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

Daphney C. Jean 1 Peter W. Baas 1 Mark M. Black 0 0 Department of Anatomy and Cell Biology, Temple University School of Medicine , 3420 North Broad Street, Philadelphia, PA 19140, USA 1 Department of Neurobiology and Anatomy, Drexel University College of Medicine , 2900 Queen Lane, Philadelphia, PA 19129, USA Doublecortin (DCX) and doublecortin-like kinase (DCLK), closely related family members, are microtubuleassociated 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. - INTRODUCTION Doublecortin (DCX) is a microtubule-associated protein first identified as a gene product associated with the neuronal migration disorder known as lissencephaly (1). Pathological mutations in DCX impair its binding to microtubules (2), indicating that the loss of microtubule functions dependent on DCX underlies lissencephaly. Initial mouse models exploring the underlying mechanisms of lissencephaly revealed limited migratory phenotypes due to genetically redundant pathways that compensate for the lack of DCX. DCX is a member of a larger gene family that includes doublecortin-like kinase 1 (DCLK). Depleting both DCX and DCLK produces a more severe phenotype than depleting either protein alone (3,4). Furthermore, double knockdowns display widespread axonal defects in mice, and this is also the case when cortical or hippocampal neurons from these animals are grown in culture (3,4). Clues about the potential functions of DCX and DCLK are suggested by their distribution in growing axons. We recently confirmed earlier observations that DCX is enriched in the growth cones of elongating axons (5 7), and further showed that DCX associates in a gradient along microtubules that increase sharply as they extend from the base of the growth cone to its periphery (8). DCLK exhibits a similar distribution, suggesting that both proteins specialize microtubules for the unique environment of the growth cone compared with the axonal shaft (9). Growth cones can be divided into three regions (10). The peripheral domain is the flattened actin-rich lamellar part of the cone, which also includes its filopodia. The central domain is contiguous with the axonal shaft and contains the majority of the microtubule mass of the growth cone. The transition zone lies at the interface of these two domains; it is a gateway through which microtubules must pass in order to enter the peripheral domain from the central domain. Such entry is essential for sustained axonal growth, and the spatial polarization of such entry is necessary for growth cone turning (10). A potential role for DCX and DCLK in the entry of microtubules into the peripheral domain is suggested not only by the enrichment of these proteins on distal regions of microtubules but also by findings that DCX can interact with actin filaments and may contribute to interactions between actin filaments and microtubules (11,12). Another possibility is that DCX and DCLK may directly impact properties of microtubules that influence their ability to invade the peripheral domain. Cryo-electron microscopy has revealed that DCX binds between the protofilaments of microtubules (13), which may be conducive to enabling the microtubule to remain relatively straight. A molecular mechanism to limit microtubule curvature might be especially important in growth cones, where microtubules are subjected to forces generated by various molecular motor proteins. Our present goal was to pursue this idea. Here, we used a combination of acute knockdown and overexpression approaches on cultured rat sympathetic neurons to explore the impact of DCX and DCLK on the properties of microtubules in growth cones and on growth cone navigation behaviors. In our previous studies, no obvious phenotypes were observed when DCX alone was depleted from these neurons (8); axons grew at normal rates and the growth cones of DCX-depleted neurons were not noticeably different from controls in terms of morphology or microtubule distribution. These findings are consistent with the results of other studies in which targeted deletion of DCX in mice produced relatively mild phenotypes (3,14). Similarly, targeting of DCLK, a closely related family member, also produced a mild phenotype (3). However, targeting of both DCX and DCLK produced a more dramatic phenotype with significant impairment in axonal growth (3,4). These findings suggest that DCX and DCLK have at least partially redundant functions and that one can largely compensate for the other in single knockdown experiments. When both proteins are depleted, the loss of their combined contributions to axonal growth becomes readily apparent. Thus, in the present studies, we have targeted both DCX and DCLK together, rather than further exploring the impact of depleting either one of them on its own. DCLK is preferentially concentrated on growth cone microtubules As shown in Figure 1, DCLK is highly enriched in the neuronal growth cone compared with the axonal shaft, confirming previous results of Burgess and Reiner (9). Figure 1 also shows that DCLK localizes to microtubules in the growth cone and its relative abundance on these microtubules increases progressively as microtubules extend from the base of the cone toward its tip. Thus, DCLK closely resembles DCX (8) in terms of its localization and microtubule association in growth cones of cultured sympathetic neurons. Double knockdown of DCX and DCLK impairs axonal growth To co-deplete DCX and DCLK (...truncated)