Related Experiment Videos
Purification of a WD repeat protein, EMAP, that promotes microtubule dynamics through an inhibition of rescue
D R Hamill1, B Howell, L Cassimeris
1Department of Biochemistry, Cell and Molecular Biology, University of Kansas, Lawrence, Kansas 66045, USA.
Abstract:
The major microtubule-associated protein in echinoderms is a 77-kDa, WD repeat protein, called EMAP. EMAP-related proteins have been identified in sea urchins, starfish, sanddollars, and humans. We describe the purification of sea urchin EMAP and demonstrate that EMAP binding to microtubules is saturable at a molar ratio of 1 mol of EMAP to 3 mol of tubulin dimer. Unlike MAP-2, MAP-4, or tau proteins, EMAP binding to microtubules is not lost by cleavage of tubulin with subtilisin. In addition to binding to the microtubule polymer, EMAP binds to tubulin dimers in a 1:1 molar ratio. The abundance of EMAP in the egg suggests that it could function to regulate microtubule assembly. To test this hypothesis, we examined the effects of EMAP on the dynamic instability of microtubules nucleated from axoneme fragments as monitored by video-enhanced differential interference contrast microscopy. Addition of 2.2 microM EMAP to 21 microM tubulin results in a slight increase in the elongation and shortening velocities at the microtubule plus ends but not at the minus ends. Significantly, EMAP inhibits the frequency of rescue 8-fold without producing a change in the frequency of catastrophe. These results indicate that EMAP, unlike brain microtubule-associated proteins, promotes microtubule dynamics.
Insights
Echinoderm microtubule-associated protein (EMAP) binds to tubulin dimers and polymers. This protein promotes microtubule dynamics by increasing elongation and shortening velocities and inhibiting rescue frequency.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Microtubule-associated proteins (MAPs) regulate microtubule dynamics.
- Echinoderms possess a major microtubule-associated protein, EMAP, a 77-kDa WD repeat protein.
- EMAP-related proteins are found in various species, including humans.
Purpose of the Study:
- To purify sea urchin EMAP and characterize its binding to microtubules.
- To investigate the effect of EMAP on microtubule assembly and dynamic instability.
- To compare EMAP's function with other known MAPs like MAP-2, MAP-4, and tau.
Main Methods:
- Purification of sea urchin EMAP.
- Analysis of EMAP binding to microtubules and tubulin dimers using varying molar ratios.
- Assessment of EMAP's effect on microtubule dynamic instability using video-enhanced differential interference contrast microscopy with axoneme fragments.
Main Results:
- EMAP binds microtubules saturably at a 1:3 molar ratio of EMAP to tubulin dimer.
- EMAP binding is resistant to subtilisin cleavage of tubulin.
- EMAP binds tubulin dimers in a 1:1 molar ratio.
- EMAP slightly increases microtubule elongation and shortening velocities at plus ends.
- EMAP significantly inhibits microtubule rescue frequency eightfold without affecting catastrophe frequency.
Conclusions:
- EMAP functions differently from brain MAPs, promoting microtubule dynamics.
- EMAP's interaction with tubulin dimers and polymers suggests a regulatory role in microtubule assembly.
- EMAP's unique properties may be crucial for microtubule functions in echinoderms.