell types (Soltys, B.