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Completed Project

Interrogating Specific Interactions in the Tau Protein Projection Domain

Amizhdini Eswaramoorthy, Vassar College ’17 and Prof. Zachary Donhauser
Tau is a microtubule associated protein (MAP) that plays an important role in regulating microtubule spacing, polymerization, and stabilization in axons. Full-length tau is a 441 amino acid protein composed of a microtubule binding domain, a positively charged proline rich region, and a negatively charged projection domain. When tau is bound to microtubules, the projection domain protrudes from the microtubule surface and is able to interact with tau proteins on neighboring microtubules. The electrostatic zipper model posits that the interactions between neighboring tau proteins are based on complementary pairing of oppositely charged regions in the projection domains and proline rich regions, and this enables tau to specifically control microtubule spacing. By this hypothesis, tau forms homodimers in solution by charge interactions. In this work, atomic force microscopy (AFM) and a form of surface quantitation were used to investigate interactions between tau proteins. A mutant form of tau (PD) containing only the projection domain and part of the proline rich region was studied. PD-wild type interactions and PD-PD interactions were measured in buffer at varied ionic strengths through solution cycling of 1mM K-Pipes buffer, 1mM K-Pipes 100 mM NaCl, and a second wash of 1 mM K-Pipes. In the AFM, tau was incubated on a mica surface with a surface charge similar to that of microtubules, and a silicon nitride tip. Interactions between tau layers on the surfaces were then measured as force as a function of tip-sample distance. In surface quantitation assays, tau was incubated on mica, washed with buffer and retrieved with liftoff buffer for quantitation with micro- BCA assay. Evidence of PD-wild type dimerization supports the electrostatic zipper model. These assays also showed that PD binds to the mica surface itself, which may have complicated the measurements. Future work will include further AFM and surface quantitation assays to test whether PD could displace wild-type tau in homodimers.