Determining the role of doublecortin X (DCX) in cytoskeleton organisation

Abstract

X-linked Doublecortin (DCX) is a 40 kDa neuron-specific microtubule (MT)-associated protein. The initial identification of inheritable dcx gene mutations and their disruption of cortical layering and brain development in lissencephaly and subcortical band heterotopia have prompted subsequent evaluation of the functions of the DCX protein. Previous biochemical studies have revealed that DCX binds between four tubulin monomers within the polymerised MT lattice to stabilise the lateral and longitudinal contacts along the MT protofilaments. Although DCX has been proposed to regulate MT-related cellular events, many aspects of its mechanism of actions and its regulation of functions are yet to be determined. The DCX protein consists of two MT-interacting doublecortin (DC) domains, DC1 (the N-terminal DC domain) and DC2 (the C-terminal DC domain), linked in tandem via a flexible unstructured region (linker) and additionally flanked by a likely unstructured N-terminal and S/P-rich C-terminal sequences. Since many DCX pathogenic mutations have been predominantly mapped to within the DC domains to thus disrupt the interaction of DCX with MTs, studies of the regulation of the DCX-MT interaction have been undertaken to improve understanding of the functions of DCX. In addition, the DCX termini that flank the DC domains may act as regulators of DCX function, but whereas the S/P-rich DCX C-terminus is known for its phosphoregulatory role in DCX’s functions, a regulatory role of the DCX N-terminus region is largely unknown.

The studies presented in this thesis address the critical roles of the DCX N- and C-termini in DCX function. Immunoprecipitation and live-imaging approaches using monkey fibroblastoma COS-1 and human neuroblastoma SH-SY5Y cells have been employed to identify the effects of the DCX N- and C-termini on the association of DCX with the cytoskeleton. The results were combined with live-imaging fluorescence recovery after photobleaching (FRAP) protocol findings to define the regulatory impact of DCX termini on dynamics of DCX in association with the cytoskeletal components, MTs and F-ACT. These studies have been the first to show the dynamic association of DCX with MTs in living cells.

The role of the DCX C-terminus has been evaluated by examining the features of DCX lacking its C-terminus. Whereas full length, wildtype DCX shows rapid and complete exchange within the MT network, the exchange of the truncated DCX protein is slowed significantly. Moreover, dynamics of exchange of the C-terminal truncated DCX was unaltered in the presence of a MT-stabilising agent, taxol, or a hyperosmotic stress stimulus, sorbitol, both of which were shown to slow wildtype DCX exchanges rate within the MT network. Thus, DCX dynamically associates with MTs in living cells and its C-terminal region plays important roles in the association of DCX with MTs.

To explore the regulatory role of DCX N-terminus, the influence of the only identified phosphorylation site within the DCX N-terminus, DCX S28, on association of DCX with MTs and F-ACT was assessed. Thus, both DCX S28A (phospho-resistant) and DCX S28E (phospho-mimetic) mutants were examined alongside wildtype DCX. For DCX S28E, decreased interaction with MTs but a shift to favour association with both F-ACT and the ACT-binding protein, spinophilin (Spn) was observed. FRAP studies showed that DCX S28E increased the dynamics of association with MTs. Compared to DCX S28E, the associations with MTs and F-ACT were reversed in the presence of DCX S28A. Therefore, these results highlight a new role for DCX S28 as a regulatory switch for cytoskeletal organisation and thus highlight a contribution by DCX-N in the phosphoregulation of DCX function.

The impact of a pathogenic mutant within the DCX N-terminus, DCX E2K, on the cytoskeletal association was also studied. Unlike most DC domain pathogenic mutants of DCX, the DCX E2K mutant protein retained its ability to interact with MTs. However, MTs in association with DCX E2K showed a reduced sensitivity to nocodazole-induced depolymerisation as well as slower α-tubulin exchanges rate. Furthermore, the DCX E2K mutant showed increased association with the F-ACT. These results highlight the importance of the N-terminus of DCX in regulating the association with, and the coordination of, the MT and F-ACT networks.

Taken together, the studies presented in this thesis have revealed several new features of the regulatory roles of the DCX termini in the association of the DCX protein with MTs and F-ACT. The findings should aid a better understanding of the DCX function in MTs and F-ACT organisation during MT-related cellular events including neuronal cell migration.