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A
role for de novo polymerization
and chromosome number in midzone complex formation
J.C. Canman
and E.D. Salmon
The microtubule
(Mt)-containing midzone complex is formed between anaphase onset
and cytokinesis (M-phase exit) and is thought to be involved in
positioning the cytokinetic furrow. The role of Mts in midzone
complex formation has remained elusive because perturbation of
Mts results in an M-phase arrest at the spindle checkpoint.
We have therefore analyzed midzone complex formation in cells
injected with anti-Mad2p antibody to overcome the spindle checkpoint
under conditions of Mt disruption. The timing of furrow
onset, lamellar extension, furrow completion, and cytoskeletal
rearrangement was similar in both anti-Mad2p-induced and control
M-phase exit. To examine the role of Mts in midzone
complex formation, PtK1 cells treated with varying
concentrations of nocodazole (NZ) were microinjected with anti-Mad2p
antibody and analyzed for the localization of F-actin, tubulin,
and DNA. Similar to uninjected cells, the extent of Mt depolymerization
in anti-Mad2p injected cells was dependent on the level of NZ
in the media. However, unlike cells injected in the absence
of NZ, anti-Mad2p injected cells in the presence of NZ contained
paired anti-tubulin-staining "dots" near the chromosomes.
Anti-Mad2p injected cells in lower concentrations of NZ (to disrupt
Mt dynamics but not grossly affect polymer levels), contained
small bundles of Mts which appeared to be stem bodies, the structures
which eventually form the midbody. Like the "dots",
the stem bodies were located in the same area as the chromosomes.
The numbers of "dots" (~22) and stem bodies (~11)
correlated with the number of chromosomes (11) in PtK1 cells.
Interestingly, when prometaphase cells in low concentrations of
nocodazole were stained with tubulin antibodies, small dots of
tubulin staining were seen at the kinetochores. The separation
distance between these "kinetotubes" and the paired "dots" seen
in anti-Mad2p antibody injected cells is similar, indicating a
possible release of these "dots" in anaphase. We conclude
that chromosomes may play a role in midzone complex formation
in PtK1 cells.
Supported by NIHGMS 24364
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