UC Davis

Microtubules are a cellular cytoskeleton network that are essential for cell division, intracellular cargo transport, cell migration and overall cellular structure. Microtubule-associated proteins (MAPs) bind along the microtubule lattice and regulate key microtubule-based processes including microtubule dynamics and direction of microtubule motors. Many MAPs were initially hypothesized to perform similar and seemingly redundant functions such as the stabilization of microtubules; however, our work provides insight into how different MAPs may adopt different regulatory binding mechanisms that allow them to execute different functions alongside each other within the microtubule landscape. In this work, we investigate the Doublecortin (DCX) superfamily. A majority of the members of the Doublecortin superfamily contain two evolutionarily conserved microtubule-binding domains linked by an in intrinsically disordered region and are implicated in various diseases. We investigate two members of this superfamily in particular: Doublecortin-like kinase 1 (DCLK1) and Doublecortin (DCX) in order to explore regulatory mechanisms that may provide deeper understanding into the molecular functions of the DCX superfamily on the microtubule lattice.In the first part of this work, we explore the role of the kinase domain of DCLK1 in tuning its affinity to microtubules. DCLK1 utilizes a multi-step process involving autophosphorylation of a residue within its C-terminal tail, resulting in the restriction of DCLK1’s kinase domain from aberrant hyperphosphorylation of its microtubule binding regions. We were able to show that hyperphosphorylation of DCLK1’s microtubule binding regions (via deletion of its C-terminal tail and subsequently increased kinase activity) resulted in drastic decreases in its microtubule-binding affinity. Microtubule binding was rescued when highly phosphorylated residues were mutated into non-phosphorylatable sites, suggesting that autophosphorylation within the C-terminal tail initiates the steps responsible for modulating DCLK1 microtubule-binding affinity. In the second part of this work, we explore the cooperative binding behavior of DCX by dissecting the role of its two microtubule binding domains as well as its intrinsically disordered linker region and how those domains may affect DCX sensitivity to variations in the microtubule lattice. We find that cooperative behavior of Doublecortin results in underlying lattice compaction and removal of the N-terminal microtubule binding domain can disrupt both cooperative binding behavior and microtubule compaction. Lastly, we explored the role of the intrinsically disordered linker region of DCX as a modulator of DCX’s direct cooperative behavior. With the study of these two MAPs belonging to the DCX superfamily, we hope to start providing new insights that may lead to future pathophysiological hypotheses involving not only DCLK1 and DCX, but also other DCX superfamily members, which require deeper investigation into their molecular function and their dysregulation in disease.