| Abstract No.: | A-A1011 |
| Country: | Canada |
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| Title: | THE ROLE OF GGMAP215 IN THE REGULATION OF MICROTUBULES IN AXONS AND GROWTH CONES |
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| Authors/Affiliations: | 1 Fiona Wong*; 1 Elise Stanley;
1 Toronto Western Research Institute, ON, Canada
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| Content: | Axon dynamics are dependent on the precise, coordinated adjustment of cytoskeletal elements that underlie its morphology. Microtubules (MTs) are critical components of the cytoskeleton that serve as structural scaffolds and tracks for axonal transport. However, the mechanisms whereby these fibers are regulated during development are complex and poorly understood. MTs can be regulated through interactions with MT-associated proteins (MAPs). The members of the evolutionarily conserved MAP215/Dis1 family are well characterized in mitotic cells, important for MT stabilization, and essential for MT spindle formation. Objective: To localize GgMAP215, the chicken member of the MAP215/Dis1 family, to the axonal cytoplasm in chick sensory ganglia. Materials and Methods: Immunocytochemical data was acquired using high resolution fluorescence microscopy and images were de-blurred offline using a constrained iterative deconvolution algorithm. Intensity correlation analysis (ICA), a test whether two proteins covary, was used to show if GgMAP215 and microtubules are associated. A MT-disrupting drug, nocodazole, was added to cultures to depolymerize MTs and investigate whether GgMAP215 distributions were altered in the growth cone. Short interfering RNAs (siRNAs) were designed against GgMAP215 and were successfully co-transfected with GFP into dissociated DRG neurons using the Helios Gene Gun. Results: GgMAP215 puncta strongly stains the nodes of Ranvier, the initial segment, and the distal axon and growth cone, which are all areas of MT stabilization in neurons. ICA analysis demonstrates that GgMAP215 and ¦Á-tubulin stains covary in de-myelinated axons, and suggests that after nocodazole treatment, GgMAP215 increases its association with reconstituting MTs during drug recovery. Immunocytochemistry demonstrated GgMAP215 knockdown with a concurrent loss of ¦Á-tubulin staining in the cell body. Conclusion: These results indicate that GgMAP215 plays a key role in MAP-dependent MT regulation in primary neurons and their growth cones. |
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