The Role of Stathmin and SCG10 in Microtubule Dynamics in the Growth Cone

Christina Bill
Colorado State University

Microtubule dynamics are an important part of neuronal development, including cell polarization, neurite extension and turning, axonal transport and synapse formation. Microtubule dynamics are regulated by capturing plus ends and stabilizing the microtubules or by enhancing rapid depolymerization through catastrophes. These events are coordinately regulated by rho family GTPases that are linked to membrane receptors. Destabilization of microtubules is regulated in part by stathmin (also called Op18 or metablastin), a ubiquitous, cytosolic protein that is inhibited through phosphorylation at several sites within its N-terminus region. The stathmin family of proteins includes the neuronal proteins SCG10, SCLIP, and RB3. SCG10 is of interest because its first 34 amino acids contain two cysteines that are palmitoylated, which targets it to the growth cone. To study the role of SCG10 in neuronal growth cone behavior, we have constructed a plasmid vector containing a coding region for the first 34 amino acids of SCG10 with and without the di-cysteine motif. In frame with this, we have inserted the coding region of wild type Xenopus stathmin, and are in the process of inserting Xenopus single, double, and triple point mutants in which ala has been substituted for the three phosphorylatable ser/thr residues. The cDNAs encoding these SCG-stathmin chimeras will then be inserted into an AdTrack shuttle vector for production of adenovirus so that these proteins can be effectively expressed in infected neurons. The effects of expressing these constructs on neuronal growth cone responses to guidance cues will then be determined using growth cone turning assays. The SCG-stathmin without the di-cysteine motif should not be localized to the growth cone, and will provide a control for growth effects that might be caused within the soma. Western blot analysis and immunostaining will also be used to quantify and verify localization of SCG-stathmin. Microtubule stabilization and interaction with the actin filament system are essential for growth cone pathfinding and outgrowth. These studies should help to elucidate the essential regulatory sites on stathmin necessary for these processes.