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

Abstract

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...

The rise and fall of the phragmoplast microtubule array

“…33]. However, the plus ends of the interdigitating MTs have to be positioned at the phragmoplast midzone to define the destination of vesicle transport.…”

“…When two genes encoding functionally redundant Kinesin-12 motors are mutated, the phragmoplast MT array no longer forms a defined midzone [31]. MT polymerization continues at the plus ends after the anti-parallel MTs are cross-linked by MAP65-3 [32,33]. However, the plus ends of the interdigitating MTs have to be positioned at the phragmoplast midzone to define the destination of vesicle transport.…”

Patterns of microtubule organization in two polyhedral cell types in the gametophyte of the liverwort Marchantia paleacea Bert.

“…The nuclear envelope may temporarily nucleate Mts at a particular stage of the cell cycle (Falconer, Donaldson & Seagull, 1988;Seagull, 1989;Vantard et al, 1990;Palevitz, 1991). A similar temporal relationship between cell plate formation and daughter nuclei reconstitution exists in the cell types examined here and the cells of the moss Sphagnum (Schnepf, 1973 ; see also Steer, 1984), an event which could explain the cortical reappearance of the cortical Mts in these plants.…”

Interaction of Antiparallel Microtubules in the Phragmoplast Is Mediated by the Microtubule-Associated Protein MAP65-3 inArabidopsis

“…This suggests that MTs are nucleated from the distal ends and polymerized toward the division site. However, in permeabilized tobacco (Nicotiana tabacum) cells and Haemanthus endosperm cells, exogenous tubulins were incorporated into the phragmoplast overwhelmingly in its midline (Vantard et al, 1990;Asada et al, 1991). These studies also showed that newly polymerized MTs overlapped and then slid outward.…”