Spindle morphogenesis and motor proteins in plants (rsw7 mutant)

While the molecular details of mitosis in animals and fungi are well understood, and copious research into the interphase array of microtubules in plants has been done, the plant mitotic spindle, and especially its molecular machinery, has been something of a blind spot for the past twenty years.

What we know from animal studies is that the spindle is organized and operated by a large number of motor proteins, which act in concert to bind the two halves of the spindle together, focus the poles, attach the microtubules to the chromosomes, and push the two halves of the spindle apart, separating the chromosomes.

A particularly important group of proteins are the kinesin-5 motors. Their job is to connect microtubules from the two halves of the spindle together, and when it comes time to divide, push them apart. In animal cells that lack these motors, the inward-pushing forces overcome any remaining outward-pushing forces, and the poles of the spindle collapse together in a big mess, and the is unable to divide.

Wild type spindles fixed in different stages of mitosis. Microtubules are labelled green, DNA is labelled red.

Clockwise, from top left: early anaphase (chromosomes just staring to separate), metaphase (chromosomes aligned at center of spindle), telophase (chromosomes have separated and new cell wall is forming between them.

This research project focuses on a mutant of the plant Arabidopsis thaliana.  The mutant, which is called rsw7, is defective in a mitotic motor protein belonging to the kinesin-5 family.  Much like animal and yeast cells defective in these proteins, mitotic spindles in rsw7 are massively disrupted and usually collapse into a monopolar spindle at anaphase (see below).  

Despite the catastrophic defect in these spindles, the cell cycle is able to continue, even when the chromosomes are not segregated and cytokinesis fails to take place.  This raises questions about the metaphase checkpoint in plants. It is generally assumed that plants have checkpoints in mitosis, and that they closely resemble those documented in animal cells. However, there is little direct evidence for this assumption, and the continuation of the cell cycle in rsw7 plants suggests that it might not be correct.

A spindle from rsw7. Microtubules are labelled green, chromosomes are labelled red. The spindle poles have collapsed together, leaving the chromosomes attached to the distal microtubule plus ends.

For more information, see paper.

Watch a movie of an rsw7 spindle collapsing.

For this project we have developed a line of plants that express both GFP-tubulin, which makes the spindles visible in living plants, and YFP-chromatin, which makes the chromosomes visible in dividing cells.  Using the confocal microscope, we can make movies of cells as they progress through mitosis. The new YFP-chromatin/GFP-tubulin line will allow us to see whether or not the chromosomes are captured by the abnormal spindle. If chromosomes do not attach to the spindle, and the cell cycle continues anyway, it will suggest that the animal model of metaphase checkpoints does not apply to plants. If the chromosomes do attach to the spindle, we will see whether or not the collapsed spindle is capable of transporting the chromosomes to the poles, even though it is turned inside out. Chromosome transport by collapsed spindles has not been documented in any other organism.

References:

Bannigan, A. and Baskin, T.I. (2007) Emerging molecular mechanisms that power and regulate the anastral mitotic spindle of flowering plants. Cell Motil. Cytoskel. 65(1):1-11

Bannigan, A., Scheible, W-R., Lukowitz, W., Fagerstrom, C., Wadsworth, P., Somerville, C. and Baskin, T.I. (2007) A conserved role for kinesin-5 in plants. J. Cell Sci. 120: 2819-2827