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* Biology Department, University of North Carolina, Chapel Hill, North Carolina 27599-3280; and The current two-state GTP cap model of microtubule dynamic instability proposes that a terminal
crown of GTP-tubulin stabilizes the microtubule lattice
and promotes elongation while loss of this GTP-tubulin
cap converts the microtubule end to shortening. However, when this model was directly tested by using a
UV microbeam to sever axoneme-nucleated microtubules and thereby remove the microtubule's GTP
cap, severed plus ends rapidly shortened, but severed
minus ends immediately resumed elongation (Walker,
R.A., S. Inoué, and E.D. Salmon. 1989. J. Cell Biol. 108:
931-937).
To determine if these previous results were dependent on the use of axonemes as seeds or were due to
UV damage, or if they instead indicate an intermediate
state in cap dynamics, we performed UV cutting of self-assembled microtubules and mechanical cutting of axoneme-nucleated microtubules. These independent
methods yielded results consistent with the original
work: a significant percentage of severed minus ends
are stable after cutting. In additional experiments, we
found that the stability of both severed plus and minus
ends could be increased by increasing the free tubulin concentration, the solution GTP concentration, or by
assembling microtubules with guanylyl-( Our results show that stability of severed ends, particularly minus ends, is not an artifact, but instead reveals the existence of a metastable kinetic intermediate
state between the elongation and shortening states of
dynamic instability. The kinetic properties of this intermediate state differ between plus and minus ends. We
propose a three-state conformational cap model of dynamic instability, which has three structural states and
four transition rate constants, and which uses the asymmetry of the tubulin heterodimer to explain many of
the differences in dynamic instability at plus and minus ends.
Biology Department,
Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0406
,
)-methylene-diphosphonate (GMPCPP).
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