Mitotic Spindle Formation in Plants

Mitotic spindles are microtubule-based structures that separate chromosomes during mitosis. In most animals and fungal cells, spindle microtubules nucleate from centrosomes or spindle pole bodies. Plant cells lack such structured microtubule organizing centers, and some of their microtubules appear to nucleate from near the nuclear envelope, but very little is known about spindle formation in plants (reviewed in Bannigan et al., 2008). Vos et al. (pp. 2783–2797) provide evidence that, despite their differences, plant mitotic spindles could form via similar molecular mechanisms to those found in animals.

Targeting Protein for Xklp2 (TPX2) is a regulator of spindle assembly in animal cells that induces microtubule nucleation and targets certain proteins to spindle microtubules. Vos et al. identified a potential TPX2 ortholog in Arabidopsis by sequence similarity. Modeling based on the crystal structure of human TPX2 with one of its binding partners shows that At TPX2 could carry out a similar interaction. The authors also found that At TPX2 can replace endogenous Xenopus laevis TPX2 to induce the formation of microtubule asters in extracts from X. laevis oocytes. In addition, At TPX2 can coimmunoprecipitate one of Xl TPX2's binding partners.

In tobacco BY-2 cells, TPX2 antibodies label the inside of the nucleus from interphase to G2, a perinuclear region early in prophase, and the spindle later in mitosis (see figure). Using various constructs and organisms, the authors found that At TPX2 has nuclear localization signals and a microtubule binding domain. Unlike other TPX2s, however, At TPX2 also has a signal for nuclear export. In addition, a movie of GFP-tagged TPX2 in BY-2 cells shows increasing TPX2 signal in the nucleus as chromatin condenses, then export of the signal from the nucleus and labeling in two foci before nuclear envelope breakdown. These data are consistent with At TPX2 functioning in the formation of the spindle, which in plants begins outside of the nucleus before nuclear envelope breakdown.

Figure 1.

Arabidopsis TPX2 is exported from the nucleus in prophase. Tobacco BY-2 cells labeled with anti-AtTPX2 antibody (left), antitubulin antibody (middle), and DAPI staining (right). In interphase (left cell), TPX2 is in the nucleus. In prophase (middle cell), the TPX2 label is found around the nucleus. After the nuclear envelope breaks down (right cell), TPX2 is in the same area as the spindle microtubules. Bar = 10 μm.

In a test of TPX2 function, the authors injected antibodies against TPX2 into Tradescantia virginiana stamen hair cells. When they were injected during metaphase, anaphase, or telophase, there was little or no effect. Antibodies introduced during prophase, however, caused either an increase in prophase duration or a complete arrest of the cell cycle. In sum, Vos et al. have provided evidence that At TPX2 is exported from the nucleus before nuclear envelope breakdown, most likely to participate in the formation of the mitotic spindle. In addition, the results suggest that certain aspects of spindle formation are likely conserved across kingdoms.