Abstract
Microtubules are dynamic polymers of GTP- and GDP-tubulin that undergo stochastic transitions between growing and shrinking phases. Rescues, the conversion from shrinking to growing, have recently been proposed to be to the result of regrowth at GTP-tubulin islands within the lattice of growing microtubules. By introducing mixed GTP/GDP/GMPCPP (GXP) regions within the lattice of dynamic microtubules, we reconstituted GXP islands in vitro (GMPCPP is the slowly hydrolyzable GTP analog guanosine-5′-[(α,β)-methyleno]triphosphate). We found that such islands could reproducibly induce rescues and that the probability of rescue correlated with both the size of the island and the percentage of GMPCPP-tubulin within the island. The islands slowed the depolymerization rate of shortening microtubules and promoted regrowth more readily than GMPCPP seeds. Together, these findings provide new mechanistic insights supporting the possibility that rescues could be triggered by enriched GTP-tubulin regions and present a new tool for studying such rescue events in vitro.
Highlights
Microtubules are self-assembling, dynamic polymers that have essential functions in eukaryotic cell structure maintenance, cell division, intracellular transport and other processes [1]
To test the hypothesis that GTP islands rescue microtubule growth, we used the slowly hydrolyzable GTP analog GMPCPP (guanosine59-[(a,b)-methyleno]triphosphate) [7] to mimic the non-hydrolyzed GTP leftover in islands within the lattices of dynamic microtubules (Fig. 1A)
The ends of microtubules assembled with GTP can be transiently stabilized against dilution-induced disassembly by capping the ends with GMPCPP-tubulin subunits [8,9,10]
Summary
Microtubules are self-assembling, dynamic polymers that have essential functions in eukaryotic cell structure maintenance, cell division, intracellular transport and other processes [1]. These GXP islands are defined as lattice regions containing a mixture of GTP-, GDP-, and GMPCPP-tubulin subunits and were identified by a different fluorescent tubulin label. GTP-tubulin into a flow channel in which rhodamine-labeled GMPCPP microtubule seeds had been bound to the cover-slip.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
