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Effects of magnesium on the dynamic instability of individual microtubules
E T O'Brien1, E D Salmon, R A Walker
1Department of Cell Biology, Duke University, Durham, North Carolina 27710.
Abstract:
We investigated the effect of magnesium ion (Mg) on the parameters of dynamic instability of individual porcine brain microtubules. Rates of elongation and rapid shortening were measured by using video-enhanced DIC light microscopy and evaluated by using computer-generated plots of microtubule length vs time. Increasing [Mg] from 0.25 to 6 mM increased the second-order association rate constant for elongation about 25% at each end. At plus ends, this resulted in a 1.5-2-fold increase in elongation rates over the tubulin concentrations explored. Rapid shortening rates were more dramatically affected by Mg. As [Mg] was increased from 0.25 to 6 mM, the average rate of rapid shortening increased about 3-fold at plus ends and 4-5-fold at minus ends. The ends had roughly equivalent average rates at low [Mg], of 30-45 microns/min. At any Mg concentration, rates of disassembly varied from one microtubule to another, and often an individual microtubule would exhibit more than one rate during a single shortening phase. Individual rates at 6 mM Mg varied from 12 to 250 microns/min. Over the concentration range explored, Mg affected the frequencies of transition from elongation to shortening and back only at minus ends. Minus ends were relatively stable at low [Mg], having 4 times the frequency of rescue than at high [Mg], and a lower frequency of catastrophe (particularly evident at low tubulin concentrations). Plus ends, surprisingly, were highly unstable at all Mg concentrations investigated, having about the same transition frequencies as did the least stable (high Mg) minus ends. Our results have implications for models of the GTP cap, again emphasizing that GTP caps cannot build up in proportion to elongation rate, and must be constrained to the tips of growing microtubules.
Insights
Magnesium ions significantly increase microtubule shortening rates, particularly at minus ends, while plus ends remain highly unstable regardless of magnesium concentration. This impacts models of microtubule dynamics and GTP cap formation.
Area of Science:
- Biochemistry
- Cell Biology
- Biophysics
Background:
- Microtubules are essential cytoskeletal components involved in cell division and intracellular transport.
- Dynamic instability, characterized by periods of growth and shrinkage, is a fundamental property of microtubules.
- Magnesium ions are crucial cofactors for tubulin polymerization and microtubule function.
Purpose of the Study:
- To investigate the effect of magnesium ion concentration on the dynamic instability parameters of porcine brain microtubules.
- To quantify the influence of magnesium on microtubule elongation and shortening rates at plus and minus ends.
- To explore the impact of magnesium on microtubule catastrophe and rescue frequencies.
Main Methods:
- Video-enhanced differential interference contrast (DIC) light microscopy was used to observe individual microtubules.
- Microtubule length changes over time were recorded and analyzed using computer-generated plots.
- Measurements of elongation and rapid shortening rates were performed at varying magnesium concentrations (0.25–6 mM).
Main Results:
- Increasing magnesium concentration (0.25–6 mM) increased microtubule elongation rates by 1.5–2 fold at plus ends.
- Magnesium significantly accelerated rapid shortening rates: ~3-fold at plus ends and ~4–5-fold at minus ends.
- Minus ends showed decreased catastrophe frequency and increased rescue frequency at low magnesium, while plus ends remained highly unstable across all tested magnesium levels.
Conclusions:
- Magnesium ions play a critical role in modulating microtubule dynamic instability, particularly enhancing depolymerization rates.
- The differential effects of magnesium on plus and minus ends highlight distinct regulatory mechanisms.
- Findings suggest that GTP caps do not scale with elongation rate and are spatially constrained, impacting models of microtubule dynamics.