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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.
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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.
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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.
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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. |
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Watch a movie of an rsw7 spindle collapsing.
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For this project we
have developed a line of plants that express both GFP-tubulin, which
makes the spindles visible in living plants, and GFP-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 GFP-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.
See a video of mitosis in a plant expressing GFP-chromatin. Watch for a lagging chromosome separation in the last division (final arrow).
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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 |
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