A novel role for doublecortin and doublecortin-like kinase in regulating growth cone microtubules

Abstract

Doublecortin (DCX) and doublecortin-like kinase (DCLK), closely related family members, are microtubule-associated proteins with overlapping functions in both neuronal migration and axonal outgrowth. In growing axons, these proteins appear to have their primary functions in the growth cone. Here, we used siRNA to deplete these proteins from cultured rat sympathetic neurons. Normally, microtubules in the growth cone exhibit a gently curved contour as they extend from the base of the cone toward its periphery. However, following depletion of DCX and DCLK, microtubules throughout the growth cone become much more curvy, with many microtubules exhibiting multiple prominent bends over relatively short distances, creating a configuration that we termed wave-like folds. Microtubules with these folds appeared as if they were buckling in response to powerful forces. Indeed, inhibition of myosin-II, which generates forces on the actin cytoskeleton to push microtubules in the growth cone back toward the axonal shaft, significantly decreases the frequency of these wave-like folds. In addition, in the absence of DCX and DCLK, the depth of microtubule invasion into filopodia is reduced compared with controls, and at a functional level, growth cone responses to substrate guidance cues are altered. Conversely, overexpression of DCX results in microtubules that are straighter than usual, suggesting that higher levels of these proteins can enable an even greater resistance to folding. These findings support a role for DCX and DCLK in enabling microtubules to overcome retrograde actin-based forces, thereby facilitating the ability of the growth cone to carry out its crucial path-finding functions.

Doublecortin Is Excluded from Growing Microtubule Ends and Recognizes the GDP-Microtubule Lattice

“…In cells, DCX-binding was greatly reduced at MT segments with high local curvature. Remarkably, this geometry-dependent binding to MTs was completely reversed in the presence of taxanes, which reconciles incompatible observations in cells [9] and in vitro [10]. We propose a model explaining DCX specificity for different MT geometries based on structural changes induced by GTP hydrolysis that decreases the spacing between adjacent tubulin dimers [11].…”

“…Conversely, as expected, TUB-EGFP fluorescence intensity did not depend on MT curvature (Figure 3D). This preference of DCX for straight MTs was surprising as the opposite was recently reported, namely that DCX specifically recognizes highly curved MTs in vitro [10] even though earlier reports also noted decreased DCX staining at bent MTs in neuronal growth cones [9]. Of note, although the vast majority of straight MTs was DCX-decorated, we observed rare occurrences in which few straight MTs did not bind DCX, which may suggest lattice defects resulting in unusual protofilament number [7, 12].…”

“…Doublecortin (DCX), a MT-associated protein (MAP) required for neuronal migration during cortical development [4, 5], binds to the same site as EBs [6], and recent in vitro studies proposed DCX localization to growing MT ends independent of EBs [7]. Because this conflicts with observations in neurons [8, 9], and the molecular function of DCX is not well understood, we revisited intracellular DCX dynamics at low expression levels. Here, we report that DCX is not a +TIP in cells, but in contrast is excluded from the EB1 domain.…”

Doublecortin-like kinase enhances dendritic remodelling and negatively regulates synapse maturation

“…Recently, similar roles of DCLK1 in growth-cone MTs of sympathetic neurons were proposed 25 . MT-binding regions of DCX and DCLK1 are composed of two tandem homologous domains, with differential affinity for assembled MTs and tubulin dimers 3,26,27 .…”

Doublecortin Recognizes the Longitudinal Curvature of the Microtubule End and Lattice

“…DCX may use curvature recognition in its interactions with microtubules in these regions. Indeed, a recent report indicated that RNAi of DCX caused aberrant microtubule curvature in growth cones [47], indicating that DCX may function to keep microtubule curvature within limits. Alternatively, DCX might recognize the longitudinal curvature of microtubule ends as a loading mechanism.…”