A novel role for doublecortin and doublecortin like kinase in growth cone steering

Daphney Carmelle Jean

doi:10.17918/00007323

Doublecortin (DCX) is a microtubule-associated protein first identified as one of the gene products associated with the neuronal migration disorder known as Lissencephaly. DCX is a member of a larger gene family that shares the microtubule-binding activity of DCX. The most closely related family member is doublecortin-like kinase (DCLK). In vivo and in vitro studies have shown that these proteins have overlapping functions in both neuronal migration and axonal outgrowth. My work focuses on the contribution of these proteins to axonal growth, during which they appear to have important functions in the growth cone, the motile structure at the tip of the elongating axon that steers it to its target. Guidance signals detected by the growth cone lead to dynamic changes in the configuration of the growth cone cytoskeleton, which consists mainly of microtubules and actin filaments, to direct growth cone shape and motility and thereby the direction of axonal growth. Three distinct cytoskeletal compartments exist in the growth cone, a peripheral actin-rich domain that includes the filopodia and lamellipodia, a central domain that contains the distal ends of axonal microtubules and a transition zone at the interface between these two domains. The crucial role in growth cone steering of microtubules hinges on their ability to invade the filopodia and stabilize growth cone motility in a specific direction. This is regulated by many factors, which include motor proteins and microtubule-associated proteins. Prior to my work, little was known about the roles played by DCX and DCLK in growth cone steering. However, their localization provides a substantial clue. DCX and DCLK are enriched in the growth cone where both proteins associate with the microtubule polymer in a gradient that increases sharply as the microtubules extend from the base of the growth cone to its periphery. Given the preferential enrichment of DCX and DCLK on microtubules that invade the peripheral domain, I hypothesized that these proteins impact aspects of microtubule configuration and function to support their role in steering the growth cone. My thesis work demonstrates that siRNA mediated-depletion of DCX and DCLK results in severe bending of growth cone microtubules. The bent microtubules appear as if they are buckling under strong forces. Indeed, inhibition of myosin-II, which generates forces on the actin cytoskeleton to push microtubules back toward the central domain, significantly decreases the extent of microtubule bending in growth cones depleted of DCX and DCLK. In contrast. overexpression of DCX and DCLK result in microtubules that are substantially straighter than usual. In addition, in the absence of DCX and DCLK, the depth of microtubule invasion into the peripheral domain is reduced compared to controls, and at a functional level, growth cone responses to substrate guidance cues are altered. These findings support a role for DCX and DCLK in fine-tuning the manner by which microtubules invade the actin-rich peripheral domain of the growth cone, thereby facilitating the ability of the growth cone to carry out its crucial pathfinding functions.

A novel role for doublecortin and doublecortin like kinase in growth cone steering

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A novel role for doublecortin and doublecortin like kinase in growth cone steering

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