Related Experiment Videos
Microtubule assembly in the presence of adenosine triphosphate
Abstract:
Microtubule (MT) assembly was investigated in the presence of ATP and Ca ions using both crude extract (CE) and purified microtubular proteins (PMP) prepared from porcine brains. ATP inhibited MT assembly from CE prepared with the reassembly buffer containing 1 mM GTP. Half-maximal inhibition occurred at an ATP concentration of 0.4-0.5 mM. Calcium ions, on the contrary, cancelled the ATP-induced inhibition, 1-2 microM calcium ions supporting maximal MT assembly. The ATP-induced inhibition in PMP was not so prominent as in CE, but occurred significantly in the presence of RNA. In PMP dissolved in the reassembly buffer containing ATP and yeast tRNA, the content of the ring fraction decreased significantly as compared with PMP containing only RNA. Furthermore, microtubule-associated proteins were found to be capable of binding ATP. The significance of the ATP-induced inhibition of MT assembly and the release of the inhibition by Ca2+ was discussed.
Insights
Adenosine triphosphate (ATP) inhibits microtubule assembly in crude brain extracts, but calcium ions reverse this effect. This calcium-mediated reversal of ATP inhibition is crucial for microtubule dynamics.
Area of Science:
- Biochemistry
- Cell Biology
- Neuroscience
Background:
- Microtubule assembly is fundamental for cellular structure and function.
- Regulation of microtubule dynamics is essential for processes like cell division and transport.
- The roles of ATP and calcium ions in microtubule assembly are complex and require further elucidation.
Purpose of the Study:
- To investigate the effects of Adenosine Triphosphate (ATP) and calcium ions on microtubule (MT) assembly.
- To compare the influence of ATP and Ca2+ on both crude extracts (CE) and purified microtubular proteins (PMP) from porcine brains.
- To explore the interaction of microtubule-associated proteins with ATP.
Main Methods:
- In vitro microtubule assembly assays using porcine brain crude extract (CE) and purified microtubular proteins (PMP).
- Varying concentrations of ATP and calcium ions were introduced to observe their effects on MT assembly.
- Analysis of protein fractions, including the ring fraction, in the presence of ATP and RNA.
Main Results:
- ATP significantly inhibited microtubule assembly from crude extract, with half-maximal inhibition at 0.4-0.5 mM ATP.
- Calcium ions (1-2 microM) effectively reversed ATP-induced inhibition, promoting maximal MT assembly.
- ATP-induced inhibition was less pronounced in purified microtubular proteins but significant in the presence of RNA, affecting the ring fraction.
Conclusions:
- ATP acts as an inhibitor of microtubule assembly, particularly in crude cellular extracts.
- Calcium ions play a critical role in regulating microtubule assembly by counteracting ATP-induced inhibition.
- Microtubule-associated proteins can bind ATP, suggesting a direct mechanism for ATP's regulatory role in microtubule dynamics.