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Characterization of the microtubule-binding domain of microtubule-associated protein 1A and its effects on
A R Vaillant1, R Müller, A Langkopf
1Department of Biology, University of Ottawa, Ottawa K1N 6N5, Canada.
Abstract:
To determine how MAP1a interacts with microtubules we expressed several 6myc-tagged MAP1a fragments in P19 EC and HeLa cells. Confocal immunofluorescence microscopy showed that the fragment consisting of amino acids (aa) 1-281 of MAP1a did not bind while the fragment consisting of aa 1-630 did, indicating that the region of MAP1a between aa 281 and 630 contains a microtubule-binding domain. Deletion of the basic repeats from aa 336-540 did not result in loss of microtubule binding, suggesting that the regions flanking the basic repeats can bind MAP1a to microtubules. These observations were confirmed using an in vitro microtubule binding assay. The levels of acetylation and detyrosination of polymerized microtubules were assessed by quantitative dot blotting in cells expressing MAP1a fragments or MAP2c. Compared with untransfected cells, the polymerized tubulin in cells expressing full-length MAP1a was more acetylated and detyrosinated, but these increases were smaller than those seen in cells expressing MAP2c. Consistent with this, the microtubules in MAP2c expressing cells were more resistant to colchicine than those in cells overexpressing MAP1a. These data implicate aa 281-336 and/or 540-630 of MAP1a in microtubule binding and suggest that MAP1a is less able to stabilize microtubules than MAP2c.
Insights
Microtubule-associated protein 1A (MAP1a) binds microtubules via amino acids 281-630. MAP1a enhances microtubule acetylation and detyrosination, but less effectively than MAP2c.
Area of Science:
- Cell Biology
- Neuroscience
- Molecular Biology
Background:
- Microtubule-associated proteins (MAPs) are crucial for microtubule dynamics and stability.
- MAP1a is a major MAP in the brain, but its precise microtubule-binding domain and stabilizing functions are not fully understood.
Purpose of the Study:
- To identify the microtubule-binding domain of MAP1a.
- To investigate the effects of MAP1a on microtubule post-translational modifications and stability.
Main Methods:
- Expression of 6myc-tagged MAP1a fragments in P19 EC and HeLa cells.
- Confocal immunofluorescence microscopy and in vitro microtubule binding assays.
- Quantitative dot blotting to assess tubulin acetylation and detyrosination.
Main Results:
- The MAP1a region between amino acids (aa) 281-630 contains the microtubule-binding domain.
- Regions flanking the basic repeats (aa 336-540), specifically aa 281-336 and/or 540-630, are implicated in binding.
- MAP1a expression increased microtubule acetylation and detyrosination, but to a lesser extent than MAP2c.
- Microtubules in MAP2c-expressing cells showed greater resistance to colchicine than in MAP1a-expressing cells.
Conclusions:
- MAP1a interacts with microtubules through specific domains, primarily aa 281-630.
- MAP1a influences microtubule post-translational modifications, contributing to stabilization.
- MAP1a exhibits a lower capacity for microtubule stabilization compared to MAP2c.