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Microtubule dynamic instability does not result from stabilization of microtubules by tubulin-GDP-Pi subunits
1Department of Biochemistry, University of North Carolina, Chapel Hill 27599-7260, USA. caplow@med.unc.edul
Abstract:
The proposal that microtubule dynamic instability results from stabilization of microtubule ends by tubulin-GDP-Pi subunits (where Pi is inorganic phosphate) [Melki et al. (1996) Biochemistry 35, 12038] was based on studies of GTP hydrolysis and microtubule assembly that showed that tubulin-GDP-Pi subunits can transiently accumulate at microtubule ends. There is no direct evidence that GDP-Pi-subunits can stabilize microtubules under conditions where dynamic instability is observed and this has been inferred from the observation that tubulin-GDP-BeFn subunits stabilize microtubules. To test if tubulin-GDP-Pi stabilizes microtubules we sought evidence for a synergism between the effect of Pi and BeFn. We found, however, that Pi antagonizes the effect of BeFn by displacing it from tubulin subunits. The alternate mechanism in which Pi inhibits BeFn stabilization of microtubules by displacing fluoride from beryllium was ruled out from the 9Be and 19F NMR spectra in the presence and absence of Pi. Further evidence that tubulin-GDP-BeFn is not an analogue of tubulin-GDP-Pi and that tubulin-GDP-Pi is not responsible for maintaining the growth phase in microtubules manifesting dynamic instability was provided by our observation that Pi did not decrease the disassembly rate under conditions where tubulin-GDP-Pi subunits are expected to have formed. Results showing that BeFn binds randomly to subunits in microtubules provided evidence that Pi dissociation from the tubulin-GDP-Pi intermediate formed during GTP hydrolysis occurs randomly rather than processively starting at the growing microtubule tip.
Insights
Inorganic phosphate (Pi) does not stabilize microtubules by forming tubulin-GDP-Pi subunits. Instead, Pi antagonizes the stabilizing effect of beryllium fluoride (BeFn), suggesting Pi dissociation is random, not processive.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Dynamics
Background:
- Microtubule dynamic instability is a key process in cell division and motility.
- Previous models proposed that tubulin-GDP-Pi subunits stabilize microtubule ends, inferred from studies with tubulin-GDP-BeFn.
- Direct evidence for tubulin-GDP-Pi stabilization under dynamic instability conditions is lacking.
Purpose of the Study:
- To directly test if tubulin-GDP-Pi subunits stabilize microtubules.
- To investigate the interaction between inorganic phosphate (Pi) and beryllium fluoride (BeFn) in microtubule stabilization.
- To determine the mechanism of Pi dissociation from tubulin during GTP hydrolysis.
Main Methods:
- Investigated potential synergism between Pi and BeFn effects on microtubule stabilization.
- Utilized 9Be and 19F Nuclear Magnetic Resonance (NMR) spectroscopy to analyze interactions.
- Measured microtubule disassembly rates under conditions where tubulin-GDP-Pi is expected to form.
Main Results:
- Inorganic phosphate (Pi) antagonizes the stabilizing effect of beryllium fluoride (BeFn) by displacing it from tubulin subunits.
- NMR data ruled out Pi inhibiting BeFn stabilization by displacing fluoride from beryllium.
- Pi did not decrease microtubule disassembly rates, indicating tubulin-GDP-Pi does not maintain the growth phase during dynamic instability.
Conclusions:
- Tubulin-GDP-BeFn is not a reliable analogue for tubulin-GDP-Pi in microtubule stabilization studies.
- Tubulin-GDP-Pi subunits are not responsible for maintaining microtubule growth during dynamic instability.
- Pi dissociation from the tubulin-GDP-Pi intermediate occurs randomly, not processively from the microtubule tip.